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Bio
07
Versatile
Luciferases
New Tools for Reporter Assays
Improving
Productivity
Through Compact Automation
MSIA™
Model system
A Universal Mass Spectrometric
Immunoassay
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Welcome
I am delighted to welcome you to our 7th edition of the Bio-Innovation
series, an educational publication focused on new and emerging trends
for every life scientist in Australia and New Zealand.This edition of BioInnovation focuses on the most recent technology platforms and products
released to market by Thermo Fisher Scientific.
Thermo Fisher Scientific recognises that the local landscape for scientists
is becoming more challenging and more competitive from a funding
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such as this we hope to assist you to increase efficiency and give you
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provide the application resources and the programs to help you achieve it.
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One of the great things about our organisation is our mission. We enable
our customers to make the world healthier, cleaner and safer. l see
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I would like to take this opportunity to thank you for choosing Thermo
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Editor
Mika Mitropoulos: [email protected]
Art & Design
Andrew Dennis: [email protected]
Tony Acciarito
Director, Scientific Business
Thermo Fisher Scientific
Contents
MagJET DNA & RNA purification kits ...................................................................................04
Effective vaccine storage ...................................................................................................06
Versatile Luciferases: New Tools for Reporter Assays............................................................08
ClipTip interlock technology: the next generation for pipettes .................................................12
Water-cooled condenser option for Thermo Scientific ULT Freezers........................................14
Novel glycan column technology........................................................................................ 16
Evaluation of the Formulatrix Tempest at EMD Serono ...........................................................19
Product focus ...................................................................................................................21
Innovation Workflow..........................................................................................................24
Become a luminary in your qPCR world................................................................................28
Large Scale Plasmid DNA Preparation.................................................................................30
Selecting Conical Tubes for Centrifugation Applications.........................................................32
Improving productivity through compact automation.............................................................36
A Universal Mass Spectrometric Immunoassay (MSIA™) Model system.................................40
Amplification of whole human mitochondrial DNA ................................................................42
Iodoacetyl tandem mass tags .............................................................................................44
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3 Nucleic acid isolation being one of the
most fundamental steps in molecular
biology applications is a prerequisite
for successful experimental workflow.
MagJET nucleic acid purification
kits utilise magnetic bead-based
technology allowing for highly efficient
nucleic acid isolation from a variety of
samples at any throughput. MagJET
kits provide high-purity DNA and
RNA ready to use in routine and
demanding downstream applications.
High yields and
robust performa
MagJET magnetic bead-based technology util
high capacity paramagnetic particles optimized
nucleic acids with superior purity and yields co
other kits on the market.
Due to large total surface area, MagJET micro
high binding capacity, resulting in superior nuc
and recovery rates typically exceeding 80%. U
size and shape ensure robust performance an
results with less sample-to-sample variation.
The MagJET micro beads are highly stable in s
respond rapidly to an applied magnetic field, m
MagJET kits an ideal choice for high-throughp
MagJET DNA & RNA purification kits
The proprietary MagJET micro beads
Designed for great
performance
MagJET magnetic bead-based technology utilises proprietary
high capacity paramagnetic particles optimised to isolate
nucleic acids with superior purity and yields compared to other
kits on the market.
Due to large total surface area, MagJET micro beads exhibit
high binding capacity, resulting in superior nucleic acid yields
and recovery rates typically exceeding 80%. Uniform bead size
and shape ensure robust performance and consistent results
with less sample-to-sample variation.
The MagJET micro beads are highly stable in solution and
respond rapidly to an applied magnetic field making the
MagJET kits an ideal choice for high-throughput automation.
Designed for great performance
Magnetite content ~60%
• High binding capacity • Fast magnetic response
• Uniform size and shape • Ideal for automation
• High recovery (>80%) • Proprietary technology
4 Bio-Innovation Issue 7 Dual magnetic core
for fast magnetic
response
~ 1 µm ± 10%
 Magnetite content ~ 60%
Robust performance
Automated and manual workflows
MagJET nucleic acid isolation technology is suitable both for
high throughput automated and manual sample processing.
(A) The automation protocols are optimised for Thermo
Scientific KingFisher instruments where magnetic beads are
transferred from well to well during the purification procedure.
(B) In the manual protocol a magnetic field applied by an
manual protocol
automated protocol
external
source
is 3used4 to capture
the 1beads
against
the wall
ofM
M
1
2
5
M
2
3
4
5
the tube. The supernatant is removed by aspiration.

Manual and automated workflows were used to purify RNA from
N. tabacum leaf samples (each 50 mg) with the Thermo Scientific MagJET
Plant RNA Kit. The Thermo Scientific™ KingFisher™ protocol was used
for the automated workflow. Isolated RNA was analyzed by agarose gel
electrophoresis (1%), M – Thermo Scientific™ RiboRuler™ High Range RNA
Ladder (Cat #SM1823).
High yields and
robust performance
MagJET magnetic bead-based technology utilizes proprietary
high capacity paramagnetic particles optimized to isolate
nucleic acids with superior purity and yields compared to
other kits on the market.
F
•
•
Due to large total surface area, MagJET micro beads exhibit
high binding capacity, resulting in superior nucleic acid yields
and recovery rates typically exceeding 80%. Uniform bead
size and shape ensure robust performance and consistent
results with less sample-to-sample variation.
•
•
•
•
The MagJET micro beads are highly stable in solution and
respond rapidly to an applied magnetic field, making the
MagJET kits an ideal choice for high-throughput automation.
Designed for great
performance
ith
Compatible w
automated pu
manual w
Dual magnetic core
for fast magnetic
response
get attracted to great results!
Greater results with
Thermo Scientific MagJET kits
~ 1 µm ± 10%
A
 Magnetite content ~ 60%
Robust performance
Nucleic acid isolation is a fundamental step in molecular
biology applications and a prerequisite for successful
experimental workflows.
Robust Performance
Manual and automated workflows were used to purify RNA
from N. tabacum leaf samples (each 50 mg) with the MagJET
Plant RNA Kit. The Thermo Scientific™ KingFisher™ protocol
was used for the automated workflow. Isolated RNA was
analysed by agarose gel electrophoresis (1%), M – Thermo
Scientific™ RiboRuler™ High Range RNA Ladder (Cat
#SM1823).
Features
• Efficient – high yields of pure
nucleic acids
B
Thermo Scientific™ MagJET™ nucleic acid purification
kits utilize magnetic bead-based technology, allowing
for highly efficient nucleic acid isolation from a variety
of samples at any throughput. MagJET kits provide
high-purity DNA and RNA ready to use in routine and
demanding downstream applications.
manual protocol
M
• Versatile – compatible with a
wide variety of sample types,
including cells and tissues, whole
blood, plants, serum, plasma,
and bacterial culture
• Flexible – protocols for
automated and manual

workflows provided
automated protocol
Thermo
Scientific
1
2
3
4
5 MagJET
M
1
2
nucleic acid isolation products
3
4
5
M

Manual and automated workflows were used to purify RNA from
N. tabacum leaf samples (each 50 mg) with the Thermo Scientific MagJET
Plant RNA Kit. The Thermo Scientific™ KingFisher™ protocol was used
Kitswas analyzed by agarose gel
for the automated workflow. MagJET
Isolated RNA
electrophoresis (1%), M – Thermo Scientific™ RiboRuler™ High Range RNA
Ladder (Cat #SM1823).
Genomic
DNA
A
Viral DNA
and RNA
Total RNA
Cells &
Tissue
Cells &
Tissue
Whole Blood
Whole Blood
Plant
Plant
Thermo Scientific MagJET
is suitable both for high-t
manual sample processin
are optimized for Thermo
where magnetic beads ar
during the purification pro
protocol a magnetic field
used to capture the beads
The supernatant is remov
Plasmid
DNA
Serum,
Plasma
Bacterial
culture
B
• Learn more about MagJET products at
thermoscientific.com/magjet
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2
5 by Dr A. Esmon
With pharmaceutical
companies investing
heavily in extensive R&D to
produce effective vaccines
for existing and emerging
health risks, there is a
growing recognition that
protecting the potency and
viability of a vaccine during
storage is essential.
Effective
vaccine storage
One of the most rapidly expanding pharmaceutical markets
today, the vaccine therapy category protects public health
globally and saves an estimated three million lives each
year [1]. However, incorrect refrigeration and storage of
vaccines can lead to costly mistakes. In clinical practice,
refrigeration problems have been cited as a major cause of
the $20 million wasted each year from ruined vaccines in the
U.S. Federal Vaccines for Children Program [2]. In addition,
a recent UK study revealed that 40% of vaccines were
kept at the incorrect temperature, which could potentially
damage a vaccine’s potency and effectiveness [3].Because
it is not apparent if a vaccine has become sub-standard
through exposure to temperature fluctuations, incorrect
storage temperatures can lead to a very real danger that
patients are given a sub-standard vaccine, leaving them
unprotected against dangerous diseases. Storing vaccines in
controlled and constant temperature conditions is therefore
the key to protecting a population at risk. Correct vaccine
refrigeration also reduces expensive wastage of high-value
and fragile biological samples, eliminating the need for costly
revaccination programmes that may have to be initiated if the
integrity of a vaccine becomes compromised.
Choosing the correct storage
There are significant differences in levels of performance and
reliability across various categories of cold storage equipment.
Critical cold storage applications demand a safe, stable
6 Bio-Innovation Issue 7 environment; the temperature fluctuations found in household
or commercial cabinets must be eliminated. Choosing the
appropriate equipment with proven performance that meets all
government standards is now an important business decision
for research, hospital, pharmacy and clinical laboratories.
Today’s specialised laboratory refrigerators and freezers are
well equipped to provide efficient high-value biological sample
storage that maintains vaccine integrity. Improvements in
design and technology ensure that advanced refrigeration
can keep vaccines at the constant conditions that clinics and
laboratories require.
Maintaining constant temperature
The majority of commonly administered vaccines need to
be stored between 2 and 8 °C and must not be exposed to
freezing temperatures, which will irreversibly reduce their titre.
However, the development of the varicella vaccine in 1995 and
the more recent introduction of the live attenuated influenza
vaccine (LAIV) have increased the complexity of vaccine
storage.
These vaccines, including MMR, Varicella (Chickenpox)
and Zoster (shingles), as well as LAIV must be maintained
in a frozen state (-18 °C is recommended) without any
freeze-thaw cycles occurring [5, 6]. Temperature fluctuations can
result from sample retrieval by multiple users, unpredictable
defrosting cycles or poor insulation. Accidental freezing of
doors and effective door seals, to offer additional vaccine
protection. Some units also supply ‘Door Ajar’ alarms that
notify users when the cabinet door is not closed properly.
Blown-in insulation that conforms to the unit’s shape prevents
cold air escaping, as well as internal temperature fluctuations
due to exposure to ambient conditions. Many modern
laboratory refrigerators and freezers also supply audio and
visual alarms that alert users to temperature deviations,
providing a real-time warning that cannot be ignored when, for
example, preventative maintenance is required. Every clinic
should be prepared with a recovery plan, in case of problems,
that includes a refrigerator with a back-up generator in the
event of, for example, a power cut or natural disaster. Today’s
refrigeration equipment can provide a full alarm function in case
of power failure and a low battery alarm that displays when the
alarm system battery backup is low.
Best practice vaccine protection
freeze-sensitive vaccines, which represent over 31% of the
vaccines on which UNICEF spent €318 million in 2005 [6], can
also irreparably damage the chemical structure of a vaccine,
rendering it ineffective. In addition, certain freeze-sensitive
vaccines contain an aluminum adjuvant that precipitates
when frozen, resulting in a loss of efficiency and potency. To
prevent temperature deviations, modern clinical refrigeration
equipment provides microprocessor-controlled in-built
monitoring systems. These can include a graphic thermometer
that confirms normal, high or low temperature conditions, with
the optional ability to automatically record temperatures over a
period of time in a chart format. Alternatively, the placement of
a US National Institute of Standards and Technology (NIST)certified thermometer in the centre of the storage unit, adjacent
to the vaccine, allows the unit’s temperature to be read and
documented twice a day. Records are then kept for a minimum
of three years, as recommended in the US CDC guidelines
[4]
. If temperatures are found to be outside the recommended
range, action must be taken immediately.
Storage equipment that features an automatic defrost system
is highly recommended, as it prevents water, ice, frost or
coolant leaks that could potentially harm vaccine samples.
Refrigeration units provide efficient compression technology
and forced-air circulation to maintain temperature uniformity
throughout the cabinet. They also provide a tight and secure
door closure through the use of spring-loaded, self-closing
The storage units selected should be of a high enough quality
to negate the need for frequent maintenance and repairs,
which can compromise vaccine quality due to cabinet
‘downtime’ and the transfer of samples to another location.
Temperature should be recorded twice daily and any deviations
from the optimum range should be reported. Studies have
demonstrated that educating at least one staff member
about correct monitoring and reporting of the refrigerator
temperature significantly improves the maintenance of storage
conditions, with fewer deviations from optimal temperature
ranges going unreported [7, 8]. It is recommended that clinics
should use dedicated refrigerators and freezers to store
vaccines. Although combined refrigerator/freezer units are
acceptable for vaccine storage if each compartment has a
separate door, inconsistencies in temperature uniformity can
arise, making combined units less suitable for the storage of
any temperature-critical samples [9]. Clinics need to make
allowances for the largest possible batch of vaccines that
they will need to store at one time. This can be problematic
as pandemics, e.g., the recent H1N1 (swine flu) outbreak, are
not easily predicted. However, since individual doses need
to remain 5 - 8 cm away from all walls, doors, drawers and
cold air vents (as these are most likely to have temperature
extremes), the footprint of the freezer needs to be chosen with
care [8].
References
1. UNICEF. Available at: www.
unicef.org/immunization/
index_coverage.html.
2. Welte M. Vaccines ruined
by poor refrigeration. USA
Today 2007
3. NHS National Patient Safety
alerts: www.nrls.npsa.nhs.
uk/alerts
4. CDC Notice to readers:
Guidelines for maintaining and
managing the vaccine cold
chain. MMWR 2003; 52(42):
1023 – 1025.
5. Thermo Scientific Laboratory
Refrigerators and Freezers
Guide for Vaccine Storage.
6. UNICEF Supply Division
annual report page. Available
at: www.unicef.org/supply/
index_report.html
7. Jeremijenko A, Kelly H,
Sibthorpe B et al. Improving
vaccine storage in general
practice refrigerators. BMJ
1996; 312: 1651 – 1652.
8. Bell KN, Hogue CJR,
Manning C et al. Risk factors
for improper vaccine storage
and handling in private provider
offices. Pediatrics 2001; 107:
e100.
9. Don’t be guilty of these
errors in vaccine storage and
handling. Immunization action
coalition: www.immunize.org
The author: Alex Esmon, PhD
Global Commercial Manager
Laboratory Refrigerators and
Freezers, Laboratory Equipment
Division, Thermo Fisher
Scientific
Conclusion
With organisations investing much time, effort and money to screen, develop
and produce potentially life-saving vaccines, it is detrimental in terms of time
and cost-efficiency if these vaccines lose effectiveness through incorrect
storage. Maintaining constant conditions for vaccine storage is key to improving
viability and protecting a population from health risks. Selecting the most
efficient clinical cold storage equipment is therefore crucial for ensuring sample
protection. The latest available refrigeration equipment enables much easier
and more reliable monitoring and maintenance of ideal temperature conditions
for vaccine storage, reducing the occurrence of temperature fluctuations
to a minimal level. These in-built technological advances in storage units, in
combination with a degree of education on quality control measures, can help
to ensure that vaccines remain viable after storage, eliminating the need for
costly losses and the implementation of revaccination programmes.
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7 Reija-Riitta Harinen, Jorma Lampinen and Arto Perälä, Thermo Fisher Scientific,
Vantaa, Finland; Janaki Narahari and Douglas Hughes, Thermo Fisher Scientific, Pierce
Protein Research, Rockford, IL, USA
The focus of this application
note is on introducing different
luciferase reporter assay types
and the requirements they
place on the luminometer. It also
shows how luciferase reporter
gene assays provide excellent
performance using low assay
volumes with normal 384-well
and shallow well 384 plates.
Versatile Luciferases:
New Tools for Reporter Assays
Luciferase genes are commonly used as reporter genes in gene regulation studies.
Reporter gene assays are used both in basic research for studying transcriptional
regulation and cell signalling, and in drug discovery for screening and validating drug
targets. The light output from luciferase reactions can be detected with
luminometers that are suitable for reporter gene assays.
Thermo Fisher Scientific has developed a family of luciferase activity assays that use
novel luciferase genes from Cypridina and Gaussia combined with the Red Firefly
luciferase gene and the green shifted Renilla gene. The assay family provides both
flash- and glow-type assays, which differ in the stability of luminescence signals and
in sensitivity. Some of the assays utilise luciferases that are secreted into the
culture media. This feature allows live monitoring of the reporter activity during cell
growth, without a need for cell lysis. In addition, the assay family includes unique
dual luciferase assays for multiplexing purposes. The dual assays include two
luciferases: one for measuring the experimental luciferase activity and the other for
measuring control activity for normalisation.
8 Bio-Innovation Issue 7 Assay Types
1. Glow-type luciferase assays
Glow-type luciferase reporter gene assays produce a very
stable luminescence light signal that lasts for approximately
one hour. As the light emission decays slowly, measurement
does not have to be performed immediately after the addition
of substrate. The stability of the light makes it possible to
pipette all the assay reagents manually without need of reagent
dispensers installed in the instrument. Glow-type assays are
generally less sensitive than flash-type assays.
2. Flash-type luciferase assays
Flash-type luciferase reporter gene assays are in general more
sensitive than glow-type assays. Luminescence signals are
transient in flash-type assays, so the signal peak is reached
soon after assay reagent addition. Therefore, flash-type assays
often require the assay reagent to be added with automatic
dispensers installed in the luminometer. Reagent dispensers in
the instrument facilitate signal monitoring right from the start of
the luminescence reaction and enable precise timing between
dispensing and measurement from sample to sample. Pipetting
the assay reagent manually increases sample-to-sample signal
level variations especially in flash-type assays and miss the
capture of true flash signal due to delay in measuring the signal
after manual reagent addition.
3. Dual luciferase assays
Thermo Scientific Dual luciferase assays are highly sensitive
flash-type assays for multiplexing. The one-step assays are
based on having two luciferases emitting luminescent light
at spectrally distinct wavelengths. The signals can easily
be separated using a luminometer equipped with high
transmission filters optimised for the assays. This kind of dual
assay eliminates the need for signal quenching between the
measurement of two luciferases. Thus, both luciferase signals
can be detected simultaneously from a single sample with a
single reagent addition. As these dual assays are flash-type
assays, the reagent dispensers installed in the luminometer act
to ensure optimal performance and sensitivity.
assays. Both the Luminoskan Ascent and Varioskan Flash
can be equipped with up to three reagent dispensers. In
addition, both instruments can be equipped with optical filters
for measuring dual luciferase assays. The filters have been
specifically optimised to separate the two luciferase signals
as efficiently as possible for the Pierce Dual Luciferase Assays
and, therefore, they ensure excellent performance.
Materials and Methods
The study of this application note focuses on performing
flash-type Luciferase Reporter Assays in low assay volumes
with normal 384 and shallow well 384 plates.
• Instrument: Varioskan Flash multimode microplate reader
equipped with a luminometric measurement module and an
onboard dispenser for automatic reagent addition
• Kits: Flash-type Luciferase Reporter Assay Kits for Gaussia,
Cypridina, Renilla and Firefly luciferases
• Microplates: white 384-well OptiPlate (Perkin Elmer,
Cat. No. 6007299) and white shallow well 384-well plate
(Thermo Scientific NUNC, Cat. No. 264706)
Luciferase samples were diluted in the Cell Lysis buffer
provided in the kit to create a concentration series of over eight
orders of magnitude. Aliquots of 4 µl (normal 384-well plate)
or 1 µl (shallow well 384 plate) of each dilution were added
into the microplate wells. Instrument control software was
programmed to add 20 µl or 5 µl of luciferase assay reagent
with a dispenser and to measure the signal kinetically for
15 seconds at a sampling rate of ten readings per second.
Each well was dispensed and measured before proceeding
to the next well. Luminescence signals were integrated from
kinetic curves for different time periods. Additionally, individual
signals and signal maxima were collected for comparison of
different time points. Assay sensitivity and dynamic range were
calculated for each data set.
Luminometers
Luminescence light output from luciferase reporter gene
assays can be quantified using, for example, Thermo
Scientific Varioskan Flash or Luminoskan Ascent microplate
luminometers (Figure 1). These luminometers have excellent
sensitivity in luminometry, both in glow- and flash-type assays.
In addition, the Varioskan Flash can measure luminescence
spectra as well as other detection technologies.
Reagent dispensers & Optical filters
Dispensers are essential in flash-type assays, which require
measurement soon after the addition of substrate, and
precise timing between dispensing and measurement from
sample to sample and from day to day. Dispensers installed
in the luminometer make the reaction triggering easy, fast and
accurate, not only for flash-type assays, but also for glow-type
Figure 1. Optimal microplate
readers for Pierce Luciferase
Reporter Assays: Varioskan
Flash (on the right) and
Luminoskan Ascent (on the left).
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9 Figure 2. Typical standard
curves of Gaussia and Cypridina
luciferase flash assays with
different plate formats
Results
The Pierce Luciferase Reporter Assays and the Varioskan
Flash provide very high performance and enable use of very
low assay volumes with normal 384-well and shallow well
384-well plates. All luciferase reporter assays show perfect
linear response over a wide concentration range with both
normal and shallow well plates. Real assay dynamics reaches
eight orders of magnitude with the Gaussia and Cypridina
flash assays (Figure 2). The Renilla flash assay also shows a
remarkably large dynamic measurement range. Flash assay
with Red Firefly luciferase shows a somewhat reduced dynamic
range, about 5 to 6 orders of magnitude.The Varioskan Flash
can automatically adjust the photomultiplier gain to increase
the assay dynamic range to eight orders of magnitude.
This unique feature means that the luciferase samples can
have any luciferase concentration over a large 100 million
fold concentration range. Thus the samples can always be
measured without additional dilution steps.
assay shows about 3% signal decay per second after reaching
maximum. The firefly flash assay represents the fastest flash
reaction where the signal decays about 20% per second during
the first couple of seconds.
Using automatic dispensers in the luminometer for reagent
addition is highly recommended to ensure exact timing in the
measurements, especially in Gaussia and Firefly assays. If the
luminescence reaction is initiated by manual pipetting, it may
lead samples to be measured in different time points of the
kinetic reaction, thus increasing sample-to-sample variations.
Assay sensitivity is dependent on the measurement integration
time, but its effect is not very strong. About a one second
measurement time provides good sensitivity, but a few
seconds more may be required for the best possible sensitivity.
The Cypridina assay produces the most stable light emission
and, therefore, it is recommended to integrate the signal
somewhat longer than with assays with a more unstable signal.
The Firefly flash reaction shows the most unstable kinetics and
therefore the recommended integration time is the shortest.
A usable integration time range is a range where changing
the integration time has only a minor effect on the assay
performance. When using shallow well 384 plates with smaller
Figure 3 illustrates the flash-type reaction kinetics of Gaussia,
Cypridina, Renilla and Firefly luciferases. All the assays reach
the light emission maximum in a few seconds after the reaction
has been started. Cypridina and Renilla assays show a fairly
stable light emission for several seconds, and the Gaussia
Recommended Integration Times
Normal 384-well plate
10 Bio-Innovation Issue 7 Shallow well 384-well plate
Luciferase
Optimal time (s)
Usable range (s)
Optimal time (s)
rUsable ange (s)
gaussia
1.5
1-1.5
7
4-7
cypridina
5
1-5
7
3-7
green renilla
2
1-2
10
4-10
red Firefly
1
0.5-1
1.3
0.5-1.3
assay volumes, it is recommended to use longer integration
times than with normal 384-well plates with higher assay
volumes (See Table 1).
Even though this application note only presents the results of
assays performed in a 384-well plate format, measurements
in a 96-well plate format provide excellent performance as
well. The performance of the Luminoskan Ascent with these
luciferase reporter assays is also comparable to the Varioskan
Flash results presented here.
Conclusions
• Pierce Luciferase assays provide an extremely wide
dynamic range for measuring both low and high luciferase
expression levels without concentrating or diluting samples.
• The Varioskan Flash detects luciferase activities over a
dynamic range of about eight orders of magnitude with the
automatic gain adjustment feature of the instrument.
• Using 384-well plates (either normal or shallow well) enables
reducing both sample and reagent consumption without
sacrificing assay performance. Use of shallow well 384
plates enables performing the assays in a total volume of
about 6 µl (1 µl sample + 5 µl assay reagent).
• Highly sensitive flash-type assays can easily be performed
with a microplate luminometer equipped with an automatic
dispenser.
• Both inter- and intra-assay variations can easily be
minimised using automatic luminometer dispensers,
which enable precise timing of the reaction start and signal
collection.
Different luciferase reporter gene assays require different
features and capabilities of the luminometer. The benefits of
using Thermo Scientific luminometers for performing Pierce
Luciferase Reporter Assays include the following:
• Excellent luminometric performance in glow- and flash-type
assays
• Onboard dispensers for reagent addition:
– follow-up of fast kinetics of flash assays
– precise timing between dispensing and
measurement in flash assays
– convenience and accuracy for flash and glow assays
• Optimised filters for measurement of dual assays
• Flexible and easy protocol setup and data processing with
PC software
Figure 3. Luciferase reaction
kinetics of flash-type assays
measured both in a normal 384well plate and a shallow well
384 plate. Renilla and Firefly
luciferases were measured
from a cell lysate. Gaussia and
Cypridina were secreted to the
growth medium and measured
directly from the medium
without a cell lysis.
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11 tech
note
Irmgard Suominen, Senior Application
Scientist, Thermo Fisher Scientific,
Vantaa, Finland. Sami Koivisto,
Technology Manager, Liquid Transfer,
Thermo Fisher Scientific, Vantaa,
Finland. Suvi Berghäll, Product
Manager, Pipetting Systems, Thermo
Fisher Scientific, Vantaa, Finland
ClipTip interlock technology: the next
generation for pipettes
The most common type of pipettes in the lab are air displacement pipettes. An airtight seal between
the pipette and the tip is crucial for the pipette´s functionality, and any compromise on the sealing
affects pipetting performance. Traditionally air displacement pipettes have relied on friction between
the pipette and the tip to form a seal. The sealing is dependent on many factors including the tip
attachment force used and might also be compromised through general use or when touching
the vessel wall during pipetting. With the new Thermo Scientific F1-ClipTip Pipetting System the
attachment of tips is accomplished with an interlock technology between pipette and tip.
2
2
Tip ejector
Tip ejector
Clip
Opener
• Aftertipattachment100µlofgreendyesolutionwas
dispensed into 96-well microplates using the reverse
• pipetting
Aftertipattachment100µlofgreendyesolutionwas
technique. After each dispensing the tips were
dispensedagainst
into 96-well
microplates
the reverse
touched
the wall
of the wellsusing
to wipe
off
pipettingdroplets
technique.
each dispensing
tips were
possible
on After
the outside
of the tip. the
It was
touched against
theoff.
wallAfter
of the
wellstwo
to wipe
off
recorded
if tips fell
filling
microplates,
possible
droplets
on
the
outside
of
the
tip.
It
was
100 µl was aspirated and the amount of liquid was
recorded ifgravimetrically.
tips fell off. After
filling two
microplates,
measured
Precision
values
(CV%) were
100 µl wasfor
aspirated
and the amount
of liquid was
calculated
each attachment
force used.
measured gravimetrically. Precision values (CV%) were
Results
calculated for each attachment force used.
1.Tip attachment
Results
Attachment force 3.8 kg was sufficient to attach tips to all
1.Tip attachment
pipettestested.However,tipsfromManufacturerBand
Attachment
force
kg was
sufficient
to 8
attach
generic
tips fell
off3.8
during
pipetting
when
rowstips
of to all
pipettestested.However,tipsfromManufacturerBand
microplatehadbeendispensed.Figure2showsClipTip
generic
tips
fell
off
during
pipetting
when
8
rows
of96-well
tips (a) and Generic pipette tips (b) after filling two
microplatehadbeendispensed.Figure2showsClipTip
microplates.
The ClipTip pipette tips did not fall off, and
tips (a) and Generic pipette tips (b) after filling two 96-well
securedanequalliquidlevelinall12tips.TheGenerictips
microplates.
Theoff,
ClipTip
pipette tips
did not
falllevels
off, and
had
one tip fall
and obtained
uneven
liquid
in
securedanequalliquidlevelinall12tips.TheGenerictips
the
remaining tips. In order to keep tips attached the force
had onetotipbefall
off, andfrom
obtained
uneven
levels
needed
increased
3.8 kg
to 4.8liquid
kg. The
tipsin
of
the remaining A
tips.
In order
to keep
manufacturer
stayed
attached,
buttips
theattached
variationthe force
needed
to
be
increased
from
3.8
kg
to
4.8
kg.
The
tips
between dispensed volumes of different channels was of
manufacturer
A stayed
attached,
variation
higher
than with
F1-ClipTip
(Fig.but
3). the
This
variation could
between
dispensed
volumes of different channels was
not
be observed
visually.
higher than with F1-ClipTip (Fig. 3). This variation could
not be observed visually.
gravimetrically. Precision values (CV%) were
Test pipettes and tips:
Clip Opener
Clip
calculated for each attachment force used.
• Thermo Scientific F1-ClipTip 12-channel
Clipfitting flange
pipette (30-300 µl) with ClipTip 300. Tip
Results
Tip
ring
Tip fitting
fitting sealing
flange 1.Tip
• 300µl 12-channel pipettes from
attachment
Tip fitting sealing ring
Manufacturers A and B with manufacturers’
Attachment force 3.8 kg was sufficient to attach
Tip
tips recommended for the particular pipette
tips to all pipettes tested. However, tips from
Tip
Figuretips.
1: ClipsThe
closed behind
the flange.
The
tip is sealed. Manufacturer B and generic tips fell off during
as well as generic
pipettes
had
friction based tip
sealing
mechanisms.
Figure
1: Clips closed
behind the flange. The tip is sealed. pipetting when 8 rows of the microplate had
In this technical note we demonstratebeen dispensed. Figure 2 shows ClipTip tips
the benefits of the revolutionary ClipTip
In this technical note we demonstrate(a) and Generic pipette tips (b) after filling two
Test method:
interlocking tip attachment technology by
the benefits of the revolutionary ClipTip
• Tip attachment was
achieved
withplate
constant
96-well microplates. The ClipTip pipette tips did
pipetting
into 96-well
as an example.
interlocking tip attachment technology by
forces using a machine
the
tested
not fall off, and secured an equal liquid level in all
pipetting to
intopress
96-well
plate
as an example.
• Aftertipattachment100µlofgreendyesolutionwas
Test set-up
Tip ejector
pipette
downward
against
the
tip
rack
box
12 tips.
dispensed
into 96-well
microplates
using
the
reverse
Test pipettes and tips:
set-upthe tips were
pipetting technique. After each•
dispensing
ThermoScientificF1-ClipTip12-channelpipette
with a lever. TheTest
lever
was
equipped
with
a
Clip Opener
pipettes
and
tips: 300.
touched against the wall of theTest
wells
to µl)
wipe
off
(30-300
with
ClipTip
• ThermoScientificF1-ClipTip12-channelpipette
directed
downward
possible weight,
droplets onwhich
the outside
of the tip.aItconstant
was
• 300µl12-channelpipettesfromManufacturersAandB
(30-300 µl) with ClipTip 300.
recorded if tips fell off. After filling
microplates,
withtwo
manufacturers’
tips recommended for the
Clip
force
to
the
test
pipette.
This
procedure
• particular
300µl12-channelpipettesfromManufacturersAandB
pipette was
as well as generic tips. The pipettes
100 µl was aspirated and the amount
of liquid
withfriction
manufacturers’
tips
recommended
for the
based tipprocedure
sealing
mechanisms.
measuredsimulated
gravimetrically.
values
(CV%)
were
thePrecision
tiphad
attachment
with
Tip fitting flange
particular pipette as well as generic tips. The pipettes
method:
calculated for each attachmentTest
force
used.
had friction
tip sealing mechanisms.
A
a known force. •The
usedbased
attachment
weight
Tip fitting sealing ring
Tipattachmentwasachievedwithconstantforcesusing
Test
method:
Results
machine
to press the tested pipette downward against
A
forces were 3.8kg,
4.8kg
and
6.3kg
(37.3N,
• the
Tipattachmentwasachievedwithconstantforcesusing
1.Tip attachment
tip rack box with a lever. The lever was equipped
machine
to press
the to
tested
downward
against
with
atoweight,
which
directed
a constant
downward
61.8N
respectively).
Attachment47.1N
force 3.8and
kg was
sufficient
attach
tips
all pipette
Tip
the tiptorack
box pipette.
with a lever.
The lever was
equipped
force
the test
This procedure
simulated
the
pipettestested.However,tipsfromManufacturerBand
with
a100
weight,µl
which
directed
downward
• After tip attachment
of green
attachment
procedure
withadye
aconstant
known force.
The used
generic tips fell off during pipettingtip
when
8
rows
of
force to the weight
test pipette.
This
procedure
simulated
the
Figure
1:
Clips
closed
behind
the
flange.
The
tip
is
sealed.
attachment
forces
were
3.8kg,
4.8kg
and
6.3kg
Figure 1: Clips closed behind the flange. The tip is sealed.
microplatehadbeendispensed.Figure2showsClipTip
solution was dispensed
into
96-well
tip attachment
a known force. The used
(37.3N,
47.1N procedure
and 61.8Nwith
respectively).
tips (a) and Generic pipette tips (b)attachment
after filling
two 96-well
weight
forces were 3.8kg, 4.8kg and 6.3kg
microplates using
the
reverse
pipetting
microplates. The ClipTip pipette tips
did not
falland
off,61.8N
and respectively).
(37.3N,
47.1N
In this technical note we demonstrate
securedanequalliquidlevelinall12tips.TheGenerictips
Test set-up
technique. After each dispensing the tips
the benefits of the revolutionary ClipTip
had one tip fall off, and obtained uneven liquid levels in
B
In this technical note we demonstrate the
were
against
the wall
of the wells
the remaining
tips.touched
In order to keep
tips attached
the force
interlocking tip attachment technology by
needed to be
increased
from
3.8 kg to 4.8
kg. The tips
ofthe outside
benefits
of
the
revolutionary
ClipTip
interlocking
to
wipe
off
possible
droplets
on
B
pipetting into 96-well plate as an example.
manufacturer A stayed attached, but the variation
tip attachment technology by pipetting into a between dispensed
of the volumes
tip. It was
recorded
if
tips
fell
off.
After
of different channels was
Figure 2. ClipTip tips (a) and Generic tips (b) after dispensing into
higher thanfilling
with F1-ClipTip
(Fig. 3). This variation
two 96-well microplates. The tip attachment force used was 3.8 kg.
96-well
two microplates,
100 µl could
was aspirated
Testplate
set-upas an example.
Figure 2.Figure
ClipTip2.tips
(a) and
tips (b)tips
after(b)dispensing
into two
ClipTip
tips Generic
(a) and Generic
after dispensing
into96-well
not
be
observed
visually.
Test pipettes and tips:
two The
96-well
microplates.force
The tip
attachment
and
the
amount
of
liquid
was
measured
microplates.
tip attachment
used
was 3.8force
kg. used was 3.8 kg.
• ThermoScientificF1-ClipTip12-channelpipette
Breakthrough ClipTip Technology
ClipTip interlock technology utilises flexible
clips positioned evenly around the top of the
tip. During attachment, the unique tip fitting
shape opens the clips allowing it to pass over
the fitting flange and return to closed position.
The clips lock the tip behind the flange creating a
complete seal with the sealing ring (figure 1). In
addition, the lock ensures the tip will not loosen
compromising the seal, or even potentially fall off
during routine pipetting or touch-off.
(30-300 µl) with ClipTip 300.
12 • 300µl12-channelpipettesfromManufacturersAandB
with manufacturers’ tips recommended for the
Bio-Innovation
Issue
7 as generic tips. The pipettes
particular pipette
as well
had friction based tip sealing mechanisms.
Test method:
but the variation between dispensed
volumes of different channels was
higher when compared to the results
of the F1-ClipTip system (Fig. 3).
Precision value of manufacturer A was 68% higher than
with F1-ClipTip system (Figure 4) after dispensing into
two 96-well microplates. The figure shows that with
F1-ClipTip the variation between channels was lower
than with a pipette with a friction based tip fitting
mechanism. With friction based mechanisms the precision
varied with the tip attachment force used.
Figure 3. Dispensed volumes of F1-ClipTip 12-channel pipette
and Manufacturer A’s 12-channel pipette after dispensing into
two 96-well microplates. The set volume was 100 µl and the tip
attachment force used was 3.8 kg.
Manufacturer A tips stayed attached,
but the variation between dispensed
volumes of different channels was
higher when compared to the results
of the F1-ClipTip system (Fig. 3).
Figure 4. Precision values with 12-channel pipettes after
dispensing into two 96-well microplates. The set volume was 100
µl and the tip attachment forces used were as 3.8 kg.
The Generic tips had one tip fall off, and
obtained uneven liquid levels in the remaining
tips. In order to keep tips attached the force
needed to be increased from 3.8 kg to 4.8 kg.
The tips of manufacturer A stayed attached,
but the variation between dispensed volumes
of different channels was higher than with
F1-ClipTip (Fig. 3). This variation could not be
observed visually.
2. Precision
Precision value of manufacturer A was 68%
higher than with F1-ClipTip system (Figure 4)
after dispensing into two 96-well microplates.
The figure shows that with F1-ClipTip the
variation between channels was lower than
with a pipette with a friction based tip fitting
mechanism. With friction based mechanisms
the precision varied with the tip attachment
force used.
Summary
An optimal pipette and tip system increases
confidence in reproducibility, reduces forces
required to attach and eject tips, and secures
the best possible accuracy and precision. In
microplate applications, demand for optimum
pipetting performance, and comfort is even
greater due to higher number of repetitions
Figure 3. Dispensed volumes
of F1-ClipTip 12-channel
pipette and Manufacturer
A’s 12-channel pipette
after dispensing into two
2. Precision
96-well microplates. The set
Precision value of manufacturer A was 68% higher than
volume was 100 µl and the
with F1-ClipTip system (Figure 4) after dispensing into
attachment
force
used
two 96-well microplates. tip
The
figure shows
that
with
F1-ClipTip the variation was
between
channels was lower
3.8 kg.
than with a pipette with a friction based tip fitting
mechanism. With friction based mechanisms the precision
varied with the tip attachment force used.
Figure 4. Precision values
with 12-channel pipettes after
dispensing into two 96-well
microplates. The set volume was
100 µl and the tip attachment
forces used were as 3.8 kg.
Figure 3. Dispensed volumes of F1-ClipTip 12-channel pipette
and Manufacturer A’s 12-channel pipette after dispensing into
two 96-well microplates. The set volume was
100 µl andcontamination
the tip
and samples. Compared to Manufacturer
severe
risk particularly in clinical
attachment force used was 3.8 kg.
A and B, the F1-ClipTip pipetting system
applications. With Manufacturer A and B more
demonstrates excellent precision and minimal
effort was needed to attach the tips firmly, and
variation between dispensed volumes of
prevent tips getting loose or dropping off.
different channels. Results show that pipetting
performance of the F1-ClipTip system is not
The complete seal between the sealing ring
affected by the tip attachment force used.
and the ClipTip tips guaranteed optimal
All tips remained attached and variation
pipetting performance as demonstrated by the
between channels was minimal. In addition, tip
excellent precision. The unique interlocking
attachment did not require banging or rocking.
tip attachment mechanism ensured that the
tips stayed firmly attached through the entire
Figure 4. Precision values with 12-channel pipettes after
With friction based tip attachment
systems
application.
dispensing
into two 96-well microplates. The
set volume was 100 These innovative features make
µl and the tip attachment forces used were as 3.8 kg.
the seal between the tip and the tip cone is
F1-ClipTip Pipetting System an excellent choice
accomplished by friction between the pipette
for microplate applications by saving valuable
and tip. In this experiment impaired precision
samples/reagents as well as time and resources
after dispensing and touching tips to the walls
while increasing confidence in pipetting results.
of a microplate was observed. This is likely due
In addition, the ergonomic benefits of this
to loosening of the tips, which affects the seal.
system reduce the risk of Repetitive Strain
The sealing may be tight enough to avoid visible
Injuries while pipetting.
leakage, but not tight enough to give optimal
pipetting results. In the worst case the tips fell
off in the middle of pipetting. In a real research
application this would mean repeating the
experiment.
Loss of samples and/or reagent, as well as time
wasted, have a large impact on daily research
and can especially be seen with microplate
applications. Dropped tips may also present a
www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
13 Water-cooled condenser option
for Thermo Scientific Ultra-Low
Temperature Freezers
Freezer farms with ultra-low freezers are very common in academic
institutions, sample storage areas, biopharma sites and many other
locations where ultra-low temperatures are required to protect
samples. Heat discharge is a normal function of compressor driven
units. The heat generated can cause significant strain on HVAC
systems. HVAC is taxed to the limit, which causes additional cost to
run the entire building system.
What is the best time or application to consider putting
water cooled condenser into a building?
• New construction projects. (Design Phase)
•Renovation
• Locations that have existing recirculating water systems or
chilled water source
• Multiple freezers or freezer farms
Advantages to water cooling condensers
• Overall energy savings potential
• 50% heat reduction to the laboratory or surroundings*
• Reduced impact on the HVAC system
• More comfortable work environment in the lab yields more
productive work
• Heat reduction in the laboratory reduces potential risk to
heat sensitive products or chemicals
How do chilled condensers function?
The air-cooled condenser looks like a small radiator (like in your
car). The hot gas from the high stage compressor is circulated
through the air-cooled condenser where a fan blows across
it to remove the heat and blow it into the room in air cooled
condenser. A water-cooled condenser goes in place of the
air-cooled condenser and instead of a fan blowing across it,
coils of water is circulated around it, which absorbs most of
14 Bio-Innovation Issue 7 REFRIGERANT IN
WATER OUT
REFRIGERANT
OUT
WATER IN
Figure 1. Graphical example of a water-cooled condenser.
the heat and circulates water back to the water tower or chilled
water reservoir system. A graphic example of a water-cooled
condenser is shown in figure 1.Ultra-low freezers using chilled
water condensers must have water flowing through the
condenser at all times. Chilled water systems likely will require
periodic maintenance. The freezers can be protected by the
connection to city or house water during the maintenance
process.The following procedure is recommended to switch
between water used for cooling. Connections must comply
with local building codes. The conversion process assumes
chillers and house water are available. Through water cooling,
the heat rejection rate of a ULT can be reduced by 70 to 80%1.
Ken Vanoster , Product Specialist, Thermo Fisher Scientific
Water cooling condensers offer an
alternative to the extra heat load on
the HVAC system.Water cooling
reduces heat to the surroundings
by approximately 50%.
Process to switch from chilled water source
to house water source
Process to switch from house
freezers using chilled
Process to switch from chilled
• Ultra-low
Check
water
pressure
in both
systems.
water source to chilled water
water
condensers must
have
water source to house
water
source
water flowing through the
source
condenser
at
all
times.
C
hilled
Pressure
value
be
x
• C heck water pressure in both
• should
C heck water pressure
in value.
both
water systems likely will require
systems. Pressure value should
systems. Pressure value should
maintenance. T he
be x value. air or
be xwater
value.
• periodic
Purge
the byhouse
line to remove
freezers
can be protected
the
• C heck the water flow rate. If
• Purge the house water line to
connection to city or house
there is no information on
remove air or particulates in
water
during
the
maintenance
particulates in the
house
water lines.
water flow rate, check the first
the house
water lines.
process.
stage discharge pressure.
• C heck the water pressure: It
• N ote the water pressure, inlet
must be at target pressure for
T he following procedure is
• recommended
Check
the waterhouse
pressure:
It must
be at target
water temperature and first
systems to protect the
to switch between
stage discharge pressure
ultra-low temperature freezers.
water used for cooling.
• Purge the chilled
water line to
• Switch the
valve to house to protect
C onnections
must comply
withhouse
pressure
for
systems
the
remove air or particulates in
water.
local building codes.
the chilled water lines.
• Turn off chilled water source.
T he ultra-low
conversion process assumes
temperature
freezers.
• C heck water pressure. M ust
• M onitor the temperature
and
chillers and house water are
be at target pressure for
water pressure on the house
systems to protect the
• available.
Switch the valveline.to house water. chilled
T hough water cooling, the heat
ultra-low temperature freezers.
rate of a U LT can be
• Switch the valve to chilled
• rejection
Turn
reduced
by 70 off
to 80%chilled water source.
water.
• Turn off house water source.
• M onitor
the temperature and
• Monitor the temperature and water
pressure
water pressure on the chilled
water line.
on the house line.
1
In addition to these
offices, Thermo Fisher
Scientific maintains
a network of representative organizations
throughout the world.
Australia
+61 39757 4300
Austria
+43 1 801 40 0
Belgium
+32 2 482 30 30
Canada
+1 866 984 3766
China
+86 21 6865 4588
France
+33 2 2803 2180
Germany
+49 6184 90 6000
Italy
+39 02 95 05 95 52
Japan
+81 3 5826 1616
Latin America
+49 6184 90 6000
Netherlands
+31 76 571 4440
Nordic
+358 9 329 100
Spain
+34 93 223 09 18
Switzerland
+41 44 454 12 12
UK
+44 870 609 9203
USA
+1 866 984 3766
www.thermofisher.com
©2011 Thermo Fisher
Scientific Inc. All rights
reserved. All trademarks are
the property of Thermo
Fisher Scientific Inc. and
its subsidiaries.
Process to switch from house water
source to chilled water source
• Check water pressure in both systems.
Pressure value should be x value.
• Check the water flow rate. If there is no
information on water flow rate, check the
first stage discharge pressure.
• Note the water pressure, inlet water
temperature and first stage discharge
pressure
• Purge the chilled water line to remove air or
particulates in the chilled water lines.
• Check water pressure. Must be at target
pressure for chilled systems to protect the
ultra-low temperature freezers.
• Switch the valve to chilled water .
• Turn off house water source.
• Monitor the temperature and water
pressure on the chilled water line.
Specifications, terms and
pricing are subject to
change. Not all products
are available in all countries.
Please consult your local
sales representative
for details.
Figure 2.Chilled water source vs. house water source.
1. Based on internal performance data. April, 2011.
www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
15 Novel glycan
column technology
for the LC-MS analysis of labelled & Native
N-Glycans Released from Proteins & Antibodies
Udayanath Aich,1 Ilze Birznieks,1
Julian Saba,2 Xiaodong Liu,1
Rosa Viner,2 Zhiqi Hao,2 Gurmil
S.Gendeh,1 Srinivasa Rao,1
Andreas Huhmer,2 Yury Agroskin,1
and Chris Pohl1 Thermo Fisher
Scientific, Sunnyvale, CA, USA;
2
Thermo Fisher Scientifi c, San
Jose, CA, USA
Methods
Results
Sample Preparation
Separation of Labelled Glycans Based on Charge, Size,
Release native glycans from glycoproteins with PNGase
and Polarity
F enzyme. Conjugate the released glycans with 2-amino
The GlycanPac AXH-1 column can be used for qualitative,
benzamide (2AB) label group using the reported procedure
quantitative, and structural analysis as well as characterisation
of Bigge et al.4 Here, 2-AB A1 (P/N GKSB 311), 2-AB A2 (P/N
of uncharged (neutral) and charged glycans present in proteins.
GKSB 312), and 2-AB A3 (P/N GKSB 314) were purchased
Figure 1 shows bovine fetuin on the GlycanPac AXH-1 (1.9
from Prozyme® (Hayward, CA). Prior to analysis, dissolve
μm, 2.1 × 150 mm) column using fluorescence detection. The
1 Ilze
1 pH
2 Xiaodong
1 Rosa
2 Zhiqi
samples
in 100% buffer
(100 mM
ammonium
formate,
=
separation
and elution ofLiu,
glycans
are based
on charge:
the Hao,2 G
Udayanath
Aich,
Birznieks,
Julian
Saba,
Viner,
2
1
4.4) and
dilute further
with acetonitrile
to makeSunnyvale,
30% buffer and CA,
neutral
glycans
elute first,Fisher
followed by
the separationSan
of acidic
Thermo
Fisher
Scientific,
USA;
Thermo
Scientific,
Jose, CA
70% acetonitrile.
2AB labelled N-glycans from monosialylated, disialylated,
trisialylated, tetrasialylated and finally pentasialylated species.
Liquid Chromatography
Glycans of each charge state are further separated based on
FIGURE 3
Overview
Results
GlycanPa
All the
glycans
were
separated
using
a
Thermo
Scientific™
their
size
and
polarity.
The
retention
time
of
each
glycan
charge
Purpose: Development of novel high-performance liquid chromatography (HPLC)
Separation of Labeled Glycans Based on Charge, Size, and Polarity
columns
for
highand
ultra
high-performance
liquid
chromatography
(UHPLC)
The
GlycanPac
AXH-1
column
can
be
used
for
qualitative,
quantitative,
and
structural
Dionex™ UltiMate™ 3000 BioRS system and either a
state was confirmed using 2AB labelled glycan standards (as
resolution separation and structural characterization of native and fluorescently
analysis as well as characterization of uncharged (neutral) and charged glycans present
labeled
N-glycans released
from various
proteins, including
antibodies.
Thermo
Scientific™
Dionex™
UltiMate™
FLD-3400RS
Rapid
shown
inFigure
Figure
2). Separation
of GlycanPac
glycansAXH-1
is based
charge,
in proteins.
1 shows
bovine fetuin on the
(1.9 μm,on
2.1 ×
150 mm)
column
using fluorescence detection. The separation and elution of glycans are based on
Methods: UHPLC with fluorescence detection (FLD) for the chromatographic analysis
Separation
Fluorescence Detector or MS detector.
size,
and
polarity,
which
provides
and
charge:
the neutral
glycans
elute first,
followed bysignificant
the separation structural
of acidic 2AB labeled
of labeled N-glycans. LC with mass spectrometry (MS) and LC-MS/MS analysis for
N-glycans from monosialylated, disialylated, trisialylated, tetrasialylated and finally
structural characterization of both labeled and native N-glycans from proteins by MS
quantitative
information.
chromatographic
profiles
pentasialylated species.
Glycans of The
each charge
state are further separated
basedshown
on
detection.
size and polarity. The retention time of each glycan charge state was confirmed
Mass
Spectrometry
intheir
Figures
1 and
2, standards
detected
by fluorescence
detection,
Results:
We have developed a high-performance, silica-based HPLC/UHPLC column
using
2AB labeled
glycan
(as shown
in Figure 2). Separation
of glycans provide
is
designed for simultaneous
(Thermo
Scientific™ GlycanPac™ AXH-1)
on charge, size, and polarity, which provides
significant structural and quantitative
2 Xiaodong
1 specifically
2 Zhiqi
2based
1 Srinivasa
MS analysis
was
performed
with
a
Thermo
Scientific™
Q Hao,qualitative
information
about
the
separation
of N-glycans.
The
Birznieks,1 Julian Saba,
Liu,
Gurmil
S. Gendeh,
Rao,by1 Andreas
H
separation
of
glycans
by charge,
size,
andRosa
polarity.
It is Viner,
designed
for high-resolution,
information.
The chromatographic
profiles shown
in Figures 1 and 2, detected
and high-throughput analysis, with unique selectivity for biologically relevant glycans,
fluorescence detection, provide qualitative information about the separation of N-glycans.
2
Benchtop
LC-MS/MS
inmethods.
negative ion mode
ofof glycans
present in each peak was determined from
ntific, Sunnyvale, CA,Exactive™
USA;
Thermo
Fisher
Sanat Jose, structure
CA,
USA
either labeled
or native, by LC-FLD
and LC-MSScientific,
The structure
glycans present in each peak was determined from the LC-MS study
using
the GlycanPac
AXH-1
(1.9 µm)
as shown inAXH-1
the following
section.
the following settings: MS scan range 380–2000. FT-MS was
the
LC-MS
study
using
thecolumn
GlycanPac
(1.9
µm) column
Introduction
acquired at 70,000 resolution at m/z 200 with AGC target of
as shown in the following section.
Glycans are widely distributed in biological systems in ‘free state’ and conjugated
FIGURE 1. Separation of 2AB labeled N-glycans from Bovine fetuin by charge, size
forms DDA
such as MS2
glycoproteins,
glycolipids,
and proteoglycans.
They are
in a
acquired
at 17,500
resolution
atinvolved
m/z 200
1e6; and
and polarity.
FIGURE 3. LC-MS analysis of 2AB labeled N-glycans from Bovine fetuin by the
FIGURE 5. L
wide range of biological and physiological processes.1 The functions, including
Results
5
GlycanPac
AXH-1
(1.9LC-MS/MS
µm) column with MS
detection. of 2AB Labelled
detected by
.
with AGC
LC-MS
and
Analysis
efficacy target
and safetyof
of 2e
protein-based
drugs such as recombinant proteins and
ce liquid chromatography (HPLC)
Separation
of Labeled
Glycans
Based
on Charge,
Size,onand
monoclonal
antibodies
(MAbs),
are often
dependent
thePolarity
structure and types of
FIGURE 4
y (UHPLC) columns for highN-Glycan Using GlycanPac AXH-1 Column
The GlycanPac
AXH-1 to
column
can be2used
for qualitative,
quantitative,
anddiverse,
structural
The structures
of glycans
are quite
glycans attached
the proteins.
commerc
ion of native and fluorescently
analysis
as welland
as characterization
of uncharged
(neutral) and
charged glycans
present
complex,
heterogeneous due
to post-translational
modifications
and physiological
Data
Analysis
The coupling of the GlycanPac AXH-1 column to MS was also
, including antibodies.
in proteins.
Figure
1 shows
bovine
fetuin on the and
GlycanPac
AXH-1
(1.9 μm, of
2.1glycans
× 150 mm)
conditions.
The
structural
characterization
quantitative
estimation
is
3 However,
column
using
fluorescence
detection. The
separation
and elution
of glycans
are based
it is on
highly
essential
in biotherapeutics
and
biopharmaceutical
projects.
®
LD) for the chromatographic analysis
SimGlycan
software
(PREMIER
Biosoft)
was
used
forlabeled
explored. This is particularly attractive as MS, with it’s ability
charge:
the neutral
glycans
elute
followed by
the
separation
of acidic
2AB
tremendously
challenging
tofirst,
comprehensively
characterize
glycan
profiles
and
(MS) and LC-MS/MS analysis for
N-glycans
from monosialylated,
trisialylated, tetrasialylated and finally
determine
the structures ofdisialylated,
glycans.
5
tive N-glycans from proteins by MS
glycan
identification
and
elucidation
data analysis.
to provide structural information, enables in-depth analysis of
pentasialylated
species. Glycans
of structural
each charge state
are further separated
based on
their size
polarity.AXH-1
The retention
each glycan charge
state was confirmed
The and
GlycanPac
columnstime
are of
high-performance
HPLC/UHPLC
columns
SimGlycan
software
accepts
raw
data
files
from
Thermo
complex glycans. 2AB labelled N-glycans from bovine fetuin
e, silica-based HPLC/UHPLC column
usingspecifically
2AB labeled
glycan standards
(as shown
in Figure
2). Separation
of glycans
is
designed
for structural,
qualitative,
and quantitative
analysis
of glycans.
ally designed for simultaneous
basedThey
on charge,
size, and
polarity,
which provides
significant
structural
and
are designed
for
high-resolution
and high-throughput
analysis,
withquantitative
unique
Scientific™
mass
spectrometers
and
elucidates
the
associated
were separated on the GlycanPac AXH-1 column and analysed
y. It is designed for high-resolution,
information.
The
chromatographic
profiles
shown
in
Figures
1
and
2,
detected
by
selectivity for biologically relevant glycans, including glycans from antibodies—either
vity for biologically relevant glycans,
fluorescence
detection,
provide
qualitative information
the Because
separation
of N-glycans.
labeled
or
native—by
LC-fluorescence
or LC-MS about
methods.
glycans
are
glycan
structure
by
database
searching
and
scoring
on a Q Exactive mass spectrometer. Data-dependant MS/
methods.
The structure
of glycansand
present
each peak hydrophilic
was determined
from the
LC-MS
study
highly hydrophilic
polar in
substances,
interaction
liquid
chromatography
using(HILIC)
the GlycanPac
µm) column
in the
following
section.are often
columnsAXH-1
based(1.9
on amide,
amine,asorshown
zwitterion
packing
materials
techniques.
MS spectra were acquired on all precursor ions (z< 2) and
Novel Glycan Column Technology for the LC-MS Analysis of Labeled
olumn Technology for the LC-MS Analysis of Labeled and Native N-Glycans Released fr
used for glycan analysis. These HILIC columns separate glycans mainly by hydrogen
bonding, resulting in size- and composition-based separation. However, identification
of the1.glycan
chargeofstate
not possible
with these
of columns
FIGURE
Separation
2ABislabeled
N-glycans
fromtypes
Bovine
fetuin bybecause
charge, size
and glycans
polarity.of different charge states are intermingled in the separation envelope, making
this approach limited. The GlycanPac AXH-1 column, which is based on advanced
mixed-mode chromatography technology, overcomes these limitations and can
separate glycans based on charge, size, and polarity configuration. In addition, each
glycan charge state can be quantified. The GlycanPac AXH-1 column provides both
greater selectivity and higher resolution, along with faster quantitative analysis.
ms in ‘free state’ and conjugated
teoglycans. They are involved in a
ses.1 The functions, including
as recombinant proteins and
ent on the structure and types of
of glycans are quite diverse,
tional modifications and physiological
uantitative estimation of glycans is
aceutical projects.3 However, it is
aracterize glycan profiles and
ance HPLC/UHPLC columns
nd quantitative analysis of glycans.
hroughput analysis, with unique
ing glycans from antibodies—either
methods. Because glycans are
ilic interaction liquid chromatography
erion packing materials are often
eparate glycans mainly by hydrogen
d separation. However, identification
ese types of columns because
d in the separation envelope, making
umn, which is based on advanced
mes these limitations and can
arity configuration. In addition, each
nPac AXH-1 column provides both
16 Bio-Innovation Issue
th faster quantitative analysis.
FIGURE 2. Comparison of 2AB labeled N-glycans standards and 2AB-N-glycans
from fetuin.
Structural A
GlycanPac
FIGURE 4. LC-MS analysis of 2AB labeled N-glycans from Bovine fetuin by a
commercial amide HILIC column (1.7 µm) with MS detection.
Antibody res
protein biothe
heterogeneit
LC-MS A
oneThe
of the
m
Glyca
in vivo
s
and and
analy
example
of no
th
glycans
where
2AA
la
during lab
column
(1.9
ambiguityµ
LC-MS/MS
a
native N-g
were
found
in
native
glyc
withammonium
minor am
provides
adv
separation
Methods
Sample Preparation
Release native glycans from glycoproteins with PNGase F enzyme. Conjugate the
released glycans with 2-amino benzamide (2AB) label group using the reported
procedure of Bigge et al.4 Here, 2-AB A1 (P/N GKSB 311), 2-AB A2 (P/N GKSB 312),
and 2-AB A3 (P/N GKSB 314) were purchased from Prozyme® (Hayward, CA). Prior
to analysis, dissolve samples in 100% buffer (100 mM ammonium formate, pH = 4.4)
and dilute further with acetonitrile to make 30% buffer and 70% acetonitrile.
Liquid Chromatography
All the glycans were separated using a Thermo Scientific™ Dionex™ UltiMate™ 3000
FIGURE 1.
BioRS system and either a Thermo Scientific™ Dionex™ UltiMate™
FLD-3400RS
Rapid Separation
Fluorescence
Detector
or MSand
detector.
FIGURE 2. Comparison
of 2AB labeled
N-glycans
standards
2AB-N-glycans
fromMass
fetuin.
Spectrometry
Q
Benchtop
MS analysis was performed with a Thermo
LC-MS/MS in negative ion mode at the following settings: MS scan range 380–2000.
FT-MS was acquired at 70,000 resolution at m/z 200 with AGC target of 1e6; and DDA
MS2 acquired at 17,500 resolution at m/z 200 with AGC target of 2e5.
Scientific™
7 Exactive™
Data Analysis
SimGlycan® software (PREMIER Biosoft) was used for glycan identification and
FIGURE 2.
LC-MS and LC-MS/MS Analysis of 2AB Labeled N-Glycan Using GlycanPac AXH-1
Column
The coupling of the GlycanPac AXH-1 column to MS was also explored. This is particularly
attractive as MS, with it’s ability to provide structural information, enables in-depth analysis
of complex glycans. 2AB labeled N-glycans from bovine fetuin were separated on the
GlycanPac AXH-1 column and analyzed on a Q Exactive mass spectrometer. Datadependant MS/MS spectra were acquired on all precursor ions (z< 2) and SimGlycan
LC-MS Analysis of Native Glycans Released from Proteins
software was used for glycan structural elucidation. A representative example of the
The GlycanPac
is 3.
well
suited
for high-performance
LC/MS
separation
analysis is AXH-1
shown column
in Figure
The
detailed
structural information
obtained
from the MS/MS
confirmati
FIGURE
A
profiles6.ar
especially
provide be
is useful fo
amount of
analysis o
Prozyme is a
International.
SimGlycan software was used for glycan structural elucidation.
A representative example of the analysis is shown in Figure
3. The detailed structural information obtained from the MS/
MS data further validated the ability of the GlycanPac AXH-1
column to separate glycans based on charge, size, and
polarity. However, coelution of different charge state glycans
(Figure 4) is common with other commercially available HILIC
columns.
f Labeled
and Native N-Glycans Released from Proteins and Antibodies
LC-MS Analysis of Native Glycans Released from
2 Gurmil S. Gendeh,1 Srinivasa Rao,1 Andreas
different
from the2 profile
fluorescently 1labelled
glycans, Pohl1
Proteins
hiqi Hao,
Huhmer,
YuryofAgroskin,
and Chris
especially higher sialic acid glycans. However, fluorescently
TheCA,
GlycanPac
an Jose,
USAAXH-1 column is well suited for high-
FIGURE 1: Separation of 2AB
labelled N-glycans from Bovine
fetuin by charge, size and
polarity.
labelled glycans generally provide better and more MS/
performance LC/MS separation and analysis of native glycans
MS fragmentation peaks. The GlycanPac AXH-1 column is
from MAbs and other proteins. Analysing unlabelled glycans
FIGURE 2: Comparison of 2AB
useful
foranalysis
the analysis
of bothfrom
native
and
labelled
N-glycans,
not
only3. eliminates
extra
reaction
and fetuin
cumbersome
FIGURE
5. LC-MS
of native N-glycans
Bovine
fetuin.
All the peaks
are
FIGURE
LC-MS analysisthe
of 2AB
labeled
N-glycans step
from Bovine
by the
N-glycansofstandards
Quantitative labelled
Determination
Glycans Based on Ch
detected by MS detection in negative ion mode.
GlycanPac AXH-1 (1.9 µm) column with MS detection.
Quantitative analysis
of each glycan
is essential for quic
and 2AB-N-glycans
from fetuin.
depending on the amount of sample available. If the amount
cleanup methods during labeling, but also retains the original
in protein batch comparisons and for comparison of dise
ural
profiles.
In
addition,
quantitative
analysis
of the sample is not extremely limited, analysis of unlabelledprovide a toolFIGURE
glycan profile without adding further ambiguity imposed by
3: LC-MS analysis of of glycans sep
resent
for calculating the relative amounts of diff
50 mm)
2AB
labelled
N-glycans
enzymatic
digestion
with
silidase
S from
and sialidase A. Figu
glycans using the GlycanPac AXH-1 is highly feasible.
the labeling reaction. Figure 5 shows the LC/MS analysis of
ased on
analysis of 2AB
labeled
based on charge the
Bovine
fetuinN-glycans
by the GlycanPac
eled
(1.9
µm)
with
fluorescence
detection.
The relative amou
native N-glycans from Bovine fetuin using the GlycaPac AXH-1
AXH-1
µm) column
withAMS
was estimated
using(1.9
a standard
curve.
standards curv
d on
the
chromatographic
analysis
of
2AB
labeled
A2 glycan
Structural Analysis of N-Glycans from Antibodies by
column (1.9 µm). The native glycans were separated based
detection.
ed
different amount of samples starting from 0.1 to 5 pmole
is
LC-MS Using GlycanPac AXH-1 Column
on charge, size, and polarity. Using an ammonium formate/
FIGURE 4: LC-MS
analysis of
of each charge s
titative
FIGURE 8: Quantitative
estimation
N-glycan from
2ABFetuin
labelled N-glycans from
Antibody
research has gained significant
interest as a part of
acetonitrile2gradient highly compatible
with MS detection,
the
1
2
2
1
1
2 Yur
lycans.
Julian Saba, Xiaodong Liu, Rosa Viner, Zhiqi Hao, Gurmil S. Gendeh, Srinivasa Rao, Andreas
Bovine fetuinHuhmer,
by a commercial
udy
the development of protein biotherapeutics. Glycosylation
separation enables excellent MS and MS/MS fragmentation
2Thermo Fisher Scientific, San Jose, CA, USA
amide HILIC column (1.7 µm)
vale, data
CA,forUSA;
of antibodies is a prime source of product heterogeneity
accurate confirmation of the glycan structure of each
with MS detection.
ge, size
with respect to both structure and function. Variation in
chromatographic peak. Native glycan profiles are significantly
chnology for the LC-MS Analysis of Labeled and Native N-Glycans Released from Prote
HPLC)
hntly
c analysis
ysis for
s by MS
C column
ous
solution,
glycans,
cans
gated
ed in a
ng
d
es of
e,
ysiological
cans is
r, it is
nd
Results
Structural Analysis of N-Glycans from Antibodies by LC-MS Using
FIGURE
3. LC-MS
analysis of 2AB labeled N-glycans from Bovine fetuin by the
GlycanPac
AXH-1
Column
FIGURE 4. LC-MS analysis of 2AB labeled N-glycans from Bovine fetuin by a
AXH-1
(1.9
µm) column
with
detection.
AntibodyGlycanPac
research has
gained
significant
interest
as aMS
part
of the development of
commercial amide HILIC column (1.7 µm) with MS detection.
Separation of Labeled Glycans Based on Charge, Size, and Polarity
protein biotherapeutics. Glycosylation of antibodies is a prime source of product
The GlycanPac AXH-1 column can be used for qualitative, quantitative, and structural heterogeneity with respect to both structure and function. Variation in glycosylation is
analysis as well as characterization of uncharged (neutral) and charged glycans present one of the main factors in product batch-to-batch variation,2-3 affecting product stability
in vivo and significantly influencing Fc effector functions in vivo. A representative
in proteins. Figure 1 shows bovine fetuin on the GlycanPac AXH-1 (1.9 μm, 2.1 × 150 mm)
column using fluorescence detection. The separation and elution of glycans are based onexample of the chromatographic separation of antibody glycans is shown in Figure 6,
charge: the neutral glycans elute first, followed by the separation of acidic 2AB labeled where 2AA labeled N-glycans from IgG were separated using the GlycanPac AXH-1
column (1.9 µm). Characterization of glycans in each peak was performed by
N-glycans from monosialylated, disialylated, trisialylated, tetrasialylated and finally
pentasialylated species. Glycans of each charge state are further separated based on LC-MS/MS and results are shown in Figure 7. Three different glycan charge states
were found in this human IgG; the majority of glycans are neutral or monosialylated,
their size and polarity. The retention time of each glycan charge state was confirmed

with minor amounts of disialylated glycans. Separation of glycans based on charge
using 2AB labeled glycan standards (as shown in Figure 2). Separation of glycans is
provides advantages compared to other commercially available HILIC columns.
based on charge, size, and polarity, which provides significant structural and quantitative

information. The chromatographic profiles shown in Figures 1 and 2, detected by
FIGURE 6. Analysis of 2AA labeled N-glycans from human IgG.
fluorescence detection, provide qualitative information about the separation of N-glycans.
FIGURE 5. LC-MS analysis of native N
detected by MS detection in negative
Conclusion
The structure of glycans present in each peak was determined from the LC-MS study
using the GlycanPac AXH-1 (1.9 µm) column as shown in the following section.
The GlycanPac AXH-1 column separates glycans with
charge, size, and polarity not possible with commercia
LC-ESI-FTMS or FT-MS/MS analysis of both native a
and antibodies were carried out successfully using Gl
 The GlycanPac AXH-1 column is useful for both highseparation and easy quantification of glycans.
 The GlycanPac AXH-1 columns are compatible with v
 These new columns have high-chromatographic effici
FIGURE 1. Separation of 2AB labeled N-glycans from Bovine fetuin by FIGURE
charge,
4. size
and polarity.
LC-MS Analysis of Native Glycans Released from Proteins
The GlycanPac AXH-1 column is well suited for high-performance LC/MS separation
and analysis of native glycans from MAbs and other proteins. Analyzing unlabeled
glycans not only eliminates the extra reaction step and cumbersome cleanup methods
during labeling, but also retains the original glycan profile without adding further
ambiguity imposed by the labeling reaction. Figure 5 shows the LC/MS analysis of
FIGURE
3.
stability.
 The GlycanPac AXH-1 column is also useful for the se
Structural
Analysis of N-Glycans fr
from proteins
and mucins.
FIGURE 4. LC-MS analysis of 2AB labeled N-glycans from Bovine fetuin by a
commercial amide HILIC column (1.7 µm) with MS detection.
GlycanPac AXH-1 Column
 The GlycanPac AXH-1 column is useful for the analys
Antibody research has gained significan
glycosylaminoglycans and glycolipids.
protein biotherapeutics. Glycosylation of
www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
heterogeneity with respect to both17 struct
one of the main factors in product batchin vivo and
significantly
influencingAllFc
ef
1. Varki, A. Biological
Roles
of Oligosaccharides:
the
References
FIGURE 5: LC-MS analysis
of native N-glycans from
Bovine fetuin. All the peaks are
detected by MS detection in
negative ion mode.
FIGURE 6: Analysis of 2AA
labelled N-glycans from
human IgG.
glycosylation
of the mainN-Glycans
factors in product batcheach charge
state
glycan was estimated
a standard
nalysis of Labeled
andis one
Native
Released
from
Proteins
andusing
Antibodies
affecting productand
stability Antibodies
in vivo and
to-batchfrom
variation, Proteins
curve. A standards curve was drawn using the data from the
-Glycans
Released
2
2
1
1
2
1
FIGURE 7: Mass spectroscopic
characterisation of glycans in
each Figure 6 peak.
2-3
sa Viner, Zhiqi 1Hao,significantly
Gurmil
S. Gendeh,
Rao,
Andreas
Huhmer,
Yury
Agroskin,
andstandard,
Chris Pohl
influencing
effector Srinivasa
functions in vivo.
A
chromatographic
analysis
of 2AB
labelled A2 glycan
2FcYury
1 and
1
FIGURE 8: Quantitative
Srinivasa
Rao,
Andreas
Huhmer,
Agroskin,
Chris
Pohl
representative
with the injection of different amount of samples starting from
Scientific,
Jose,
CA, USAexample of the chromatographic separation
estimation ofSan
each charge
state
of antibody glycans is shown in Figure 6, where 2AA labelled
0.1 to 5 pmole).
N-glycans from IgG were separated using the GlycanPac
AXH-1 column (1.9 µm). Characterisation of glycans in each
FIGURE 3. LC-MS analysis of 2AB labeled N-glycans from Bovine fetuin by the
FIGURE 5. LC-MS analysis of native N-glycans from Bovine fetuin. All the peaks are
Quanti
GlycanPac
AXH-1
(1.9 µm) column with
MS detection.
detected
by MS
detection in AXH-1
negative ion
mode. separates glycans with
The
GlycanPac
column
peak
was
LC-MS/MS
are shown
Quantit
FIGURE
5. performed
LC-MS analysisby
of native
N-glycansand
from results
Bovine fetuin.
All the peaks are • Quantitative
ans from Bovine fetuin by the
Determination of Glycans
Based on Charge
Size, and Polarity
in prote
by MS detection in negative ion mode.
tion.
Quantitative
analysis of each
glycanon
is essential
for quick
of glycan
variation profiles
unique selectivity
based
charge,
size,assessment
and polarity
not
in detected
Figure 7.
Three different glycan charge states were found
ive, quantitative, and structural
in protein batch comparisons and for comparison of diseased to normal cell glycosylation provide
tral) and charged glycans present
possible
with quantitative
commercial
HILIC
columns.
in this human IgG; the majority of glycans are neutral or
profiles.
In addition,
analysis
of glycans
separated based on charge state alsoenzyma
nPac AXH-1 (1.9 μm, 2.1 × 150 mm)
provide a tool for calculating the relative amounts of different sialic acid linkages after
nd elution of glycans are based on
analysi
• enzymatic
LC-ESI-FTMS
orsilidase
FT-MS/MS
analysis
of both
native
and
monosialylated, with minor amounts of disialylated glycans.
digestion with
S and sialidase
A. Figure
8 shows
the quantitative
separation of acidic 2AB labeled
(1.9 µm
analysis of 2AB labeled N-glycans based on charge the using GlycanPac AXH-1 column was es
d, tetrasialylated and finally
labelled glycans from proteins and antibodies were carried
Separation of glycans based on charge provides advantages
References based on
(1.9 µm) with fluorescence detection. The relative amount of each charge state glycan
are further separated
the chro
n charge state
wasA.confirmed
was
using a standard
A standards
curve was
drawn using the data from differen
1. Varki,
Biological Roles
outestimated
successfully
usingcurve.
GlycanPac
AXH-1
columns.
compared to other commercially available HILIC columns.
re 2). Separation of glycans is
the chromatographic analysis of 2AB labeled A2 glycan standard, with the injection of
of Oligosaccharides: All
nificant structural and quantitative
amount of samples
starting
from 0.1 to
pmole). for both
• different
The GlycanPac
AXH-1
column
is 5useful
FIGUR
Theories Are
gures 1 and the
2, detected
byCorrect.
N-glyca
high-resolution
charge-based
and easy
Quantitative Determination of Glycans Based on Charge
about the separation
N-glycans.
FIGURE
8: Quantitative
estimation of eachseparation
charge state glycan
in 2AB labeled
Glycobiologyof1993,
3, 97–130.
ermined from the LC-MS study
N-glycan from Fetuin
quantification of glycans.
Quantitative analysis of each glycan is essential for quick
n in the following
section.
2. Bertozzi,
C.R.; Freeze, H.H.;
• The GlycanPac AXH-1 columns are compatible with
assessment of glycan variation in protein batch comparisons
Varki, A.; Esko, J.D. Glycans
Biotechnology
and thesize
om Bovineinfetuin
by charge,
various MS instruments.
and for comparison of diseased to normal cell glycosylation
Pharmaceutical Industry,
•
These new columns have high-chromatographic efficiency
profiles. In addition, quantitative analysis of glycans separated
Structural Analysis of N-Glycans from Antibodies by LC-MS Using
Essentials of Glycobiology,
GlycanPac
Column
and AXH-1
excellent
column stability.
based
on charge
provide
a tool
for calculating
Second Edition; Cold Spring
FIGURE
4. LC-MS
analysisstate
of 2ABalso
labeled
N-glycans
from Bovine
fetuin by a
Structural Analysis of N-Glycans from Antibodies by LC-MS Using
Antibody research has gained significant interest as a part of the development of
commercial amide HILIC column (1.7 µm) with MS detection.
Harbor Laboratory Press: New
•
The
GlycanPac
AXH-1
columnisisa also
usefulof for
the
the
relative
amounts
of
different
sialic
acid
linkages
after
GlycanPac
AXH-1
Column
protein
biotherapeutics.
Glycosylation
of antibodies
prime source
product
ans from Bovine fetuin by a
York, 2009; Chapter 51.
heterogeneity with respect to both structure and function. Variation in glycosylation is
Antibody research has gained significant interest as a part of the development of
S detection.
separation
of
reduced
O-glycans
from
proteins
and
enzymatic
digestion
with
silidase
S
and
sialidase
A.
Figure
2-3
one of the main factors in product batch-to-batch variation, affecting product stability
protein biotherapeutics. Glycosylation of antibodies is a prime source of product
2 structure
1 and
1 significantly influencing Fc effector functions in vivo. A representative
3. Guidance for Industry,
in vivo mucins.
and
deh,1 Srinivasa
Rao,1 Andreas
Huhmer,
Yuryand
Chris
Pohl
heterogeneity
respect to both
function.
Variation in
glycosylation
is
8 shows
thewith
quantitative
analysis
ofAgroskin,
2AB
labelled
N-glycans
example of the chromatographic separation of antibody glycans is shown in Figure 6,
one of the main factors in product batch-to-batch variation,2-3 affecting product stability
Scientific Considerations in
where
2AA
N-glycans from
IgG were
separated
using thefor
GlycanPac
AXH-1
in vivo on
and charge
significantlythe
influencing
effector functions
in vivo.column
A representative
• Thelabeled
GlycanPac
AXH-1
column
is useful
the analysis
of
based
usingFc
GlycanPac
AXH-1
(1.9
Demonstrating Biosimilarity
column (1.9 µm). Characterization of glycans in each peak was performed by
example of the chromatographic separation of antibody glycans is shown in Figure 6,
LC-MS/MS
and results
areneutral
shown in Figure
7. Three different glycan charge
charged
and
glycosylaminoglycans
and states
glycolipids. Conc
µm)
with
fluorescence
relative
of AXH-1
to a Reference Product, Draft
where
2AA
labeled N-glycansdetection.
from IgG wereThe
separated
using amount
the GlycanPac
glycan in 2AB labelled N-glycan
from Fetuin
Conclusion
e N-Glycans Released from Proteins and Antibodies
column (1.9 µm). Characterization of glycans in each peak was performed by
LC-MS/MS and results are shown in Figure 7. Three different glycan charge states
were FIGURE
found in5.this
human
IgG; the
majority
of glycans
are
neutral
or monosialylated,
LC-MS
analysis
of native
N-glycans
from
Bovine
fetuin.
All the peaks are
with minor
amounts
disialylated
glycans.
Separation
of glycans based on charge
detected
by MSof
detection
in negative
ion
mode.
provides advantages compared to other commercially available HILIC columns.
Guidance; U.S. Department of
Health and Human Services
led N-glycans from Bovine fetuin by the
Food and Drug Administration,
MS detection.
February
2012 [Online] www.
1
fda.gov/downloads/Drugs/
standards GuidanceCompliance
and 2AB-N-glycans
RegulatoryInformation/
Guidances/UCM291128.pdf
(accessed Jan. 18, 2013).
were found in this human IgG; the majority of glycans are neutral or monosialylated,
with minor amounts of disialylated glycans. Separation of glycans based on charge
provides advantages compared to other commercially available HILIC columns.
Quantitative
Determination
of Glycans
Based on Charge
 The GlycanPac
AXH-1
column separates
glycans with unique selectivity based on
Quantitative
analysis
each
glycan
is essential
quick
assessment
of glycan
variation
charge,
size, of
and
polarity
not
possiblefor
with
commercial
HILIC
columns.
FIGURE
6. Analysis
of
2AA
labeled
N-glycans
from
human IgG.
Conclusion
ive N-Glycans Released from Proteins and Antibodies
ndeh, Srinivasa
Rao,1
Andreas
Huhmer,2
Yury
Agroskin,1
and Chris Pohl
in protein batch
and for comparison of diseased to normal cell glycosylation
1 comparisons
 LC-ESI-FTMS
or FT-MS/MS
analysis
of both
native
and on
labeled
from proteins
profiles.
In addition, quantitative
analysis
of glycans
separated
based
chargeglycans
state also
antibodies
werethe
carried
successfully
using
GlycanPac
AXH-1
FIGURE 6. Analysis of 2AA labeled N-glycans from human IgG.
provideand
a tool
for calculating
relativeout
amounts
of different
sialic
acid linkages
aftercolumns.
enzymatic digestion with silidase S and sialidase A. Figure 8 shows the quantitative
 The GlycanPac AXH-1 column is useful for both high-resolution charge-based
analysis of 2AB labeled N-glycans based on charge the using GlycanPac AXH-1 column
separation and easy quantification of glycans.
(1.9 µm) with fluorescence detection. The relative amount of each charge state glycan
wasestimated
using a standard
A standards
curve was
drawn
usingMS
the instruments.
data from
The GlycanPac
AXH-1 curve.
columns
are compatible
with
various
the chromatographic analysis of 2AB labeled A2 glycan standard, with the injection of
 These
new
columnsstarting
have high-chromatographic
efficiency and excellent column
different
amount
of samples
from 0.1 to 5 pmole).
labeled N-glycans from Bovine fetuin by the
FIGURE 5. LC-MS analysis of native N-glycans from Bovine fetuin. All the peaks are
Quantitative
stability. Determination of Glycans Based on Charge
4. Bigge, J.C. et al.,
with MS detection.
detected by MS detection in negative ion mode.
FIGURE
8: Quantitative
of each
charge for
state
glycan
in 2AB of
labeled
LC-MS Analysis
of Native Glycans Released from Proteins
Quantitative
analysisestimation
of each glycan
is essential
quick
assessment
glycan variation
 Thefrom
GlycanPac
AXH-1 column is also useful for the separation of reduced O-glycans
N-glycan
Fetuin
Non-Selective and Efficient
in
protein batch
comparisons and for comparison of diseased to normal cell glycosylation
The GlycanPac AXH-1 column is well suited for high-performance LC/MS separation
from
proteins
and
mucins.
profiles. In addition, quantitative analysis of glycans separated based on charge state also
Fluroscent
Labeling
of
Glycans
and
analysis
of
native
glycans
from
MAbs
and
other
proteins.
Analyzing
unlabeled
om Proteins
a tool for calculating
the relative
amounts
sialic
linkages
 provide
The GlycanPac
AXH-1 column
is useful
for of
thedifferent
analysis
of acid
charged
andafter
neutral
Using
2-Amino
Benzamide and glycans not only eliminates the extra reaction step and cumbersome cleanup methods
enzymatic
digestion with silidase
S and sialidase A. Figure 8 shows the quantitative
performance
LC/MS
separation
glycosylaminoglycans
and glycolipids.
during labeling, but also retains the original glycan profile without adding further
analysis
of
2AB
labeled
N-glycans
based
on
charge
the
using
GlycanPac
AXH-1
column
proteins. Analyzing
Anthranilicunlabeled
Acid. Anal. Biochem. ambiguity imposed by the labeling reaction. Figure 5 shows the LC/MS analysis of
(1.9 µm) with fluorescence detection. The relative amount of each charge state glycan
nd cumbersome cleanup methods
native N-glycans from Bovine fetuin using the GlycaPac AXH-1 column (1.9 µm). The
1995, 230, 229–238.
was estimated using a standard curve. A standards curve was drawn using the data from
ofile without adding further
native glycans were separated based on charge, size, and polarity. Using an
the chromatographic analysis of 2AB labeled A2 glycan standard, with the injection of
shows the LC/MS analysis of
FIGURE
7. Mass
spectroscopic
characterization
glycans
in each Are
Figure
6 peak.
1.different
Varki,
A.
Biological
Rolesstarting
of Oligosaccharides:
All the Theories
Correct.
ammoniumStructural
formate/acetonitrile
gradient
highly
compatible
with
MS
detection,
the
amount
of samples
from 0.1 to 5 of
pmole).
5. Apte, A; Meitei, N.S.
Analysis of N-Glycans from Antibodies by LC-MS Using
Pac AXH-1 column (1.9 µm). The
Bioinformatics
e,
polarity.
Using
an in Glycomics:
ledand
N-glycans
from
Bovine
fetuin by a
atible
detection,
the with
m)
withwith
MS MS
detection.
Glycan
Characterization
ntation data for accurate
Mass Spectrometric Data Using
ographic peak. Native glycan
SimGlycan.
Methods
Mol. Biol.
uorescently
labeled
glycans,
Glycan
Using
GlycanPac
AXH-1
scently labeled
generally
2010,glycans
600, 269–81.
s. The GlycanPac AXH-1 column
as also explored. This is particularly
-glycans, depending
on the
Acknowledgements
ormation, enables
in-depth analysis
mple
is not
extremely
limited,the
e fetuin
were
Weseparated
would
like toonthank
Mark
XH-1
is
highly
feasible.
ve mass spectrometer. Data-
 LC-E
and a
 The G
separ
 The G
 These
stabil
 The G
from
 The G
glyco
Refe
1. Varki,
Glyco
2. Berto
Pharm
Glycobiology 1993, 3, 97–130.
Harbo
8: Quantitative
estimation
of each
charge
state
glycanininBiotechnology
2AB labeled and the
2.FIGURE
Bertozzi,
C.R.; Freeze,
H.H.; Varki,
A.; Esko,
J.D.
Glycans
3.
Guida
N-glycan
from Fetuin
Pharmaceutical
Industry, Essentials of Glycobiology, Second Edition; Cold Spring
Refer
Harbor Laboratory Press: New York, 2009; Chapter 51.
Food
3. Guidance for Industry, Scientific Considerations in Demonstrating Biosimilarity to a
Guida
Reference Product, Draft Guidance; U.S. Department of Health and Human Services
Jan. 1
Food and Drug Administration, February 2012 [Online] www.fda.gov/downloads/Drugs/ 4. Bigge
GuidanceComplianceRegulatoryInformation/Guidances/UCM291128.pdf (accessed
2-Am
Jan. 18, 2013).
5. Apte
4. Bigge, J.C. et al., Non-Selective and Efficient Fluroscent Labeling of Glycans Using
Spec
The GlycanPac AXH-1 column separates glycans with unique selectivity based on
2-Amino Benzamide and Anthranilic Acid. Anal. Biochem. 1995, 230, 229–238.
charge, size, and polarity not possible with commercial HILIC columns.
5. Apte, A; Meitei, N.S. Bioinformatics in Glycomics: Glycan Characterization with Mass
LC-ESI-FTMS
or FT-MS/MS
analysis
of both native
and labeled
glycans
from600,
proteins
Spectrometric
Data Using
SimGlycan.
Methods
Mol. Biol.
2010,
269–81.
and antibodies were carried out successfully using GlycanPac AXH-1 columns.
We woul
The GlycanPac AXH-1 column is useful for both high-resolution charge-based
from The
separation and easy quantification of glycans.
instrume
WeGlycanPac
would like
to thank
Markare
Tracy,
Yoginder
Wang, and Patrick K. Bennett
The
AXH-1
columns
compatible
with Singh,
various Jessica
MS instruments.
from Thermo Fisher Scientific for their help and permission to use their UHPLC and MS
These new columns have high-chromatographic efficiency and excellent column
instruments.
References
separation GlycanPac
enables excellent
and MS/MS fragmentation data for accurate
AXH-1 MS
Column
confirmation of the glycan structure of each chromatographic peak. Native glycan
Antibody
research
has gained significant
interest as of
a part
of the development
of 6 peak.
FIGURE
7. Mass
spectroscopic
characterization
glycans
in each Figure
profiles areprotein
significantly
different
from
the profile of fluorescently labeled glycans,
biotherapeutics. Glycosylation of antibodies is a prime source of product
especially higher
sialic acid
fluorescently
labeled
glycans
generally is
heterogeneity
with glycans.
respect toHowever,
both structure
and function.
Variation
in glycosylation
2-3 affecting
provide better
fragmentation
peaks. The
GlycanPac
AXH-1
column
product
stability
one and
of themore
mainMS/MS
factors in
product batch-to-batch
variation,
is useful forinthe
of both native
and Fc
labeled
N-glycans,
depending
on the
vivoanalysis
and significantly
influencing
effector
functions in
vivo. A representative
amount of example
sample available.
If the amountseparation
of the sample
is notglycans
extremely
limited,
of the chromatographic
of antibody
is shown
in Figure 6,
2AA labeled
IgG wereAXH-1
separated
using feasible.
the GlycanPac AXH-1
analysis ofwhere
unlabeled
glycansN-glycans
using thefrom
GlycanPac
is highly
column (1.9 µm). Characterization of glycans in each peak was performed by
LC-MS/MS and results are shown in Figure 7. Three different glycan charge states
of N-Glycans
from
Antibodies
byor
LC-MS
Using
were Structural
found in thisAnalysis
human IgG;
the majority of
glycans
are neutral
monosialylated,
GlycanPac
AXH-1
Columnglycans. Separation of glycans based on charge
with minor
amounts
of disialylated
Yoginder
Singh, Jessica
labeled
from
Bovine
fetuin by a
2) and
SimGlycan
sor
ionsN-glycans
(z<Tracy,
provides
advantages
compared
to other
commercially
available
HILIC
columns.
Antibody
research
has
gained
significant
interest
as
a
part
of
the
development
of
Prozyme
is
a
registered
trademark
of
ProZyme,
Inc.
SimGlycan
is
a
registered
trademark
of
PREMIER
Biosoft
1.7
µm) withWang,
MS
detection.
and Patrick
K. Bennett
epresentative
example
of the
International. All other
trademarks
are the property
of Thermo Fisher
andis
itsasubsidiaries.
protein
biotherapeutics.
Glycosylation
of Scientific
antibodies
prime source of product
nformation obtained
from
the Scientific
MS/MS
FIGURE
6. Analysis
of respect
2AA
N-glycans
from
human
IgG.infringeinthe
from Thermo
Fisher
heterogeneity
with
to both
structure
function.
Variation
glycosylation
This information
is not
intended
to encourage
uselabeled
of these
products
in anyand
manners
that might
intellectual is
-1 column to separate glycans
property rights of others.
one of the main factors in product batch-to-batch variation,2-3 affecting product stability
egistered trademark
of PREMIER
Biosoft
for their
help
andglycans
permission
nScientific
of different
state
1/13S
in vivo and significantly influencing Fc effector functions in vivo.PO70513_E
A representative
and itscharge
subsidiaries.
e HILIC columns.
to use their UHPLC and MS
example of the chromatographic separation of antibody glycans is shown in Figure 6,
n any manners that might infringe the intellectual
where
2AA
labeled
N-glycans
from
IgG
were
separated
using
the
GlycanPac
AXH-1
instruments.
column (1.9 µm). Characterization of glycans in each peak was performed by
PO70513_E 1/13S
LC-MS/MS and results are shown in Figure 7. Three different glycan charge states
were found in this human IgG; the majority of glycans are neutral or monosialylated,
with minor amounts of disialylated glycans. Separation of glycans based on charge
provides advantages compared to other commercially available HILIC columns.
eleased from Proteins
ed for high-performance LC/MS separation
18 Bio-Innovation
Issue
and
other proteins.
Analyzing unlabeled
tion step and cumbersome cleanup methods
al glycan profile without adding further
 The G
charg
7 FIGURE 6. Analysis of 2AA labeled N-glycans from human IgG.
Conclusion





Ackn
Acknowledgements
Conclusion
stability.
 GlycanPac
The GlycanPac
AXH-1
column
separates
with unique
selectivity
based on
 The
AXH-1
column
is also
useful forglycans
the separation
of reduced
O-glycans
charge, size,
and polarity not possible with commercial HILIC columns.
from proteins
and mucins.

LC-ESI-FTMS
or
FT-MS/MS
analysis
of
both
native
and
labeled
glycans
 The GlycanPac AXH-1 column is useful for the analysis of charged and neutralfrom proteins
and antibodies were
out successfully using GlycanPac AXH-1 columns.
glycosylaminoglycans
andcarried
glycolipids.
 The GlycanPac AXH-1 column is useful for both high-resolution charge-based
separation and easy quantification of glycans.
References
flexible low volume
liquid handling
Evaluation of the Formulatrix Tempest at EMD Serono: A flexible low
volume liquid handler with two axis gradient dispensing capability
Vikram Shankar*, Adam Shutes,
Bill Griffin, Brian Healey Lead
Discovery Technologies, EMD
Serono Research Institute,
Rockland, MA, 02370. *vikram.
[email protected]
Liquid handling instrumentation which provides precise and
flexible dispensing over a range of volumes and different
components is core to a fully functional assay development,
hit discovery and characterisation lab. At EMD Serono, we use
liquid dispensing (50 µl to 200 nl) in a limited number of tasks,
such as regular assay component dispensing (around 15-70 µl),
as well as low volume dispensing (200 nl). Other aspects of assay
development & biochemical characterisation of inhibitors are
performed in a manual and labour intensive manner.
Introducing the Formulatrix Tempest in our workflow has allowed
us to improve the current technique not only in terms of efficiency
but also accuracy and precision. Unique to the Tempest is its new
microfluidic technology that relies on independent channel control over
all the 96 nozzles, thereby allowing one to dispense any volume from any
input into any well. We evaluated this diversity for a wide range of assays
from those requiring factorial dispensing, like assay design and modality
of inhibition studies, to applications like assay miniaturisation that use fixed
volume dispensing.
Tempest
5ul Assay
The Tempest is a flexible and compact liquid dispenser that
uses state of the art microfluidics to dispense any volume
from any input into any well.
It uses positive displacement to dispense liquids with minimal
waste. It has a dispensing range of 200nl to no upper limit
and a very low non-recoverable dead volume (~40µl). Typical
dead volumes are ~400µl and can be reduced to ~100µl
using tip dispensing. It is accurate, precise, fast and is
compatible with 96, 384, 1536 well micro plates.
Tempest accuracy
We analysed the accuracy of the Tempest in two formats
A fixed volume miniaturised assay comprising of 2µl enzyme & 3µl
substrate.A variable volume assay that requires different volumes
of 8 different enzymes.With both formats, we found the CV’s for
the whole plate to be within the acceptable range of 5%.
Variable Volume
Assay (CV’s)
Avg
Std dev
CV
0.390
0.003
0.818
Kinase I
1.318
0.388
0.003
0.896
Kinase P
1.122
0.390
0.003
0.739
Kinase A1
1.133
0.389
0.003
0.805
Kinase F
1.296
0.392
0.004
0.993
Kinase I
1.508
0.390
0.004
1.018
Kinase P
1.550
0.390
0.004
0.950
Kinase A1
2.519
0.387
0.004
0.974
Kinase F
2.374
0.390
0.003
0.754
Kinase FL
1.885
0.390
0.003
0.714
Kinase R
3.109
0.389
0.003
0.801
Kinase S
2.109
0.389
0.003
0.789
Kinase V
1.905
0.387
0.003
0.761
Kinase FL
1.820
0.386
0.003
0.755
Kinase R
1.584
0.388
0.004
1.011
Kinase S
1.721
0.386
0.003
0.866
Kinase V
2.085
www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
19 Workflow comparison
Modality of inhibition
Our current workflow for establishing assay parameters
involves 3 separate steps.
a) Titration to determine the enzyme concentration for a 30%
conversion. (Fig 1)
b) Kinetic run to determine the ATP Km of the enzyme. (Fig 2)
c) Final titration to confirm the established parameters. (Fig 3)
We evaluated the Tempest’s efficiency in modality of inhibition
experiments. By dispensing a gradient of ATP down the plate
we studied the response of two in-house competitive inhibitors.
Figure 2.
1.5
0.4
0.3
0.8
0.8
Kinase A Linearity
31.25
3.90625
0.1
0.1
0.3
500
500
250
250
125
125
62.5
31.25
0.4
15.625
7.8125
3.90625
1.953125
0.3
0.9765625
0.1
0.2
31.25
15.625
0.0
-7.5
7.8125
3.90625
1.953125
0.48828125
0.2
1.953125
0.9765625
0.48828125
0.1
0.1
0.2
0.5
0.5
2.0
1.0
1.0
2.5
1.5
1.5
2.02.0
2.52.5
Enzyme
Enzymeconc
conc(ng/ul)
(ng/ul)
Enzyme conc (ng/ul)
0
1000
2000
3000
4000
Time (s)
0
5000
5
0
0.0
4000
1000
Time (s)
5000
2000
6000
3000
7000
4000
5000
6000
7000
-5.0
-2.5
0.0
2.5
5.0
Time (s)
5.0
2.5
7.5
7.5
5.0
Figure 4.
0.1
7.5
vmax 0.7459
KM 15.06
0.25
0.25
0.25
200
0.4
0.6
0.8
0.4
0.6
0.2
Enzyme
conc
0.8 (ng/ul)
0.6
Enzyme conc (ng/ul)
0.8
0.00
200
100
100
0
0
50
100
150
0
200
-5.0
5
200
0
200
100
100
0
05
10
0
5 10
0
S/Km
S/Km
15
0.50
3000
3000
S/Km
20
5 0.00
2000
2000
2000
1000
1000
1000
0
1520
20
0
10
0.00
S/Km 0.25
15
20
0.50
0.75
1.00
0
0
0
5
1.25
0 5
5 10
10 15
0
S/Km S/Km
15 0
10
15 20
20 5
S/Km
% Conversion
Inhib A IC50 (nM)
ATP (uM)
Inhib B IC50 (nM)
0.75
258
250
1953
0.50
151
125
1300
0.25
91
62.5
882
65
31.25
530
36
15
402
27
7.5
337
21
3.75
258
10
1.75
119
0.50
0.75
1.00
1.25
Table 1.
Figure 10.
Cell dispensing
We further evaluated whether operation of the diaphragm during dispensing had an
effect on cells. As shown in Fig 10 & 11, we saw no morphological changes in cells
dispensed with the Tempest when compared to manual dispense.
Figure 11
Summary
Low volume pipetting technology is key to our workflow and is of utmost
importance. Here we have highlighted the improved efficiency the Tempest
provides to our workflow. Other key features include an easy USB connectivity,
a user friendly software and automation capability. ELISA, PCR, HTS, DNA/RNA
research are some of the other applications that the Tempest can support.
20 Bio-Innovation Issue 7 20
10
S/Km
conc of Enz (ng/ul)
Enzyme conc (ng/ul)
conc of Enz (ng/ul)
Table 1: IC50 values of both
inhibitors at various ATP
concentrations
2000
1.00
0.25
using Tempest
3000
1000
1015
11: A375
7.5 cells dispensed
2.5Figure5.0
0.0
7.5
3000
0.75
0.25
-2.5
Figure 5.
0.00
0.00
Figure 10: A375 cells dispensed
manually
Figure 9.
Figure 8.
0
0.2
ATP inhibition
Figure 9:15uM
Competitive
250uM
ATP B
of Kinase
A by Inhibitor
250uM
ATP
250uM
250uM
ATP ATP
0.0
-7.5
7.5
IC50 nM
0.50
Figure 8 : Competitive inhibition
of Kinase A by Inhibitor A
15uM
ATP
15uM
ATP
15uM ATP
0.1
0.0
0.0
0.0
2.5 7.57.5
5.0
0.0 2.5 2.5
0.0 5.05.0
-7.5
-5.00.0 -2.5
-5.0 -5.0
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product focus
Thermo Scientific™ Barnstead™ Type 1
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Why is UV intensity monitoring important for ultrapure water?
UV intensity monitoring is an innovative technology designed to
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29 www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
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The tools & technologies of pr
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Today’s molecular biologist relies on the
plasmid, a closed, circular, double stranded
form of DNA that is propagated in bacteria.
Large Scale
Plasmid DNA Preparation
Hui Li, Department of
Pharmacology and Molecular
Toxicology, University of
Massachusetts Medical Center,
Worcester, MA
Many different protocols can produce plasmid DNA in large quantities that are sufficiently pure for general cloning,
enzyme digestion, sequencing, cellular transfection, in vitro transcription/ translation, protein expression and other
purposes. For instance, DNA can be isolated in cesium chloride gradients, but this method requires an ultracentrifuge and
utilises ethidium bromide, which must be handled with care.1 Alternatively, gravity or spin column kits effectively purify
plasmid DNA in superspeed and microcentrifuges. This brief highlights Thermo Scientific Sorvall superspeed centrifuges
and rotors that can be used in concert with the commercially available DNA preparation kits and also provides a low-cost
method to obtain plasmid DNA in large-scale (about 1 mg) without using a kit.
Procedure
4. Add 15 mL buffer B, mix by inversion 5-6 times and let sit at
PROTOCOL 1: Cost-Efficient, Large-Scale DNA Plasmid Prep
using only floor standing Superspeed Centrifuges from start to
finish.
room temperature for 5 min. Solution should become more
clear and viscous.
5. Add 15 mL buffer C, mix by inversion 5-6 times. A heavy
precipitate will form; swirl to break up precipitate.
6. Centrifuge at 20,000 x g for 20 min at 4 °C.
7. Pour supernatant through cheese cloth and collect in a
clean bottle. Note: An additional spin for 10 min may pellet
any excessive debris not cleared in the first spin.
8. Add an equal amount of isopropanol to the bottle and swirl
gently to mix. Perform centrifugation with the following
parameters: 18,000 x g for 30 min at 4 °C. Note: Use
a lab pen to mark the outer edge of the bottle before
centrifugation to help locate the somewhat clear and
translucent pellet (also applies to subsequent steps).
9. Carefully remove the supernatant. Add >50 mL of 70%
ethanol to the 250 mL bottle and agitate to resuspend and
wash the DNA. Perform centrifugation with the following
parameters: 18,000 x g for 10 min at 4 °C.
10.Remove all the supernatant and dry the pellet in a vacuum
dessicator for 30-60 min. Over-drying the pellet can
prevent effective DNA resuspension in subsequent steps.
11.Dissolve the pellet in a total of 2 mL TE containing RNAase
at a final concentration of 100 µg/mL. If dissolved in
higher volumes, the DNA may be too dilute to effectively
precipitate in subsequent steps.
12.Transfer to a 15 mL or similar volume tube and incubate at
37 °C for 1 hr.
Hui Li, Department of Pharmacology and Molecular Toxicology,
University of Massachusetts Medical Center, Worcester, MA.
This procedure describes the isolation of plasmid DNA using
a floor model superspeed centrifuge from start-to-finish. The
Thermo Scientific Sorvall LYNX 6000 superspeed centrifuge
employs a diverse array of rotors and can accommodate a
wide variety of applications.
1. Using the appropriate antibiotic selection, grow 500 mL or
more of bacterial culture (usually overnight at 37 °C, with
shaking).
2. Divide culture by pouring into 250 mL bottles. Pellet
bacteria by performing centrifugation in a Sorvall® LYNX
6000 superspeed centrifuge with a Thermo Scientific
Fiberlite carbon fiber rotor (such as the Fiberlite® F146x250y, F12-6x500 LEX, F10-4x1000 LEX, or F9-6x1000
LEX rotor) with the following parameters: 6,000 x g for 15
min at 4°C.
3. Decant supernatant and thoroughly resuspend bacteria in
one of the bottles with 15 mL buffer A. Transfer bacteria to
the other bottle and resuspend the combined pellets.
There should be no “clumps” remaining.
30 Bio-Innovation Issue 7 13. Precipitate the DNA for 30 min on ice with 0.5 volume 20%
PEG 8000, 2.5 M NaCl or with one volume of 13% PEG
8000, 1.6 M NaCl.
14.Perform centrifugation in the Thermo Scientific Fiberlite
F14-14x50cy rotor with 15 mL conical adapters or in the
Thermo Scientific A21-24x15c rotor with the following
parameters: 20,000 x g for 20 min at 4 °C.
15.Carefully remove all the supernatant. The pellet may be
very translucent and smeared along the outer edge of the
tube. It is critical to remove all the PEG.
16. Dissolve the DNA in 700 µL of TE or 10 mM Tris and transfer
to a high performance 1.5 mL micro-centrifuge tube.
17.Add an equal volume (700 µL) of phenol, vortex 30 sec and
perform centrifugation in the Thermo Scientific Fiberlite
F27-48x1.5 rotor with the following parameters: 19,000 x g
for 5 min at 4 ºC.
18.Remove the upper aqueous phase and transfer to another
high performance microtube containing 350 µL phenol and
350 µL chloroform.
19.Vortex and perform centrifugation with the following
parameters: 19,000 x g for 5 min at 4 °C.
20.Perform a final extraction in 700 µL chloroform. Repeating
the phenol/ chloroform extraction may increase DNA
purity. A back extraction once with 700 µL of TE can
increase yield.
21.Precipitate the DNA by the addition of 0.8 volume of
isopropanol and 0.1 volume of 3M NaOAc, pH 4.5-5.5. Mix
several times by inversion. Perform centrifugation in the
Fiberlite F27-48x1.5 rotor with the following parameters:
19,000 x g for 15 min at 4 °C.
22.Decant supernatant and carefully wash the DNA pellet with
1 mL of 70% EtOH.
23. Air dry pellet and dissolve in 500-1000 µL Tris or TE.
24.Measure the OD260 and OD260/280 and calculate DNA
concentration (DNA concentration in g/L = (OD260)(50)
(Dilution)) and assess purity. Note: An OD260/280 below
1.8 suggests impurities. Double phenol/chloroform
extraction can improve the ratio.
large volume capabilities, such as the Sorvall LYNX 6000
superspeed centrifuge. The initial steps in DNA preparation
involves the pelleting of bacteria in which the plasmid of
interest has been propagated. Individual bacterial cultures with
volumes up to 6 L can be processed using the Sorvall LYNX
6000 superspeed centrifuge. Centrifugation at 6,000 x g for
10-15 min at 4 °C is sufficient to pellet bacteria grown in a 500
mL or larger culture. For smaller volume cultures, the time and
speed for pelleting is reduced; centrifugation at 5,000 x g for
10 min at 4 °C is sufficient for a 150 mL bacterial culture. Table
2 lists a selection of rotors the Sorvall LYNX 6000 superspeed
centrifuge that will serve to accommodate small and large scale
pelleting (up to 6L) needs prior to DNA purification.
Table 2:
Thermo
Scientific Rotor
Capacity
Max Speed
(place x mL) (rpm)
Max RCF
(x g)
Fixed Angle Rotors
Fiberlite F9-6x1000 LEX
Fiberlite F10-4x1000 LEX
Fiberlite F12-6x500 LEX
Fiberlite F14-6x250
6 x 1000
4 x 1000
6 x 500
6 x 250
9,000
10,500
12,000
14,000
17,568
20,584
24,471
30,240
Swinging Bucket Rotors
BIOFlex HC
BIOFlex HC with adapter
BIOFlex HC with adapter
BIOFlex HS
4 x 1000
4 x 500
8 x 250
4 x 400
5,500
5,500
5,500
7,000
7,068
7,068
7,068
10,025
Table 1. Solutions for DNA
preparation
Table 2. Rotors available for
DNA preparation in the Sorvall
LYNX 6000 superspeed
centrifuge.
Table 3. High speed 15 mL and
50 mL conical tube rotors and
adapters available for DNA
preparation in the Sorvall LYNX
6000 superspeed centrifuge.
References
1. Noles, S.R., Schwartz,
M.W., Fischer, R.A., Martin, R.,
Schneider, W., Jagadeeswaran,
P., Rao, K.J. 2008.
Traditional Methods for CsCl
Isolation of Plasmid DNA by
Ultracentrifugation.
Following the initial pelleting of bacterial culture, DNA
purification steps require centrifugation at >15,000 x g. For
ease of use and to ensure the integrity of samples, the use of
sterile 15 mL to 50 mL conical tubes is desired, however, these
conical tubes are typically limited to <7,000 x g. The unique
Thermo Scientific conical tube rotors and conical tube adapters
allow for the use of disposable sterile conical tubes at RCFs up
to 63,409 x g. Table 3 lists a selection of rotors and adapters for
the Sorvall LYNX 6000 superspeed centrifuge that will serve to
accommodate the forces required by most commercial kits.
Table 1:
Solutions Needed
Table 3:
Buffer A: 50 mM Tris-HCl, pH 8.0; 10 mM EDTA
Buffer B: 0.2 M NaOH; 1% SDS
Buffer C: 3 M Potassium Acetate, pH 5.5
Isopropanol
70% Ethanol
RNAase A stock solution: 10 mg/mL
PEG/NaCl solution: 20% PEG 8000, 2.5 M NaCl or 13% PEG 8000, 1.6
M NaC
Phenol, Tris buffered to pH 8.0
Chloroform
10 mM Tris or TE
3 M Sodium acetate, pH 4.5-5.5
Thermo
Scientific Rotor
Fixed Angle Rotors
Fiberlite F14-14x50cy
Fiberlite F14-14x50cy with adapter
A21-24x15c
Swinging Bucket Rotors
TH13-6x50 with adapter 75007322
PROTOCOL 2: Using Superspeed Centrifuges for Plasmid
DNA Preparation with Commercially Available Kits
Protocol 1 describes the process for plasmid DNA preparation
without using a commercially available kit.
Capacity
Max Speed Max RCF
(place x mL) (rpm)
(x g)
14 x 50
14 x 15
24 x 15
14,000
14,000
21,500
33,746
33,746
63,049
6 x 50
13100
30,314
Conclusion
This application brief describes the wide variety of
superspeed offerings provided by Thermo Fisher Scientific
to accommodate the needs of molecular biologists during
plasmid DNA preparation. Thermo Scientific equipment
accommodates small and large volume bacterial cell pelleting
and offers efficient, versatile, lightweight, and reliable rotors.
Many commercially available kits for plasmid DNA preparation
that call for the use of a centrifuge with high speed and
31 Selecting Conical Tubes
for Centrifugation Applications
By Stephanie Carter, Wenxiao Lu, Marci Moore, Joe Granchelli & Cindy Neeley
Laboratory centrifuges are common, everyday instruments.
While centrifuge usage is widespread, the selection of the
proper centrifuge and rotor system, along with the proper
centrifuge tube consumable, can often be a challenging
experience.
There are several factors to consider when selecting the correct
conical tube for centrifugation. These include the relative
centrifugal force required, the fit of the particular tube in the
rotor, sample volume, and the compatibility of the sample to
the tube material.
Understanding the requirements of a centrifugation application
before selecting a conical tube can prevent sample leakage or
loss, allow for easy sample recovery, and reduce the risk for
potential damage to the centrifuge and rotor.
32 Bio-Innovation Issue 7 Factors to consider when choosing
a conical centrifuge tube
A. RPM vs. RCF
All centrifuge tubes have a maximum
speed rating determined by the
manufacturer. Vessels used at speeds
higher than the recommended rating can
fail, resulting in sample loss and potential
damage to the centrifuge and rotor.
Most protocols specify speed in either
revolutions per minute (RPM) or relative
centrifugal force (RCF). It is crucial to
understand the difference between
RPM and RCF. The rotor revolves at the
specified RPM and the force applied
to the contents is dependent on the
rotor’s radius, with larger radii applying
greater force on the sample. RCF
represents the gravitational force being
applied to the sample. It determines the
centrifugation outcome independent of
rotor size. RCF is measured in force x
gravity, or g-force, and is more relevant
to the actual impact and outcome of the
centrifugation than RPM. Users should
verify the required RCF by their specific
applications to ensure that the required
centrifugation force does not exceed the
manufacturer’s specified g-force rating.
2
Thermo Scientific Centrifuge Model
Thermo Scientific Rotor Model
Thermo Scientific Adapter Catalogue #
Legend™ 1XR
BIOShield™ 720
75003678
3500
Legend 1XR
Fiberlite F15-6x100y
75003095
24652*
Legend 1XR
TX-400
75003682
4696*
Legend 1XR
TX-200
75003771
5580*
Multifuge™ X3R
TX-750
75003639
4816*
Multifuge X3R
BIOShield 1000A
75003642
5000
Multifuge X3R
Fiberlite F15-8x50cy
010-0378-06
24446*
Multifuge X3R
BIOLiner
75003673
2739*
Evolution™
Fiberlite F13-14x50cy
010-0378-06
15000
4696*
5580*
24652*
75003095
Legend 1XR
75003771
TX-400
Legend 1XR
75003682
Fiberlite F15-6x100y
TX-200
Legend 1XR
Maximum RCF (x g)
3500
75003678
Thermo Scientific
Adapter Catalog #
Thermo Scientific Rotor Model
BIOShield™ 720
m
tto
bo
al
nic
Co
Maximum RCF (x g)
Thermo Scientific Centrifuge
Model
on
po
rti
on
rti
po
er
Lo
w
Table 1. Recomended Maximum RCF for Thermo Scientific™ Nunc™ 15 mL Conical Tubes (Catalogue # 339650, 339651)
Figure 2. Fixed angle rotor and
tube fit illustration. Centrifugal
forces are exerted on the sides
of the tube and transferred to
the rotor/adapter.
Legend™ 1XR
ion
ort
rp
pe
Figure 2. Fixed angle rotor and tube fit illustration. Centrifugal forces are exerted on the sides of the tube and transferred to the
rotor/adapter.
g force
good contact between the
tube and the rotor where
the g force applies
Figure 2.
Table 1. Recommended Maximum RCF for Thermo Scientific™ Nunc™ 15 mL Conical Tubes (Catalog # 339650, 339651)
Conical bottom portion
types of centrifuge units. In most cases, a rotor adapter is
needed to ensure good fitting of the tube to the rotor.
Lower portion
Figure 1. Swing-out bucket
rotor and tube fit illustration.
Centrifugal forces are exerted
on the conical and lower portion
of the tube. The imperfect fit
of the lower portion of the tube
to the rotor leaves a gap which
may cause tube damage
Table 1 and Table 2 display the recommended maximum
RCF for the Thermo Scientific Nunc 15 mL and 50 mL
conical tubes based on tests of 9 models of rotors in 3
g force
rotor
Upper portion
3
D. Sample volume
Before selecting a centrifuge tube size,
the desired sample volume should
be determined. The sample volume
is limited to the available rotors in the
laboratory and the protocol being used.
Generally, a centrifuge tube should
be filled at least 75%. In some cases,
such as with ultracentrifuge tubes, it
is required that tubes are completely
filled to prevent failure. Using centrifuge
tubes less than half filled to capacity
can lead to high levels of material stress
and can result in tube failure. If the
available sample volume is smaller than
the available rotor capacity, adapters
are available for most rotors from
the manufacturer (Table 1, Table 2).
Adapters allow a smaller-volume tube
to be used at the appropriate fill volume.
This is essential when the recommended
maximum RCF is going to be applied for
centrifugation.
Figure 1. Swing-out bucket rotor and tube fit illustration. Centrifugal forces are exerted on the conical and lower portion of the
tube. The imperfect fit of the lower portion of the tube to the rotor leaves a gap which may cause tube damage.
gap between
the tube and
the rotor
When using a fixed angle rotor in a centrifuge unit, the
side of the tube makes contact with the rotor/adapter and
most of the centrifugal force exerted on the tube can be
transferred to the rotor (Figure 2). Consequently, the
conical tube in a fixed angle rotor can be spun at much
higher RCF in comparison to a swing-out bucket rotor.
does not allow the lower portion of the tube to make
contact with the rotor/adapter. The centrifugal forces on
the side wall of the conical tube cannot be transferred to
the rotor and may cause damage to the lower portion of
the tube (Figure 1).
The Nunc conical centrifuge tubes are
made of premier quality polypropylene.
Table 3 displays information on chemical
compatibility of common reagents to
polypropylene centrifuge tubes.
Up
C. Chemical compatibility
In addition to the maximum RCF,
when evaluating centrifuge tubes it
is important to make sure that the
sample components will not harm the
plastic tube material. Chemicals can
affect the strength, flexibility, surface
Figure 1.
rotor
When using a fixed angle rotor in a
centrifuge unit, the side of the tube
makes contact with the rotor/adapter
and most of the centrifugal force exerted
on the tube can be transferred to the
rotor (Figure 2). Consequently, the
conical tube in a fixed angle rotor can be
spun at much higher RCF in comparison
to a swing-out bucket rotor.
texture, colour, and shape of the plastic.
Chemical resistance is influenced by
temperature, duration and frequency of
exposure, chemical concentration, and
centrifugal force. Physical and chemical
changes which may be caused by
chemical exposure include:
• Absorption of solvents, resulting in
softening or swelling of the plastic
• Stress-cracking of the plastic
• Permeation of solvent in the sample
through the plastic
• Dissolution of polymer in the sample
B. Fit of the conical tube in the centrifuge rotor
It is important to note that the centrifuge tube
performance largely depends on how well the tube fits in
the rotor (or rotor adapter). For optimal tube
performance it is imperative that there is contact between
the tube and the rotor/adapter so that centrifugal force
can be distributed to the rotor rather than the tube. Forces
exerted directly on the conical tube may cause stress lines,
bulging, or cracking. For example, when using a swingout bucket rotor in a centrifuge unit, the direction of the g
force centers on the conical bottom of the tube locking the
tube in the center position of the rotor. In some cases, this
B. Fit of the conical tube in the
centrifuge rotor
It is important to note that the centrifuge
tube performance largely depends
on how well the tube fits in the rotor
(or rotor adapter). For optimal tube
performance it is imperative that there
is contact between the tube and the
rotor/adapter so that centrifugal force
can be distributed to the rotor rather
than the tube. Forces exerted directly
on the conical tube may cause stress
lines, bulging, or cracking. For example,
when using a swing-out bucket rotor
in a centrifuge unit, the direction of the
g-force centres on the conical bottom
of the tube locking the tube in the centre
position of the rotor. In some cases, this
does not allow the lower portion of the
tube to make contact with the rotor/
adapter. The centrifugal forces on the
side wall of the conical tube cannot be
transferred to the rotor and may cause
damage to the lower portion of the tube
(Figure 1).
Table 1 and Table 2 display
the recommended maximum
RCF for the Thermo Scientific
Nunc 15 mL and 50 mL
conical tubes based on tests
of 9 models of rotors in 3
types of centrifuge units. In
most cases, a rotor adapter is
needed to ensure good fitting
of the tube to the rotor.
* Maximum speed for the centrifuge/rotor system is reached.
www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
33 Table 2. Recommended Maximum RCF for Thermo Scientific Nunc 50 mL Conical Tubes (Catalogue # 339652, 339653)
Thermo Scientific Centrifuge Model
Thermo Scientific Rotor Model
Thermo Scientific Adapter Catalogue #
Maximum RCF (x g)
Legend 1XR
BIOShield 720
75003677
6500
Legend 1XR
Fiberlite F15-6x100y
75003103
23500
Legend 1XR
TX-400
75003683
4696*
Legend 1XR
TX-200
75003803
5580*
Multifuge X3R
TX-750
75003638
4816*
Multifuge X3R
BIOShield 1000A
75003643
6500
Multifuge X3R
Fiberlite F15-8x50cy
not necessary
20000
Multifuge X3R
BIOLiner
75003674
2739*
Evolution
Fiberlite F13-14x50cy
not necessary
15000
* Maximum speed for the centrifuge/rotor system is reached.
Summary
As in most endeavors, gathering the
correct information ahead of time is
essential. In this study, we demonstrated
the important factors to consider
when selecting the conical tubes for
centrifugation so that sample leakage or
loss is prevented. Choosing the correct
centrifuge consumables also ensures
proper execution of centrifugation, and
reduces the risk for potential damage to
the centrifuge and rotor.
The factors we discussed in this article
include the RCF required by the protocol,
the fit of the particular conical tube
in the rotor, sample volume, and the
chemical compatibility of the sample to
the tube material. Users should verify the
maximum RCF of the centrifuge tube to
ensure that the required speed does not
exceed the manufacturer’s rating. Here,
we recommended the maximum RCF
of the Nunc 15 mL and 50 mL conical
tubes in several Thermo Scientific
centrifuge and rotor systems while taking
these factors into consideration.
Key: Table 3.
References
Goodman, Tammy (Thermo
Scientific). Selecting Centrifuge
Consumables. American
Laboratory April 2009.Thermo
Scientific, NALGENE Centrifuge
Tubes and Bottles Your
Complete Guide to Centrifuge
Ware, 2001
34 Bio-Innovation Issue 7 S = Satisfactory
S1 = Satisfactory, may cause discolouration.
M = Marginal; may be satisfactory for use in
a centrifuge, depending on length of exposure
and speed.Testing under operating conditions is
suggested before actual run.
U = Unsatisfactory; not recommended.
Table 3. Chemical Compatibility of Common Reagents
to Polypropylene Centrifuge Tubes
Classification
Alcohols
Cryopreservation Agents
Detergents
Fixatives
Other
Chemical
Rating
Butanol, pure
S
Ethanol, 100%
S
Isopropanol, 100%
S
Methanol, 15%
S
Methanol, 98% x-750
S1
Propanol, 100%
S
Dimethyl sulfoxide (DMSO), pure
S
Glycerol
S
Sodium Dodecyl Sulfate (SDS), pure
S
Triton X-100, pure
S
Tween-20
S
Acetone, 50%
S
Acetone, pure
M
Formaldehyde, 10%
S1
Formaldehyde, 30%
M
Formalin, 10%
S1
Formalin, 30%
M
Gluteraldehyde, pure
S
Paraformaldehyde, pure
M
Beta- Mercaptoethanol, pure
M
Cell culture media and sera
S
EDTA, pure
S
RNAzol
S
Trypsin
S
Toluene, pure
U
Xylene, pure
U
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one step to stronger
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Improving productivity
through compact automation
Many modern analytical laboratories face increasing workloads from a
broad range of sample types with a simultaneous drive for faster results and
more complex sample characterisation needs.
Many routine QC/QA laboratories can perform material
analyses with single range, basic Fourier transform-infrared
(FT-IR) instrument configurations. However, modern analytical
laboratories face increasing workloads from a broad range
of sample types with a simultaneous drive for faster results
and more complex sample characterisation needs. Flexibility
to analyse multiple sample types becomes mandatory when
rapidly responding to these different application requests.
Such diversity requires laboratory instruments to be
reconfigured for specific measurements multiple times per
day, taking time away from other critical activities. This also
implies that laboratory personnel possess the necessary skills
and experience to make decisions on how best to configure
the instrument for a given application. In addition, frequent
handling of delicate optics components presents a costly risk
for instrument failure. As a result, many industrial laboratories
choose to outsource complex analyses. These limitations
inevitably slow the laboratory’s ability to respond to urgent
business needs.
References
1. Heavy atoms or groups of
atoms shift the IR wavenumber
value lower, according to the
relationship where ˜v is the
IR wavenumber (cm-1) and μ
is the reduced mass. As the
mass (μ) increases, the IR peak
shifts to lower wavenumbers.
Glossary
CaF2 – calcium fluoride
DLaTGS – deuterated
L-alanine doped triglycene
sulphate
InGaAs – Indium gallium
arsenide
KBr – potassium bromide
36 The Thermo Scientific™ Nicolet™ iS™50 FT-IR spectrometer
alleviates many of these productivity concerns by automating
setup of the FT-IR system for multi-spectral range experiments
(>20,000 cm-1 to 80 cm-1) and for integrating techniques like
FT-Raman, near-IR and mid/far-IR attenuated total reflectance
(ATR) into a single workflow. Intelligent design behind the
Nicolet iS50 spectrometer permits unattended, risk-free
operation, increasing lab efficiency, sample throughput, and
operational consistency between users. This capability is
delivered in an economical, compact system (63 cm of linear
bench space) enabling any laboratory to employ multiple
techniques for their analysis.
Flexibility and Value-added Activities
Working labs need analytical flexibility to respond to a variety
of situations where answers are critical for decision-making.
Examples include deformulating mixtures to build a case
for patent infringement, identifying counterfeit materials
for product safety alerts, analysing forensic samples for
criminal investigations, performing failure analysis to minimise
Bio-Innovation Issue 7 production run delays, assessing process scale-up options
for a new product launch, or troubleshooting customer
complaints. Such diversity of applications requires the
selection and installation of the correct instrument accessory
as well as choosing the optimal source, beamsplitter, detector,
optical path, and experimental conditions. Manually changing
components and sampling parameters requires skill and may
risk exposure of expensive optics to the external environment
(i.e., dust, fingerprints or water vapor). In addition, changing
these parameters can result in extensive wait times to
equilibrate the instrument before the next measurement.
These manual reconfigurations provide little added value
to the laboratory workflow. Users must plan and set up
batch experiments to minimise the number of steps. This
creates bottlenecks, limiting access to the full capability of
the instrument. As a result, labs are less able to address
“emergency situations” without interrupting the batch run and
resetting the instrument parameters. For instance, analysis of a
polymer with additives requires mid-IR and far-IR plus Raman
spectroscopy. This would entail three beamsplitter changes
with associated risks in handling expensive components and
instrument recovery times between changes.
The productivity improvements with the Nicolet iS50 FT-IR
spectrometer come from two main sources. First, the internally
mounted iS50 ABX Automated Beamsplitter Exchanger
uses one-button simplicity (described as a Touch Point) to
perform instrument setup and operation, providing a “one
touch and done” workflow. The removal of manual handling
and exposure of the optics to the environment means instant
readiness. Second, the user need no longer care about which
optics are installed. As seen in Table 1, the potential for error
in manual operations is apparent when the array of possible
component combinations is considered. With the Nicolet iS50
spectrometer, however, a user simply presses the Touch Point
on the instrument to automate the configuration and ready
the instrument for the experiment. For example, pressing the
Touch Point on the iS50 NIR module automates the setup
without requiring any understanding of which optics are used.
What matters is performing NIR analysis – not worrying about
These manual reconfigurations provide little added value
to the laboratory workflow. Users must plan and set up
batch experiments to minimize the number of steps. This
creates bottlenecks, limiting access to the full capability of
the instrument. As a result, labs are less able to address
“emergency situations” without interrupting the batch run
and resetting the instrument parameters. For instance,
analysis of a polymer with additives requires mid-IR and
far-IR plus Raman spectroscopy. This would entail three
beamsplitter changes with associated risks in handling
expensive components and instrument recovery times
between changes.
Figure 1 describes the analytical power the user can
achieve with the iS50 spectrometer to obtain answers
needed for time-sensitive decisions. With a single user
interaction, the instrument can perform multiple
measurements and analyses, resulting in a final report,
even when unattended. The Thermo Scientific OMNIC ™
software provides a user-friendly interface to set up
applications quickly and generate spectra for definitive
answers. By adding powerful analytical tools like the
Thermo Scientific OMNIC Specta™ software with a
library of over 30,000 spectra and multi-component
searching (or the TQ Analyst™ software for chemometrics),
a complete analytical workflow from sampling to results
can often be achieved in less than 60 seconds.
The productivity improvements with the Nicolet iS50
FT-IR spectrometer come from two main sources. First,
the internally mounted iS50 ABX Automated Beamsplitter
Exchanger uses one-button simplicity (described as a
Touch
Point)
to performThe
instrument
setup
andcare
operation,
choosing the right
components.
instrument
takes
of
providing a “one touch and done” workflow. The removal
this step.
of manual handling and exposure of the optics to the
environment means instant readiness. Second, the user
Integrationneed
of the
with which
its modules
nospectrometer
longer care about
opticsand
are installed. As
components
the1,user
to expand
increasing
seenallows
in Table
the potential
forcapabilities,
error in manual
operations
productivity
with toolswhen
suchthe
as:array of possible component
is apparent
is considered.
With
the and
Nicolet
iS50
• Up tocombinations
three detectors
(such as near-,
midfar-IR)
spectrometer,
however,
a user simply
presses the Touch
• The iS50
Raman sample
compartment
module
Point on
the instrument
to sampling
automate station
the configuration and
• The built-in
diamond
iS50 ATR
ready the instrument for the experiment. For example,
• The iS50 NIR module with integrating sphere or fibre
pressing the Touch Point on the iS50 NIR module
Figure 1: Nicolet iS50 analysis workflow
opticsautomates the setup without requiring any understanding
• The iS50
GC-IRoptics
module
of which
are used. What matters is performing
Automated Multi-spectral Analysis: Mid- and
• A sample
gravimetric
analysis-IR
This paper will demonstrate how the integration and
NIRcompartment
analysis – not thermal
worrying
about choosing
the right
Far-IR
ATR plus Near-IR
(TGA-IR
Interface) The instrument takes care of this step.
components.
automation of the Nicolet iS50 spectrometer leads to new
Most FT-IR users understand the utility of the mid-IR spectral
Integration of the spectrometer with its modules and
levels of productivity, while minimizing risk to costly
range
for qualitative
and
quantitative
analyses.
Less well
components
the power
user tothe
expand
capabilities,
components.
Unlike
most
spectrometers,
operating
the
Figure 1 describes
theallows
analytical
user can
achieve
known,
theiS50
far-IR
region can
providesimpler
new and
unique
increasing
productivity
with
tools
such
as:
Nicolet
instrument
becomes
as
modules
are
with the iS50 spectrometer to obtain answers needed for
information.
Simply
put,
as thesteps
mass
ofremoved
atoms involved
in
added
and
as
more
manual
are
even
when
• Up decisions.
to three detectors
(such user
as near-,
mid- and
far-IR)
time-sensitive
With a single
interaction,
the
1
unattended.
vibrations
increases,
the
wavenumber
decreases.
The perform
iS50 Raman
sample
compartment
module
instrument• can
multiple
measurements
and
analyses,
Thus, for materials like organometallics or metal oxides, the
iS50
ATR sampling
resulting in• aThe
finalbuilt-in
report,diamond
even when
unattended.
The station
Thermo
IR absorption shifts below 400 cm-1 and below the range of
•
The
iS50
NIR
module
with
integrating
sphere
or
Scientific OMNIC™ software provides a user-friendly interface
standard KBr optics. Numerous polymers, sugars, and other
fiber optics
to set up applications quickly and generate spectra for
• The iS50 GC-IR module
large molecules also have far-IR information which may be
definitive answers. By adding powerful analytical tools like the
• A sample compartment thermal gravimetric
useful or definitive to the analyst. Traditionally, collecting FT-IR
Thermo Scientific
OMNIC
Specta™
software with a library
analysis-IR
(TGA-IR
Interface)
spectra in both the mid-IR and far-IR region entailed significant
Figure 1: Nicolet iS50
analysis work
of over 30,000 spectra and multi-component searching (or
the TQ Analyst™ software for chemometrics), a complete
analytical workflow from sampling to results can often be
Experiment
Source
achieved in less than 60 seconds.
™
sample preparation. This included changing hygroscopic
optics and multiple detectors, and risking altered system
Beamsplitter
Detector
Accessory
performance from water
vapor. The Nicolet iS50
spectrometer
Mid-IR Transmission
Thermo Scientific Polaris
KBr
KBr-DLaTGS
Standard
enables rapid analysis over the full mid-IR and wellCells
into the
Far-IR Transmission
Polaris
Solid Substrate
Polyethylene
DLaTGS
Cellsequipped
w/Far-IR Windows
80 cm-1) when
with the
far-IR region (4,000
cm-1 to
This paper will demonstrate how the integration and
ABX, iS50 ATR, InGaAs
and the correct beamsplitters.
Near-IR Transmission
White Light
CaFiS50
Cuvettes
2
automation of the Nicolet iS50 spectrometer leads to
The typical, multi-range
opening
Mid-IR ATR
Polaris
KBr
DedicatedFT-IR
DLaTGSapplication requires
iS50 ATR
new levels of productivity, while minimising risk to costly
the spectrometerDedicated
to swap
beamsplitters. ThisiS50
requires
Far-IR ATR
Polaris
Solid Substrate
DLaTGS
ATR care in
components. Unlike most spectrometers, operating the Nicolet
costly components
Raman InGaAsand exposes the
iS50internal
Raman optics
FT-Raman
Raman Laser
CaFhandling
2
iS50 instrument becomes simpler as modules are added and
to the environment by disrupting purge or desiccation. This
Table
1:
Experiments
made
possible
with
the
Nicolet
iS50
FT-IR
Spectrometer
as more manual steps are removed even when unattended.
activity adds a recovery period to re-equilibrate the instrument
before quality data can be collected. These wait times add
www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
37 Table 1: Experiments made
possible with the Nicolet iS50
FT-IR Spectrometer
Table 2: Far-infrared analysis:
Analysis:
Typical versus Nicolet iS50
Near-IR
process *With the iS50 ATR
present, the far-IR background
he utility(BKG)
ofisthe
mid-IR
collected,
the iS50 ABX
swaps beamsplitters,
and the
d quantitative
analyses.
mid-IR background is collected
ion can inprovide
new and
<1.5 minutes. The sample
as the ismass
oftheatoms
loaded and
spectra are
collected in sequence. All times
the wavenumber
are approximate.
ike organometallics or
shifts below 400 cm-1
d KBr optics. Numerous
ge molecules also have
useful or definitive to the
g FT-IR spectra in both
tailed significant sample
ng hygroscopic optics and
tered system performance
S50 spectrometer enables
IR and well into the
cm-1) when equipped with
he correct beamsplitters.
Experiment
Source
Beamsplitter
Detector
Accessory
Mid-IR Transmission
Thermo Scientific Polaris™
KBr
KBr-DLaTGS
Standard Cells
Far-IR Transmission
Polaris
Solid Substrate
Polyethylene DLaTGS
Cells w/Far-IR Windows
Near-IR Transmission
White Light
CaF2
InGaAs
Cuvettes
Mid-IR ATR
Polaris
KBr
Dedicated DLaTGS
iS50 ATR
Far-IR ATR
Polaris
Solid Substrate
Dedicated DLaTGS
iS50 ATR
FT-Raman
Raman Laser
CaF2
Raman InGaAs
iS50 Raman
no value to operations, wasting the analyst’s precious time.
Integration and automation on the spectrometer eliminate
non-productive wait times, improving efficiency.
As an example, Table 2 compares the steps needed to perform
a full spectral analysis from far-IR to near-IR between the
manual method (Typical) and the Nicolet iS50 method with
built-in iS50 ATR and iS50 NIR module. This represents three
spectral ranges in one sampling operation, a unique power of
the instrument. Most important the built-in iS50 ATR optics and
detector permit spectral data collection in both the mid- and
far-IR regions. The analysis time decreases from around 30
minutes to less than seven.
With the Nicolet iS50 spectrometer, the user is able to load
two sampling locations (the built-in ATR and the Integrating
Sphere module), start the macro and walk away, while in the
manual operation, continuous attention is needed to swap the
beamsplitters at the right moments.
This seemingly hidden improvement allows unattended
operation, permitting productivity through automation. Figure
2 shows just the mid- and far-IR spectra collected from
acetylferrocene analysed using an OMNIC macro-controlled
workflow. The additional information from the far-IR spectra is
clear – the low end triplet verifies that the iron is sandwiched
between the cyclopentadiene rings. The NIR data is not shown,
but the entire process required seven minutes, including
collection of the mid- and far-IR backgrounds. Automation also
reduced the total hands-on time of the user (pressing buttons,
loading sample) to ≈20 seconds.
Process Step
Sample Preparation
Mid-IR Background
Typical
Grind, Mix
Collect BKG
Time
(minutes)
10
0.5
Mid-IR Collect
Load Sample,
Collect Spectrum
2
Change Optics
Manual Exchange
0.5
Recovery Time
Wait for Purge
5–10
Collect BKG
0.5
Far-IR Background
Far-IR Collect
Load Sample,
Collect Spectrum
2
Change Optics (NIR)
Manual Exchange
0.5
Recovery Time
Collect Background
Collect Sample
Data Analysis (Search)
Total Time
Wait for Purge
Collect BKG
5
0.5
Load Sample,
Collect SAM
1
Perform Search
2
29.5–34.5
Multiple Techniques and Multi-range Analysis:
Enhanced Flexibility
The Nicolet iS50 spectrometer can be configured with
FT-Raman, NIR, and wide-range diamond ATR. Switching
between these experiments raises concerns of instrument
recovery time (purge), exposure/handling of optics, and
potential confusion or user error. The experiments are
often seen as independent activities for these reasons. The
spectrometer with iS50 ABX simplifies this apparently complex
situation to one step – initiation of a macro. The Nicolet iS50
instrument shown in Figure 3 is configured with the iS50 NIR,
iS50 Raman, iS50 ATR and the iS50 ABX modules and shows
how easy sample loading and analysis can be done.
For operating one module at a time, the user need only
press the associated Touch Point. Seen more closely in
Figure 4, Touch Points make one-button operation effortless
when switching between modules (sampling stations) and
automating optics exchange. Rather than thinking through the
components needed (light source, beamsplitter, optical path
and detector) to run an experiment, the user simply presses the
Touch Point to switch from an ATR to an NIR measurement and
waits until the instrument indicates that it is ready to begin.
This error-free operation is done in 30 seconds. The Nicolet
iS50 analytical power in Figure 1 becomes clear when the four
data collections – mid-IR and far-IR ATR, NIR, and Raman – are
performed in one workflow.
Collecting spectra from each of these modules using a
conventional manual approach required about 50 minutes,
including sample loading, optical changes, time for
equilibration, and optimisation
of the Raman signal. The analyst
Nicolet iS50
Time
with Built-in ATR
(minutes)
needed to be present throughout
None
0
the experiment to perform the
Collect BKG (2nd)*
1.
beamsplitter changes and collect
various backgrounds for each
Load Sample,
1
Collect Spectrum
sampling station. At the end of the
50 minutes, four spectra and their
Automated
0.5
analyses were available. Actual
No Recovery Time
0
data collection took 5 minutes
Collect BKG (1st)*
0.5
and total hands-on time was 45
Load Sample,
1
minutes, representing inefficient
Collect Spectrum
use of the analyst’s time.
Automated
0.5
In contrast, the results shown in
No Recovery Time
0
Figure 5 emerged from a single
Collect BKG
0.5
OMNIC-macro operation. The
macro was programmed to begin
Collect SAM
0.5
by collecting backgrounds for
the mid- and far-IR ATR, and
Automated Search
0.5
then switched to the iS50 Raman
6.5
module. Next the samples were
Table 2: Fa r-infrared analysis: Typical versus Nicolet iS50 process
* With the iS50 ATR present, the far-IR background (BKG) is collected, the iS50 ABX swaps beamsplitters, and the
application requires
mid-IR background is collected in <1.5 minutes. The sample is loaded and the spectra are collected in sequence.
38 Bio-Innovation
Issue
7 ap beamsplitters. This
All times are approximate.
components and exposes
Figure 2:
Figure 2: Mid-IR and far-IR
Time
Nicolet iS50
TimeThe
loaded
on the ATR,Multi-spectral
NIR, and Raman sampling
stations. After optimising the signal using the autofocus feature of the
spectra of Acetylferrocene.
Automated
Analysis:
Process Step
Typical
(minutes)
with Built-in ATR
(minutes)
far-IR
optics
permit
collection
Raman
the macro
wasplus
initiated,
and the analyst walked away. From starting the macro to completion of the
Mid-module,
and Far-IR
ATR
Near-IR
Sample Preparation
Mix
10
None to 1700 cm-1, which may0 be
final report, the analysis took less than 12 minutes, representing a time
savings of over 70%. TheGrind,
actual
data collection
sufficient (fingerprint and far-IR)
Most
understand
of the
mid-IR
Mid-IR
Collect BKG
Collect BKG (2nd)*
time
was FT-IR
again 5users
minutes,
however, the
totalutility
hands-on
time
for the analyst
wasBackground
only 2 minutes – a highly
efficient use of0.5
the
for many applications. 1.
spectral
range
for
qualitative
and
quantitative
analyses.
Mid-IR
Collect
Load
Sample,
2
Load
Sample,
1
analyst’s (and the instrument’s) time.
Figure 3: The Thermo Scientific
Less well known, the far-IR region can provide new and
Collect Spectrum
Collect Spectrum
Nicolet iS50 FT-IR spectrometer
unique information. Simply put, as the mass of atoms
configured for FT-Raman,nearChange Optics
Manual Exchange
0.5
Automated
0.5
Conclusion
IR, and mid/far-IR ATR with
involved in vibrations increases, the wavenumber
Recovery Time
Wait for Purge
5–10
No RecoverytheTime
0
automated beamsplitter
Many
forces contribute
to materials
new pressures
on industrial analytical
1
decreases.
Thus, for
like organometallics
or laboratories: increased sample loads, decreased
exchanger.
Far-IR
Background
Collect
BKG
0.5
Collect
BKG
(1st)*
0.5
staffing,
retirement
of
experts,
and
shrinking
budgets.
The
Thermo
Scientific
Nicolet
iS50
FT-IR
spectrometer
makes
metal oxides, the IR absorption shifts below 400 cm-1
Figure 4: Touch Points on
a significant
contribution
challenges
through automation
in a multi-tasking,Load
single
platform
Far-IR Collect
Sample,
2
Load Sample,
1 the
and below
the rangeto
ofalleviating
standardthese
KBr optics.
Numerous
Nicolet iS50 spectrometer
Collect Spectrum
Collect Spectrum
instrument.
The
Nicolet
iS50
spectrometer
greatly
simplifies
and
streamlines
workflows
by
decreasing
the
number
polymers, sugars, and other large molecules also have
employ one-button switching
of steps
one-button
ease may
and macro
operations
performed
analyst.
addition, risks
inherent
in manual0.5
Change
OpticsIn(NIR)
Manual
Exchange
Automated
0.5iS50
between modules and the
far-IRwith
information
which
be useful
or definitive
to theby the
ABX automates optics set-up
operations
(e.g.,
user
error,
environmental
exposure)
and
long
recovery
times
are
eliminated.
Analysts
of
any
skill
level
Recovery Time
Wait for Purge
5
No Recovery Time
0
analyst. Traditionally, collecting FT-IR spectra in both
canthe
successfully
obtain
meaningful
results with
minimal hands-on
Touch Point A – NIR module
mid-IR and
far-IR
region entailed
significant
sample time.
Collect Background
Collect BKG
0.5
Collect BKG
0.5
Touch Point B – Raman module
preparation. This included changing hygroscopic optics and Collect Sample
Load Sample,
1
Collect SAM
Touch Point C – Built-in0.5
Technology
designed
to
improve
workflow
can
be
found
in
the
iS50
ABX
and
task-specific
modules
(i.e.,
Raman,
NIR,
multiple detectors, and risking altered system performance
Collect SAM
diamond ATR
TGA-IR
The
TouchThe
Point
operation
accessenables
to the full range of capabilities by automatically configuring
Component D – ABX Automated
frometc.).
water
vapor.
Nicolet
iS50simplifies
spectrometer
Data Analysis (Search)
Perform Search
2
Automated Search
0.5
Beamsplitter Exchanger
therapid
opticsanalysis
(near-, midand
far-IR)
and
switching
between
sampling
stations
(modules)
in
seconds
for
enhanced
over the full mid-IR and well into the
Total Time offers a powerful new tool that goes
29.5–34.5
6.5
-1
-1 the Nicolet iS50 FT-IR spectrometer
Figure 5: Multi-technique
productivity.
For the
modern
lab,
far-IR region
(4,000
cmindustrial
to 80 cm
) when equipped with
data for a recyclable plastic
Tableand
2: Fafar-IR),
r-infrared
analysis:
Typical
Nicolet activities
iS50 process
beyond
routine
FT-IR
to more
analyses
(e.g., FT-Raman
adding
value
toversus
laboratory
the iS50
ABX,
iS50
ATR,comprehensive
and the correct
beamsplitters.
component using the
Multiple Techniques and Multi-range Analysis:
in a compact, easy-to-operate platform.
* With the iS50
ATR present, the far-IR background (BKG) is collected, the iS50 ABX swaps beamsplitters,
the in
spectrometerand
pictured
Enhanced
Flexibility
The typical, multi-range FT-IR application requires
mid-IR background is collected in <1.5 minutes. The sample is loaded and the spectra are collected
sequence.
Figure 3.inInset
shows NIR
The
Nicolet
iS50
spectrometer
can
be
configured
with
opening the spectrometer to swap beamsplitters. This
All times are approximate.
independently for clarity.
FT-Raman, NIR, and wide-range diamond ATR. Switching
requires care in handling costly components and exposes between these experiments raises concerns of instrument
the internal optics to the environment by disrupting purge recovery time (purge), exposure/handling of optics, and
potential confusion or user error. The experiments are
or desiccation. This activity adds a recovery period to
often seen as independent activities for these reasons.
re-equilibrate the instrument before quality data can
be
Figure 3.
The spectrometer with iS50 ABX simplifies this apparently
collected. These wait times add no value to operations, complex situation to one step – initiation of a macro. The
wasting the analyst’s precious time. Integration and
Nicolet iS50 instrument shown in Figure 3 is configured
automation on the spectrometer eliminate non-productive with the iS50 NIR, iS50 Raman, iS50 ATR and the iS50
ABX modules and shows how easy sample loading and
wait times, improving efficiency.
analysis can be done.
As an example, Table 2 compares the steps needed to
perform a full spectral analysis from far-IR to near-IR
between the manual method (Typical) and the Nicolet iS50
method with built-in iS50 ATR and iS50 NIR module. This
represents three spectral ranges in one sampling operation,
a unique power of the instrument. Most important the
built-in
ATR
detector
permit
spectral
Figure 3:
The ThermoiS50
Scientific
Nicoletoptics
iS50 FT-IRand
spectrometer
configured
for FT-Raman,
near-IR,data
and mid/far-IR
ATR with in
the automated
beamsplitter
exchanger.
collection
both the
mid- and
far-IR regions. The analysis
time decreases from around 30 minutes to less than seven.
With the Nicolet iS50 spectrometer, the user is able to
load two sampling locations (the built-in ATR andFigure
the4.
Integrating Sphere module), start the macro and walk away,
while in the manual operation, continuous attention is
needed to swap the beamsplitters at the right moments.
This seemingly hidden improvement allows unattended
D
A permitting productivity through
operation,
automation.
Figure 2 shows just the mid- and far-IR spectra collected
from acetylferrocene analyzed using an OMNIC macrocontrolled workflow. The additional information from
C
the far-IR spectra is clear – the low end triplet verifies
that the iron is sandwiched between the cyclopentadiene
B
rings. The NIR data is not shown, but the entire process
required seven minutes, including collection of the
mid- and far-IR backgrounds. Automation also reduced
the total hands-on time of the user (pressing buttons,
Figure 4: Touch Points on the Nicolet iS50 spectrometer employ one-button switching between modules
and theloading
iS50 ABX automates
optics set-up
sample)
to ≈20 seconds.
Touch Point A – NIR module
Touch Point B – Raman module
Touch Point C – Built-in diamond ATR
Component D – ABX Automated Beamsplitter Exchanger
For operating one module at a time, the user need only
press the associated Touch Point. Seen more closely in
Figure 4, Touch Points make one-button operation
effortless when switching between modules (sampling
stations) and automating optics exchange. Rather than
thinking through the components needed (light source,
beamsplitter, optical path and detector) to run an
experiment, the user simply presses the Touch Point to
switch from an ATR to an NIR measurement and waits
until the instrument indicates that it is ready to begin.
This error-free operation is done in 30 seconds.
The
Nicolet
iS50 analytical
1 becomesThe far-IR optics permit collection to 1700 cm-1,
Figure
2: Mid-IR
and far-IR power
spectrainofFigure
Acetylferrocene.
clear
when
data collections
– mid-IR
far-IRapplications.
which
maythe
befour
sufficient
(fingerprint and
far-IR)and
for many
ATR, NIR, and Raman – are performed in one workflow.
Collecting spectra from each of these modules using a
conventional manual approach required about 50 minutes,
including sample loading, optical changes, time for
equilibration, and optimization of the Raman signal. The
analyst needed to be present throughout the experiment
to perform the beamsplitter changes and collect various
backgrounds for each sampling station. At the end of the
50 minutes, four spectra and their analyses were
available. Actual data collection took 5 minutes and
total hands-on time was 45 minutes, representing
inefficient use of the analyst’s time.
In contrast, the results shown in Figure 5 emerged from
a single OMNIC-macro operation. The macro was
programmed to begin by collecting backgrounds for the
mid- and far-IR ATR, and then switched to the iS50
Raman module. Next the samples were loaded on the
ATR, NIR, and Raman sampling stations. After optimizing
the signal using the autofocus feature of the Raman
module, the macro was initiated, and the analyst walked
FigureFrom
5: Multi-technique
for amacro
recyclableto
plastic
component using
spectrometer
away.
startingdatathe
completion
ofthethe
final pictured in Figure 3. Inset shows NIR independently for clarity.
report, the analysis took less than 12 minutes, representing
a time
savings of over 70%. The actual data collectionfor enhanced productivity. For the modern industrial lab,
Conclusion
time
wasforces
againcontribute
5 minutes,
however, total hands-on time
the Nicolet iS50 FT-IR spectrometer offers a powerful
Many
to new pressures on industrial
www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
foranalytical
the analyst
was only
2 minutes
– aloads,
highly
efficient new tool that goes beyond routine FT-IR to more
laboratories:
increased
sample
decreased
usestaffing,
of the retirement
analyst’s of
(and
the instrument’s)
time. The comprehensive analyses (e.g., FT-Raman and far-IR),
experts,
and shrinking budgets.
39 Immunoaffinity capture and enrichment
of protein and peptide targets is a widely
utilised method for sample cleanup prior to
mass spectrometric analysis. Termed the
Mass Spectrometric Immunoassay (MSIA) 1,
this hybridised combination of micro-scale
purification with highly sensitive detection
has repeatedly demonstrated the ability to
detect much lower abundance proteins from
a given proteome as compared to other
fractionation methods2,3.
Eric E. Niederkofler, Senior
Scientist, David A. Phillips,
Technician, Dobrin Nedelkov,
Manager of Research and
Development and Urban A.
Kiernan, Senior Scientist,
Thermo Fisher Scientific,
Tempe, Arizona. Bryan Krastins,
Senior Applications Scientist,
Scott Peterman, Senior
Applications Scientist, Alejandra
Garces, Senior Applications
Scientist and Mary F. Lopez,
BRIMS Center Director, BRIMS
Center, Thermo Fisher Scientific,
Cambridge, Massachusetts.
Figure 1. Protein A/G
MSIA-Tip Workflow: Protein
A/G MSIA-Tips are used by
repetitive pipetting of solutions,
allowing for efficient interaction
between capture and analyte
reagents within the porous
monolithic solid supports.
First, antibody is loaded onto
the Protein A/G surface. The
loaded antibody is then used to
immuno-capture and enrich its
targeted analyte from biological
samples. After rinsing, retained
analyte is eluted and subjected
to reduction, alkylation and
standard tryptic digestion and
LC-MS/MS protocols.
Figure 2. An 8-point IGF1
calibration curve was generated
from standard samples
consisting of 1, 5, 10, 25, 100,
500, 1000, and 1500 ng/mL
of IGF1 and 500 ng/mL of the
internal standard (LR3-IGF1).
MS area ratios of IGF1:LR3IGF1 are plotted against the
IGF1 concentrations and used to
calculate the concentrations of
IGF1 in unknown samples based
on the samples’ area ratios.
Replicate analyses of a single
plasma donor demonstrated
CV of 8.5%.
40 A Universal Mass Spectrometric
Immunoassay (MSIA™) Model system
based on human insulin-like growth factor 1 (IGF1)
Even though this approach to sample purification is now the
staple in proteomics methods; more robust, consistent, and
versatile methods for performing this sample purification and
enrichment step are a growing necessity. To further improve
upon this front-end sample processing, a new technology,
Thermo Scientific™ MSIA™, was tested. In the form of a
functional pipettor tip, their performance was evaluated (see
Application Notes: MSIA1002 and MSIA1003) and bench
marked against other technologies. However, to perform these
tests, a model system translatable to various ligand surfaces
including Protein A, Protein G and Protein A/G was needed.
Presented are the results of a developed model system for the
Protein A/G MSIA-Tips based on Insulin-Like Growth Factor
1(IGF1), a clinically relevant endocrine and oncology protein
biomarker 4,5,6.
Materials
•
•
•
•
•
•
Thermo Scientific Protein A/G MSIA-Tips
Thermo Scientific Versette Liquid Handling Platform
Anti-human IGF1 antibody
Human recombinant IGF1 (IGF1 standard)
Recombinant LR3-IGF1 (Internal reference standard)
Thermo Scientific Pierce BupH Phosphate Buffered Saline
(PBS)
• Antibody dilution buffer - 10mM MES/0.1% polysorbate
20, pH 5
• EDTA plasma, human donor
•Trypsin
• LC-MS grade water
• Thermo Scientific Optima grade Formic Acid (FA)
• Thermo Scientific Optima grade Acetonitrile (ACN)
• Standards dilution buffer - 10 g/L BSA in PBS pH 7.2
Bio-Innovation Issue 7 •
•
•
•
•
•
Sample dilution buffer - PBS/0.3% SDS
Elution buffer - 33% acetonitrile/0.4% trifluoroacetic acid
Reduction buffer - 10mM DTT in 30% isoproponal/0.1M
ammonium bicarbonate pH 8.0
Alkylation reagent - 0.5M Iodoacetamide/0.1M
ammonium bicarbonate pH 8.5
Thermo Scientific TSQ Vantage™ Triple Stage Quadrupole
Mass Spectrometer
Thermo Scientific Hypersil GOLD™ C18 column (50 mm x
2.1 mm, 1.9 μm particle size)
Methods
Samples: An 8-point IGF1 calibration curve was prepared
by serial dilutions of recombinant human IGF1 into standards
dilution buffer (concentration range 1-1500 ng/mL IGF1).
Replicate samples (n = 12) from a single EDTA plasma donor
and IGF1 calibration curve samples were prepared by diluting
40 μL plasma/IGF1 standards with 20 μL 0.5 mg/L internal
reference standard (in standards dilution buffer) followed by
100 μL PBS/0.3%SDS and incubated at room temperature
for 30 minutes prior to extraction and enrichment with
Protein A/G MSIA-Tips. Antibody Loading of Protein A/G
MSIA-Tips: Protein A/G MSIA-Tips were loaded with 100
μL rabbit anti-human IGF1 antibody (0.01 mg/mL) following
protocols provided in the user manual (total processing time
30 minutes). IGF1 Extraction and Enrichment: Co-extraction
and enrichment of IGF1 and LR3-IGF1 were performed using
a single Protein A/G MSIA-Tip (loaded with antibody) per each
sample following the protocols provided in the user manual
(total processing time 30 minutes). After extraction, IGF1 and
LR3-IGF1 were digested and analysed by SRM as described
below.
were performed using a single Protein A/G MSIA-Tip
(loaded with antibody) per each sample following the
protocols provided in the user manual (total processing
time 30 minutes). After extraction, IGF1 and LR3-IGF1
were digested and analyzed by SRM as described below.
Sample Elution and Trypsin Digestion
Captured IGF1 and LR3-IGF1 were co-eluted from the
Protein A/G MSIA-Tips by repetitively mixing (20 cycles
of aspirating and dispensing 30 μL volumes) 50 μL of
elution buffer within the wells of a 96-well plate using the
MSIA-Tips with captured IGF1 and LR3-IGF1, thus
eluting IGF1 and LR3-IGF1 into the 50 μL elution buffer
within each well. Samples were lyophilized to dryness and
then re-suspended in 30 μL of reduction buffer and
allowed to reduce for 30 minutes at 37°C. Reduced
samples were then alkylated by adding 2.4 μL alkylation
reagent and incubating in the dark at room temperature
for 30 minutes. Reduced and alkylated samples were
diluted with 92.5 μL of warm (50°C) 0.1M NH4HCO3/
5mM CaCl2 and then digested by adding 25 μL 4 mg/L
trypsin to each sample. Samples were allowed to digest for
2 hours at 50°C and then stopped by adding 5.3 μL of
acid solution (3 μL 100% formic acid and 2.3 μL 1mg/mL
glucagon). Injection volumes of 155 μL of the digests were
injected into the LC-MS for SRM.
3
Sample Elution and Trypsin Digestion: Captured IGF1 and
LR3-IGF1 were co-eluted from the Protein A/G MSIA-Tips by
repeatedly mixing (20 cycles of aspirating and dispensing 30
μL volumes) 50 μL of elution buffer within the wells of a 96-well
plate using the MSIA-Tips with captured IGF1 and LR3-IGF1,
thus eluting IGF1 and LR3-IGF1 into the 50 μL elution buffer
within each well. Samples were lyophilised to dryness and
then re-suspended in 30 μL of reduction buffer and allowed to
reduce for 30 minutes at 37°C. Reduced samples were then
alkylated by adding 2.4 μL alkylation reagent and incubating
in the dark at room temperature for 30 minutes. Reduced and
alkylated samples were diluted with 92.5 μL of warm (50°C)
0.1M NH4HCO3/5mM CaCl2 and then digested by adding
25 μL 4 mg/L trypsin to each sample. Samples were allowed
to digest for 2 hours at 50°C and then stopped by adding 5.3
μL of acid solution (3 μL 100% formic acid and 2.3 μL 1mg/
mL glucagon). Injection volumes of 155 μL of the digests were
injected into the LC-MS for SRM.
SRM Methods: SRM methods were developed on a Thermo
Scientific TSQ Vantage Triple Stage Quadrupole Mass
Spectrometer with a Thermo Scientific Accela™ pump, a CTC
PAL® auto-sampler, and a Thermo Scientific Ion Max source
equipped with a high-flow metal needle. A mass window of full
width at half maximum of 0.7 (unit resolution) was used in the
SRM assays because immuno-enriched samples have a very
high signal-to-noise ratio. Reversed-phase separations were
carried out on a Hypersil GOLD™ C18 column (50 mm x 2.1
mm, 1.9 μm particle size) with a flow rate of 240 μL/minute.
Solvent A was 0.2% formic acid, and solvent B was 0.2%
formic acid in acetonitrile.
Results and Discussion
A model system for the Protein A/G MSIA-Tips, based on
Insulin-Like Growth Factor 1(IGF1), was developed to serve
as a template for future LC-MS/MS methods that perform
quantitative immuno-affinity proteomics. The Protein A/G
MSIA-Tip workflow (Figure 1) proved to be simple and fast,
requiring as little as 1 hour to provide immuno-purified IGF1.
simple and fast, requiring as little as 1 hour to provide
immuno-purified IGF1. Using the Versette liquid handler
equipped with these tips, up to 96 samples were able to be
processed in parallel with minimal user interaction.
Figure 1:
STEP 1
STEP 2
STEP 3
STEP 4
Antibody
Incubation
Sample
Incubation
Washes
Elution
Protein A/G
Antigen
Binding
Antibody binding
Analyte
Rinse & Elute
Internal reference
Antibody Solution
Analytical Sample
1. Buffer
2. Water
Dispense Eluate
into microplate,
dry down
LC-MS/MS
Reconstitution
Reduction
Alkylation
Tryptic digestion (2h @ 50°C)
Figure
A/Gliquid
MSIA-Tip
Workflow:
Protein
MSIA-Tips
Using 1.
theProtein
Versette
handler
equipped
with A/G
these
tips,
are
used
by
repetitive
pipetting
of
solutions,
allowing
forwith
efficient
up to 96 samples were able to be processed in parallel
interaction between capture and analyte reagents within the
minimal user interaction.
porous monolithic solid supports. First, antibody is loaded onto
the Protein A/G surface. The loaded antibody is then used to
The resulting IGF1
MSIA-SRM,
using Protein
A/G
MSIA-Tips,
immuno-capture
and enrich
its targeted
analyte
fromA/G
biological
The resulting IGF1
MSIA-SRM,
using
Protein
demonstrated
a
wide
linear
dynamic
range
(Figure
and the
samples.
Afterdemonstrated
rinsing, retained
analyte
is eluted
and2)subjected
MSIA-Tips,
a wide
linear
dynamic
range
ability
to
reproducibly
quantify
unknown
IGF1
concentrations
to
reduction,
alkylation
and standard
tryptic digestion
(Figure
2) and
the ability
to reproducibly
quantifyand
from plasma
samples (Table A). Loaded with IGF1 antibody,
LC-MS/MS
protocols.
unknown IGF1 concentrations from plasma samples
the Protein A/G MSIA-Tips enabled for the efficient capture
(Table A). Loaded with IGF1 antibody, the Protein A/G
and enrichment of femtomole amounts IGF1 (Table A) with no
MSIA-Tips enabled for the efficient capture and
additional sample preparation or depletion.
enrichment of femtomole amounts IGF1 (Table A) with
no additional sample preparation or depletion.
Figure 2:
References
1. Nelson, R.W.; Krone, J.
spectrometric immunoa
1153– 1158.
2. Niederkofler, E.E.; Kier
Detection of Endogenou
Very Low Concentration
Cir. Heart fail. 2008, 4,
3. Lopez, M.F.; Rezai, T.;
Reaction Monitoring-M
Responsive to Parathyro
Clin. Chem. 2010, 56, 2
References
4.Nelson,
Monzavi,
R.;
1.
R.W.; Krone,
J.R.;Cohen, P.
disease.
Best
Pract. Res.
Bieber,
A.L.; Williams,
P. Mass
spectrometric
immunoassay
433-447.
Anal. Chem. 1995, 67,
5. Hankinson,
S. E.; Wille
1153–
1158.
Circulating concentratio
Figure 2. An 8-point IGF1 calibration curve was generated from
standard samples consisting of 1, 5, 10, 25, 100, 500, 1000, and
Reproducibility,
1500 ng/mL
and 500
ng/mL of the
(LR3LLOD of IGF1 Assay
Range
IGF1internal standard
% CV (n = 12)
IGF1). MS area ratios of IGF1:LR3-IGF1 are plotted against
the IGF1
- 1500 to
ng/mL
concentrations
calculate the concentrations of IGF1
1 ng/mL (5.2 and1used
- 7800
114 %
8.5 %
femtomole)*samples(5.2
in unknown
based on the samples’ area ratios. Replicate
femtomole)*
analyses of a single plasma donor demonstrated CV of 8.5%.
*Amounts based on a 40μL plasma sample volume.
Table A. Protein A/G MSIA-Tips IGF1-SRM assay characteristics.
LLOD
Assay Range
1 ng/mL
1 - 1500 ng/mL
Conclusion
IGF1
Recovery
Reproducibility,
%CV (n = 12)
% model system,
8.5 %
(5.2
- 7800
We described
the(5.2
development
of a114
novel
femtomole)*
femtomole)*
targeting human IGF1, to test the performance of Protein
A/G
MSIA-Tips.
Using
thisplasma
test system,
MSIA-Tips
*Amounts
based on
a 40μL
sample the
volume.
demonstrated
the
ability
to
reproducibly
perform
quantitative
Table A. Protein A/G MSIA-Tips IGF1-SRM assay characteristics.
measurements using SRM detection. These results clearly
show
how these devices can provided a mechanism for the
Conclusion
highly
specific and
reproducible enrichment
a target
analyte
We described
the development
of a novel of
model
system,
from
plasma.
Not
only
does
this
described
SRM
assay
provide
targeting human IGF1, to test the performance of Protein
a
uniform
approach
to
technology
evaluation,
it
also
serves
A/G MSIA-Tips. Using this test system, the MSIA-Tips as a
template
for thethe
development
of future LC-MS/MS
based MSIA
demonstrated
ability to reproducibly
perform
methods.
quantitative measurements using SRM detection. These
2. Niederkofler, E.E.; Kiernan,
and risk of breast cance
U.A.; O’Rear, J.; et al. Detection
of
Endogenous
6. Wolk, B-Type
A.; Mantzoros, C
Natriuretic Peptide at Very Low
Insulin-like growth fact
Concentrations in Patients With
population- based, caseHeart Failure. Cir. Heart fail.
1998, 90, 911-915.
2008, 4,258-264
3. Lopez, M.F.; Rezai, T.;
Sarracino, D.A.; et al. Selected
Reaction Monitoring-Mass
Spectrometric Immunoassay
Responsive to Parathyroid
Hormone and Related Variants.
Clin. Chem. 2010, 56, 281-290.
4. Monzavi, R.; Cohen, P. IGFs
and IGFBPs: role in health and
disease. Best Pract. Res. Clin.
Endocrinol. Metab. 2002, 16,
433-447.
5. Hankinson, S. E.; Willett,
W. C.; Colditz, G. A., et al.
Circulating concentrations of
insulin-like growth factor-I and
risk of breast cancer. Lancet.
1998, 351, 1393-1396.
6. Wolk, A.; Mantzoros, C.
S.; Andersson, S. O., et al.
Insulin-like growth factor 1
and prostate cancer risk: a
population- based, case-control
study. J. Natl. Cancer Inst. 1998,
90, 911-915.
results clearly show how these devices can provided a
www.thermofisher.co.nz/bio-innovation | www.thermofisher.com.au/bio-innovation
mechanism
for the highly specific and reproducible
enrichment of a target analyte from plasma. Not only does
41 Amplifcation
of whole human mitochondrial DNA
Here we compare the performance of five different polymerases when amplifying
whole human mitochondrial DNA. Thermo Scientific Phusion High-Fidelity
Polymerases were compared to three enzyme mixes recommended for
amplification of long templates by the manufacturers. Phusion High-Fidelity
DNA Polymerases performed the amplification in less time and with fewer
units of enzyme than the other polymerases. Reliable amplification of human
mitochondrial DNA can be accomplished more quickly and cost-effectively with
Phusion High-Fidelity DNA Polymerases.
42 Bio-Innovation Issue 7 Introduction
human
h Thermo Scientific
NA Polymerases
Materials and Methods
Phusion High-Fidelity DNA Polymerases consist of a
Pyrococcus-like enzyme with a double-stranded DNA-binding
domain, which gives the fusion polymerase high processivity
and fidelity. The performance of Phusion High-Fidelity DNA
Polymerases and three polymerase mixes from two other
vendors were compared by amplifying the whole human
mitochondrial DNA. Total DNA isolated from human blood
was used as a template. The amplified product was 16.5 kb.
M
1
2
3
4
5
M
s
a-
ses
Conclusions
The performance of five different polymerases was compared
when amplifying whole human mitochondrial DNA. Phusion
DNA Polymerases completed the entire PCR reaction in
less than 5 hours, while the other polymerases required
almost 8 hours. Compared to the three other polymerases
tested, which are recommended by the manufacturers
for amplification of long genomic targets, Phusion DNA
Polymerases provided high yields with lower enzyme amounts.
An important advantage of Phusion DNA Polymerases are
their extremely low error rate. It is 50-fold lower than that of Taq
polymerase. Two different buffers are provided with Phusion
DNA Polymerases: Phusion HF Buffer (error rate 4.4 x 10-7)
and Phusion GC Buffer (error rate 9.5 x 10-7). HF Buffer should
be used as the default buffer for high fidelity amplification.
However, GC Buffer can improve the performance on
some difficult or long templates. Due to their high accuracy,
Phusion DNA Polymerases can reliably be used for studying
mitochondrial DNA point mutations. In conclusion, Phusion
DNA Polymerases perform accurate and fast amplification of
mitochondrial DNA.
Te ch Note
Human mitochondrial DNA is a 16.6 kb circular doublestranded molecule. Mutations (deletions, duplications and
point mutations) in the mitochondrial genome leading to
mitochondrial dysfunction are increasingly recognised as a
contributor to a wide range of human diseases. Mitochondrial
dysfunction is involved in diseases such as diabetes, cancer,
heart diseases and migraine. In addition, neurodegenerative
disorders such as Parkinson’s disease and Alzheimer’s disease
are associated with mitochondrial dysfunction. Amplifying
the whole mitochondrial genome using PCR has been found
to be an efficient method for detecting mitochondrial DNA
deletions involved in human diseases1. Amplifying the whole
mitochondrial genome is useful for detecting relatively large
mitochondrial deletions; for other mutations (e.g. point
mutations), shorter target fragments are usually amplified.
The primers used for the amplification anneal to mtDNA at the
following positions: forward 10-40; reverse 16,494 –16,4631.
All reactions were conducted using conditions recommended
by the manufacturers. The amounts of enzymes in units and
total cycling times are shown in Figure 1. The reactions were
set up on ice and run on a thermal cycler.
The conditions for Phusion High-Fidelity DNA Polymerase
and Phusion Hot Start DNA Polymerase were as follows:
• 50 ng template • 0.5 μM primers
• 200 μM dNTPs • 1 x GC buffer
• 1 U enzyme Total volume 50 μL
The cycling conditions for Phusion High-Fidelity DNA
Polymerases were as follows:
Figure 1. Amplification of whole mitochondrial genome
Polymerase
Amount
Cycling time
using
Thermo Scientific Phusion DNA
Polymerases
and three
1. Thermo Scientific Phusion High- Fidelity DNA Polymerase
1U
4 h 48 min
polymerases
from
two
other
suppliers.
Phusion
DNA
2. Phusion® Hot Start High-Fidelity DNA Polymerase
1U
4 h 48 min
Polymerases
provided higher yield with
shorter
cycling times
3. Enzyme mixture from supplier R
2.6 U
7 h 40 min
with
less
enzyme
than
the
other
polymerases.
4. Enzyme mixture from supplier T
2.5 U
7 h 49 min
5. Enzyme mixture from supplier T, hot start version
Polymerase
1. Thermo Scientific Phusion HighFidelity DNA Polymerase
2.5 U
7 h 49 min
Amount
1U
Temperature
Time
98 ºC
30 s
1
98 ºC
10 s
30 cycles
72 ºC
8 min 15 s
"72 ºC 4 ºC"
"10 min hold"
Number of cycles
1
Acknowledgements
We thank Docent Anu
Wartiovaara, Research
programme of Neurosciences,
University of Helsinki, Finland for
the primer sequences.
Reference
Tengan C.H. and Moraes C.T.
(1996) Detection and analysis of
mitochondrial DNA deletions by
whole genome PCR. Biochem
Mol Med 58: 130-134.
Cycling time
4 h 48 min
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43 Iodoacetyl tandem mass tags
for cysteine peptide modification, enrichment & quantitation
Ryan D. Bomgarden - Thermo Fisher Scientific, Rockford, IL, USA, Rosa I. Viner - Thermo Fisher
Scientific, San Jose, CA, USA, Karsten Kuhn - Proteome Sciences, Frankfurt, Germany, Ian Pike Proteome Sciences, Frankfurt, Germany, John C. Rogers - Thermo Fisher Scientific, Rockford, IL, USA
Overview
Purpose:
To develop an iodoacetyl Tandem Mass Tag (iodoTMT)
reagent for irreversible cysteine peptide labeling, enrichment
and multiplexed quantitation.
Methods:
Reduced sulfhydryls of protein cysteines were labelled with iodoTMTzero and/or
iodoTMTsixplex reagents. Labelled peptides were enriched using an immobilised anti-TMT
antibody resin before mass spectrometry (MS) analysis.
Results:
We developed an iodoTMT reagent set to perform duplex isotopic or sixplex isobaric mass spectrometry (MS) quantitation of cysteine-containing
peptides. IodoTMT reagents showed efficient and specific labeling of peptide cysteine residues with reactivity similar to iodoacetamide. Using an anti-TMT
antibody, we characterised the immunoaffinity enrichment of peptides labelled with iodoTMT reagents from complex protein cell lysates and for detection
of S-nitrosylated cysteines.
Introduction
Thermo Scientific Tandem Mass Tag (TMT) Reagents enable concurrent identification and multiplexed
quantitation of proteins in different samples using tandem mass spectrometry.1 Previously, we described
a cysteine-reactive, isobaric Tandem Mass Tag (cysTMT™) reagent that utilised a dithiopyridine reactive
group to selectively label cysteine sulfhydryls. Although this labeling chemistry is highly specific and
efficient, it results in a reversible di-sulfide linkage between the peptide and isobaric tag. Here, we report
the development of an irreversible, cysteine-reactive TMT™ reagent containing an iodoacetyl reactive group
(iodoTMT™). Due to the irreversible labeling of the iodoTMT reagent, it can be used for quantifying cysteine
modifications such as S-nitrosylation, oxidation and disulfide bridges.
44 Bio-Innovation Issue 7 Methods
Sample Preparation
Preparation of iodoTMT-labelled proteins: Proteins and/or
cell lysates were solubilised at 2 mg/mL in 50 mM HEPES pH
8.0, 0.1% SDS and reduced with 5 mM TCEP for 1 hour at
50°C. Reduced proteins were labelled with 5-10 mM iodoTMT
reagent (~10 molar excess) for 1 hr at 37°C protected from
light. Excess iodoTMT reagent was removed by acetone
precipitation of samples at -20°C for 4-20 hrs.
Proteins were enzymatically digested at 37°C for 4 hrs and
desalted before liquid chromatography (LC)-MS/MS analysis or
enrichment.
Enrichment of iodoTMT-labelled peptides: Labelled peptides
(25–100 μg) were resuspended in TBS at 0.5 μg/μL and
incubated with an immobilised anti-TMT antibody resin (20–100
μL) overnight with end-over-end shaking at 4°C. After collection
of the unbound sample, the resin was washed 4X with 4 M Urea/
TBS, 4X with TBS, and 4X with water. Peptides were eluted 3X
with 50% acetonitrile/0.4% TFA, frozen, and then dried under
vacuum before LC-MS/MS analysis.
mM neocuporine, 1% SDS). Free sulfhydryls were blocked
with methyl methane thiosufate (MMTS) for 20 min at room
temperature and desalted to remove excess blocking reagent.
S-nitrosylated cysteine sulfhydryls were selectively labelled
using 0.4mMiodoTMT reagent in the presence of 20 mM
sodium ascorbate. Excess iodoTMT reagent was removed
by acetone precipitation of samples at -20°C for 4-20 hrs.
Unlabelled cysteines were reduced and alkylated with 20
mM iodoacetamide. Proteins were enzymatically digested at
37°C for 4 hrs and desalted before LC-MS/MS analysis or
enrichment.
LC-MS/MSAnalysis: A nanoflow high-pressure liquid
chromatography system with a Thermo Scientific PepMap C18
column (75 μm ID x 20 cm) was used to separate peptides using
a 5%-40% gradient (A: water, 0.1% formic acid; B: acetonitrile,
0.1% formic acid) at 300 nL/min over 60 min. A Thermo Scientific
LTQ Orbitrap XL ETD linear ion trap mass spectrometer was used
to detect peptides using a top-3 CID, -3 HCD experiment for
peptide identification and reporter ion quantitation.
Selective labeling of S-nitrosylated proteins: Proteins
and cell lysates were solubilised at 2 mg/mL in modified
HENS buffer (100 mM HEPES pH 8.0, 1 mM EDTA, 0.1
FIGURE 1. iodoTMT reagents
and labeling reaction.
(A) Mechanism of iodoTMT
reagent reaction with cysteine
containing proteins or peptides.
(B) Structure of iodoTMTsixplex
reagents for cysteine labeling,
enrichment, and isobaric MS
quantitation
Data Analysis: MS spectra were searched using Thermo Scientific Proteome Discoverer software v1.3
A
Figure 1.
B
Figure 2.
Figure 3.
A
B
FIGURE 2. Schematic of
iodoTMTsixplex reagent
workflow. Six different sample
conditions can be prepared
for iodoTMT reagent labeling.
Labelled proteins are combined
before iodoTMT peptide
enrichment using immobilised
anti-TMT antibody resin and
subsequent LC-MS/MS analysis
of isobaric reporter ions.
FIGURE 3. iodoTMT reagent
labeling specificity and
efficiency. A) Reduced BSA
(100 μg) was labelled with
increasing concentrations of
iodoTMT reagent. IodoTMT
reagent labeling efficiency
was determined by peptide
signal (XIC) of modified of
cysteines compared to total
cysteine-containing peptides
signal. B) Reduced BSA (100
μg) was labelled with 10 mM
iodoTMT reagent. IodoTMT
reagent labeling specificity
was determined by comparing
modified peptide signal to total
peptide signal for different
amino acids. Labeling specificity
and efficiency were also
assessed by peptide spectral
counting which gave similar
results (data not shown).
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45 FIGURE 4. iodoTMTsixplex
reagent relative quantitation of
BSA peptides. Reduced BSA
(100 μg) was labelled with 10
mM iodoTMTsixplex reagent
and combined in fixed ratios
(126:127:128:129:130:131
= 10:3:1:1:3:10 &
1:10:1:10:1:10) before protein
digestion. Graph of peptide
relative quantitation for all quant.
Figure 4.
Figure 5.
A
FIGURE 5. Anti-TMT enrichment
of iodoTMT-labelled peptides.
A) Percent of iodoTMT-labelled
peptide modifications identified
before and after enrichment. B)
Comparison of unique proteins
identified from unique peptides
from A549 cell lysates before
and after anti-TMT antibody resin
enrichment.
B
Figure 6.
A
B
FIGURE 5. cysTMT and iodoTMT
reagent labeling of S-nitrosylated
proteins. A549 cell lysates (A)
and BSA (B) were blocked with
MMTS (lanes 1-8, 10-15) or
untreated (lanes 9, 16) before
500 mM nitro-glutathione (NO)
treatment and TMT reagent
labeling in the presence or
absence of ascorbate and copper
sulphate (CuSO4). Proteins were
separated by SDS-PAGE and
analysed by anti-TMT antibody
Western blotting or Coomassie
stain.
References
1. Thompson, A., et al.
Tandem mass tags: a novel
quantification strategy for
comparative analysis of
complex protein mixtures by
MS/MS. Anal Chem. 2003, 75,
1895-204.
2. Forrester, M.T., et
al. Detection of protein
S-nitroylsation with the biotin
technique. Free Radic Biol Med.
2009, 46(2), 119-126.
3. Gygi, S.P., et al. Quantitative
analysis of complex protein
mixtures using isotope-coded
affinity tags. Nat Biotech. 1999,
17, 994-999.
4.Murray, C.I, et al. Identification
and quantification of
S-nitrosylation by cysteine
reactive tandem mass
tag switch assay. Mol Cell
Proteomics. 2012, 11(2):
M111.013441.]
Acknowledgments
The authors would like to thank
Dr. Jennifer Van Eyk and Dr.
Christopher Murray (Johns
Hopkins) for sharing preliminary
data on S-nitrosylation
switch assay protein labeling
conditions.
46 Results
We have developed and used an iodoacetyl TMT reagent
(iodoTMT) to irreversibly label sulfhydryls of cysteine-containing
peptides for multiplex quantitation by LC-MS (Figure 1).
Compared to the cysTMT reagent workflow, the iodoTMT
reagent workflow is simpler since reducing agents are
not removed from protein samples before labelling (Figure
2). The iodoTMT reagents showed efficient and specific
labeling of peptide cysteine residues with reactivity similar to
iodoacetamide (Figure 3A & 3B). IodoTMT reagents were also
used for sixplex isobaric quantitation of cysteine-containing
peptides (Figure 4). We also characterised an anti-TMT
antibody developed against the reporter region of the TMT
reagent for immuno-enrichment of iodoTMT-labelled peptides
(Figure 5) and Western blot detection of iodoTMT-labelled
proteins (Figure 6A).
We used the iodoTMT reagent as a probe for labeling
S-nitrosylated cysteines in a modified S-nitro switch
assay (Figure 6). IodoTMT reagents successfully labelled
S-nitrosylated cysteines after selective reduction using
ascorbate; however, labelling efficiency was less than cysTMT
reagents. This result is consistent with different efficiencies of
the sulfhyrdyl-reactive groups (dithiopryridine vs. iodoacetyl) of
each reagent. In addition, we discovered that addition of 1mM
copper sulfate to the switch reaction buffer inhibited iodoTMT
reagent labeling but not cysTMT reagent labeling.
Bio-Innovation Issue 7 Addition of copper sulfate is thought to facilitate S-NO bond
reduction during the labeling reaction; however, in the presence
of ascorbate, Cu2+ is readily reduced to Cu1+, which can
generate free radicals.2 The free radicals generated resulted
in protein degradation (Figure 6B: lanes 5, 8, 12 and 15) and
possible loss of iodine from the iodoacetyl reactive group.
Overall, using the combination of iodoTMT labeling with
anti-TMT enrichment has several advantages over previously
described cysteine-reactive workflows3,4 for labeling, enrichment
and quantitation of cysteine-containing peptides and cysteine
modifications such as S-nitrosylation
Conclusions
• Iodoacetyl Tandem Mass Tags (iodoTMT) are novel reagents
for specific irreversible labeling of cysteine residues pre- or
post-digestion.
• iodoTMT reagents can be used as either isotopic pairs or
as an isobaric set for MS- or MS/MS-based multiplexed
quantitation.
• An antibody to the TMT reagent reporter region allows
specific detection, capture, and enrichment of iodoTMTlabelled proteins and peptides.
• iodoTMT reagents can be used for detection of cysteine
modifications such as S-nitrosylation.
Ajax horiz-half-page_01-RL_1360187487.indd 1
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