Issue 4 Summer 2002 - Applied Biosystems

Biosystems Solutions
E d i t o r i a l
Applied Biosystems, Lingley House, 120 Birchwood Boulevard, Warrington, Cheshire WA3 7QH, UK.
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E d i t o r
Kay L Hill
C o n t r i b u t o r s - A p p l i e d B i o s y s t e m s
Peter Boogaard, Tony Hardware, Martin Heinrich, Paul Johnson, Karsten Lueno, Barbara Maniglia, Wolfgang Mayser, Sue Ann Molero, Tristan Moore,
Sabine Moter, Michael O'Neill, Pierre Paroutaud, Victoria Parr, Steve Picton, Thomas Schild, Henri Snijders, Nico Stom and Dave Watts
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T r a d e m a r k s
Applera Corporation is committed to providing the world's leading technology and information for life scientists.
Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses.
AB (Design), Applera, PrepMan, BioAnalyst, GeneMapper, Biospectrometry, Assays-on-Demand, Voyager-DE, Q TRAP, VISION, oMALDI, SeqScape, API 3000, API 150EX,
TurboIonSpray, Oracle, Factura, GenBase, Interrogator, POP-4, POP-5, POP-6, POP-37 and SQL*LIMS are trademarks and Applied Biosystems, ABI PRISM, BigDye,
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AmpliTaq, AmpliTaq Gold, GeneAmp and TaqMan are registered trademarks of Roche Molecular System, Inc.
ICAT is a trademark of the University of Washington, exclusively licensed to Applied Biosystems Group of Applera Corporation.
BlastMachine is a registered trademark of Paracel Inc.
The ABI PRISM 3100 Genetic Analyzer and the ABI PRISM 3100-Avant Genetic Analyzer include patented technology licensed from Hitachi, Ltd.
as part of a strategic partnership between Applied Biosystems and Hitachi, Ltd., as well as patented technology of Applied Biosystems.
The Applied Biosystems 3730 and 3730xl DNA Analyzers include patented technology licensed from Hitachi, Ltd. as part of a strategic partnership
between Applied Biosystems and Hitachi, Ltd., as well as patented technology of Applied Biosystems.
The PCR process and the 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.
Applied Biosystems/MDS SCIEX is a joint venture between Applera Corporation and MDS Inc., the instrumentation technology division of MDS Inc.
MDS and SCIEX are trademarks of MDS Inc.
The InteraX system is protected under US Patent No. 6,342,345
Certain aspects of the technology described herein are covered under current or pending US and/or international patents.
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©2002 Applied Biosystems. All rights reserved. Printed in the UK 06/02
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Biosystems
Solutions
European Edition, Issue 4 - Summer 2002
Q TRAP LC/MS/MS System
More metabolites, more proteins,
more confidence in your results
Science that changes lives

proteomics
The Discovery Moment
> If you work in proteomics,
you know complexity firsthand.
Your next proteomics discovery awaits.
Applied Biosystems, the company that enabled the sequencing of the human
genome, is pioneering new proteomics workflows with next-generation systems.
Now you can look deeper into the proteome and perform protein ID, quantitation,
and characterisation at new levels of productivity. With technology breakthroughs
like the Applied Biosystems 4700 Proteomics Analyzer and ICAT reagents,
we deliver the innovations to get you to your next discovery moment.
Visit us at: europe.appliedbiosystems.com
You’ve just got to find the
order in the chaos.
Maybe you do it by unlocking
an insight about your research
when you least expect it.
So your work starts to
take on a new direction.
Toward a discovery.
A new drug target or pathway.
Even a cure.
contents
technical communications
07-17
The BlastMachine System
The InteraX System
Choosing the Right Target!
Revolution in 5' Nuclease Assay
15 Years in a Thin Glass Tube
Analysis of Gene Expression Pattern in Asthma Research
new product review
18-35
Q TRAP LC/MS/MS System for Protein Analysis
SQL*LIMS 4.1 Software
PrepExpress NT Software
Affinity Depletion Cartridges
Moving Forward in Genomic Research...
Improved Software Tools for Data Analysis
PhotoSpray Source
TaqMan ® Assays for Food Testing
promotions
36-39
Free Subscription to BioBeat!
Start off your Gene Expression Studies!
customer relations
40-48
SQL*LIMS Installed to Streamline Product Release
Global Oligonucleotide Operations
Proteomics Research Center Embraces RIS Implementation
German Society Grants ‘Life Science Award’
Collaborative Study on High-throughput Genotyping
Genotyping in the Genomic Era
Training Dates
08
A Sequence to Die For!
Fugu Genome Draft
Announced
18
Pro ICAT Software
Automatic Interpretation
of Proteomic Data
24
3100-Avant System
Introducing a New
Genetic Analyzer
cover story - 05
Q TRAP LC/MS/MS System
More metabolites, more proteins,
more confidence in your results
38
Europe Online
Visit the New
European Website
3

4
O
Corporate review
Introducing the Next Generation of Production
Level DNA Analysis Systems
n April 23, 2002 Applied Biosystems announced
the introduction of the Applied Biosystems 3730
and 3730xl DNA Analyzers. These next generation
production scale systems are expected to improve
data quality and increase productivity by a factor
of two or more compared to current technology
platforms. They bring powerful new tools for rapid,
accurate, and cost-effective DNA analysis to
discovery researchers worldwide studying human
and other genomes.
The addition of the 3730 and 3730xl DNA Analyzers
to the Applied Biosystems existing portfolio of
industry-leading DNA analyzers provides researchers
with a wider range of platforms to address their
throughput and application needs. The 3730
analyzer, a 48-capillary electrophoresis system,
combines the core technology of the highly successful
ABI PRISM ® 3100 Genetic Analyzer with new advances
in automation and optics, to meet the needs of higher
throughput individual researchers and core facilities.
The 3730 platform can also be upgraded to the
3730xl DNA Analyzer, the 96-capillary successor to
the ABI PRISM ® 3700 DNA Analyzer.
While researchers and production facilities are
expected to primarily use the 3730xl DNA Analyzer
for production sequencing – both de novo and
resequencing – the 3730 DNA Analyzer is expected
to be used for a wider range of sequencing and
fragment analysis applications including de novo and
comparative sequencing and genotyping. The new
systems run both ABI PRISM ® BigDye ® Terminator
chemistries for sequencing and five-dye chemistries
for fragment analysis, as do all Applied Biosystems
DNA analyzers.
Throughput, Automation, Efficiency Improvements
The 3730 and 3730xl DNA Analyzers can
reduce sequencing project costs and increase
efficiency by providing longer read lengths,
enhanced instrument sensitivity and efficiency
and the highest 24-hour unattended capacity of any
DNA analyzer. Sequence read lengths of greater
than 1,000 base pairs and improved basecalling
could reduce the number of samples needed
to complete de novo genome sequencing and
resequencing projects by 20-40% respectively.
Applied Biosystems: Pioneers in Genetic Analysis Systems
Both new systems are part of the pioneering line of
capillary electrophoresis instruments for DNA
analysis from Applied Biosystems. The ABI PRISM ®
310 Genetic Analyzer, the first commercially
available capillary sequencer, was introduced in
1995. That was followed in 1998 by the introduction
of the 96-capillary ABI PRISM ® 3700 DNA Analyzer.
Government and commercial researchers in the
United States, Europe and Asia used the 3700
system to sequence the human genome.
Researchers worldwide currently use the 3700
system for the industrial-scale analysis of genomic
information. The 16-capillary ABI PRISM ® 3100
Genetic Analyzer, introduced in 2000 was designed
to provide the flexibility of the 310 system with
added throughput for medium-to-high throughput
laboratories. The ABI PRISM ® 3100-Avant Genetic
Analyzer, introduced in April 2002, is a four-capillary
electrophoresis system that can be upgraded to the
16-capillary 3100 system (see page 14).
Applera Announces New Knowledge Business for
Celera Discovery System (CDS)
Applera Corporation announced earlier this year that
its Applied Biosystems Group will become the
exclusive distributor of the Celera Discovery System
(CDS) operated by its Celera Genomics Group and
that Applied Biosystems will integrate CDS and other
genomic and biological information into a new
Knowledge Business.
For the future, Applied Biosystems has plans for
its Knowledge Business to include genomic assays
and related content (see page 29), as well as other
information-rich products, services, and analytical
tools to meet the needs of its life sciences
customers. The Knowledge Business products and
services will be marketed, in part, on an expanded
information portal.
For more information on
3730/3730xl DNA Analyzers enter: No. 401
Q TRAP LC/MS/MS System
More Metabolites, More Proteins, More Confidence in Your Results
Prof. Gérard Hopfgartner, University of Geneva, Switzerland
O
ne of the first steps in the investigation of the
biotransformation of a new potential drug is the
characterisation of its metabolites in in vitro systems.
To cope with new demands of the pharmaceutical industry,
such as reducing the discovery and development time of new
drugs, powerful tools are needed to complete the various tasks.
This is particularly true for the bioanalytical support for
drug metabolism and pharmacokinetics studies. Many in vitro
samples can be generated using hepatocytes and microsomes
from different species and liquid chromatography combined
with mass spectrometry (LC/MS) already plays a very important
role in this field. Once these metabolites have been
characterised in vitro it is important to monitor their presence
in vivo and to follow their pharmacokinetic profile. The goal is
to obtain as much information as possible regarding the
structure of the metabolites present in plasma and their
quantity in a very short period of time. Often in vivo samples
contain very low concentrations of the drug and its metabolites
and only limited sample volume is available. Sensitivity in the
cover story
low nanogram range is required and the challenge can be
compared to the search for ‘needles in a haystack’
Currently there is no unique mass spectrometer which has
all the desired features required for this type of work and
most laboratories use the combination of various types of
mass spectrometers (MS) including triple quadrupole, ion trap,
quadrupole time-of-flight (QqTOF) MS with relative long
analysis times. Good chromatographic separation remains an
important element. Selective scan modes such as neutral
loss scan and precursor scan experiments which can be
performed on triple quadrupole instruments, are very important
to determine the biotransformation products in complex
matrices. Phase I metabolites such as oxidative products or
phase II metabolites (glucuronides or sulphates) can be rapidly
identified using this technology.
On triple quadrupole instruments the sensitivity in product ion
scan mode is often not sufficient to obtain good spectra quality.
QqTOF technology can overcome this lack of sensitivity with
additional accurate mass information.
page 6
5

6
cover story
The limitation is that true precursor and neutral loss scan
experiments and reliable quantitative analysis are not possible
on QqTOF mass spectrometry. The same applies for the ion
trap technology. Ion trap MS however allows sensitive multiple
MS (MSn ) experiments to clarify the fragmentation process
which simplifies the spectral interpretation.
Ideally one would like to perform all different experiments in
one single LC/MS analysis. This can be done with the use
of powerful software tools such as information dependent
acquisition (IDA). The gain in time is considerable, but suffers
from the slow scan speed of typical triple-quadrupole MS and
moderate sensitivity when performing LC/MS analysis.
The new Q TRAP system from Applied Biosystems is an
LC/MS/MS linear ion trap mass spectrometer. This instrument
is based on a triple-quadrupole ion path and is capable of all
of the conventional tandem quadrupole scan modes, as well as
several high sensitivity ion trap mass spectrometer scans, using
the final quadrupole as a linear ion trap mass spectrometer.
The linear ion trap can be operated either in the classical triple
quadrupole mode or in the linear ion trap mode with several
powerful scan (single MS, MS/MS and MS3 ) operating modes.
In the triple quadrupole mode it shows particular strength for
accurate and precise quantation in the multiple reaction
monitoring scan mode. For maximum performance the LINAC
collision cell technology is part of the Q TRAP system as well.
The different scan modes can be combined in the same LC/MS
run without compromising the chromatographic performance.
It also allows the generation of either quadrupole- or ion-trap
like collisionally activated dissociation spectra, including MS3 spectra. These capabilities are demonstrated in figure 1
where in one single LC/MS analysis of a human urine sample,
quadrupole like product ion spectra and MS3 spectra with
Figure 1.
sufficient data points over the peaks are obtained. Only 5µl
of urine was directly injected onto the HPLC column.
The following IDA experiment was used; i) Enhanced single
MS (EMS) for the selection of precursor using also an inclusion
list ii) Enhanced Product Ion (EPI) iii) MS3 .
Figure 1A shows the TIC of the EPI trace. The EPI spectrum
of the glucuronide of the parent (precursor at m/z 613)
is depicted in figure 1B. Whilst figure 1C shows the MS3 spectrum of the selected fragment at m/z 404. In drug
metabolism one can theoretically calculate the masses of
potential metabolites and then set up a corresponding specific
MS/MS experiment to search for those metabolites in very
complex matrices.
The very fast duty cycle of the Q TRAP system opens new
doors for metabolite analysis. Figure 2 shows the analysis
of Tolcapone in urine using 8 EPI experiments. Using this
approach almost all major metabolites can be identified in a
single LC/MS/MS run. The EPI spectra of tolcapone and it’s
acid metabolite are depicted in figures 2B and 2C.
Figure 2.
The Q TRAP system is complementary to the API QSTAR ®
Pulsar system. For very challenging compounds accurate mass
measurement remains very important for reliable structural
elucidation, based only on mass spectrometric techniques.
The combination of both instruments on a similar software and
interface platform make it particularly attractive and powerful
for drug discovery and early drug characterisation.
See also page 20 for related article
For more information on:
Q TRAP LC/MS/MS System enter: No. 402
technical communications
The BlastMachine System
Optimised BLAST System for Sequence Similarity Analysis
P
aracel’s BlastMachine system is a turnkey software
and hardware solution running a Paracel-optimised
version of the NCBI BLAST algorithm on a pre-packaged
Linux computer farm, for large-scale sequence
similarity analysis.
In the past, BLAST users who needed immediate deployment,
scalability and support have had to rely on costly multiprocessor
UNIX systems. Those with less immediate need or
tighter budgets could take the time to develop their own Linux
farms. Paracel's BlastMachine system represents significant
investments of time, resources and expertise resulting in a
total, immediately available, cost-effective BLAST solution.
Paracel has rewritten portions of the NCBI BLAST algorithm to
improve speed and to accommodate longer query sequence
lengths and larger databases. BlastMachine software,
integrated with a Linux farm by Paracel, scales gracefully
through efficient distribution of data and computing across
multiple processors, all without compromising the quality of
search results.
Included with the purchase of a BlastMachine system is the
Paracel Filtering Package (PFP), a full-featured cleaning,
filtering and masking utility for DNA and protein sequence
data. BlastMachine customers can run PFP on their existing
computer systems to remove repeats and contaminants from
their internal data prior to a BLAST search to achieve improved
results and search efficiency.
With the launch of our BlastMachine system, joining our
GeneMatcher technology for accelerated dynamic sequencesearch
algorithms, Paracel offers high-throughput commercial
implementations of all major comparison algorithms for
genomic analysis and annotation. Paracel also offers software
products for large-scale sequence clustering and assembly.
Optional Professional Services may be offered for your
Biologists, System Administrators and Software Engineers to
help you optimise the performance and accuracy of your
bioinformatics systems and drug discovery pipeline.
For more information on:
BlastMachine system enter:
No. 403
Paracel products enter: No. 404
7

8
technical communications
A Sequence to Die For!
Fugu Genome Draft Announced
by Michael D. O'Neill, BioBeat ® Online Magazine (www.biobeat.com)
O
n October 26, 2001, at the Genome Sequencing and
Analysis Conference (GSAC) in San Diego, California,
an international team of investigators, including
legendary molecular biologist Dr. Sydney Brenner,
announced completion of a draft sequence of the Fugu
rubripes genome.*
Dr. Sydney Brenner at the Gordon Conference on Human Molecular
Genetics in Newport, Rhode Island, August 1999.
(Photo by Michael D. O'Neill, BioBeat Online Magazine).
Fugu, a pufferfish, is a delicacy in Japan, but it can be a risky
meal, as it contains a deadly neurotoxin, and if not properly
prepared, can prove fatal to the diner. The Fugu genome is
particularly interesting to scientists because it contains very little
of the so-called ‘junk’ DNA that lies between gene segments in
other vertebrate genomes, particularly the human genome.
The Fugu genome is made up almost exclusively of
coding sequence, i.e., DNA that codes for proteins, and of
controlling regions that influence the expression of genes.
Consequently, study of the Fugu genome offers investigators a
potentially more direct route to the identification of genes and
the analysis of gene function, by allowing them to avoid the
complications of sifting through all the non-coding sequence
that is generally present in the genomes of higher organisms.
The Fugu genome is the smallest known vertebrate genome
(350-400 million DNA base pairs), but it is believed to contain
approximately the same number of genes as the much larger
human genome (3,000 million DNA base pairs) i.e., the Fugu
genome is more than eight times as gene-dense as the human
genome. In addition, the Fugu and the human genomes are
thought to share very similar sets of genes.
The porcupine fish (Cyclichthys orbicularis) belongs to a family (Diodontidae)
that is closely related to the pufferfish family (Tetraodontidae).
(Images courtesy of Jeff Jeffords, www.divegallery.com)
For these reasons, it is believed that sequence comparisons
between the Fugu genome and the human genome may
accelerate the identification of human genes, because genes
that can be relatively quickly identified in the gene-dense Fugu
genome can be used to find the corresponding genes in the much
less gene-dense human genome. Looking for a gene in the human
genome can be likened to looking for a needle in a haystack.
Looking for the corresponding gene in the Fugu genome may
allow the researchers to immediately eliminate a lot of the hay,
and thus simplify and accelerate the gene search.
A portion of the Fugu genome sequencing project was carried out
on ABI PRISM ® 3700 DNA Analyzers at Celera Genomics.
In addition, sequencing of cosmids from Fugu genomic libraries
was carried out at the Institute of Molecular and Cell Biology
(IMCB) in Singapore, which presently has two ABI PRISM 3700
DNA Analyzers and two ABI PRISM 377 DNA Sequencers.
Dr. Alice Tay, leader of the DNA Sequencing & Analysis Facility at the
Institute of Molecular and Cell Biology (IMCB) in Singapore.
(Image courtesy of Dr. Tay).
technical communications
IMCB Sequencing Leader Lauds Performance
of Applied Biosystems Sequencers
Dr. Alice Tay, leader of the DNA Sequencing & Analysis
Facility at the IMCB, said, “We are very pleased with the
performance of the DNA Sequencers from Applied Biosystems,
especially the quality of the data from the 3700 system.
Generally, the sequences are so clean that we are able to obtain
at least 500 bases for each read with almost no manual editing.”
Dr. Tay said the IMCB is presently focused on the finishing
phase of the Fugu genome sequencing project.
Additional information on the instruments used in this
sequencing project, as well as on other products from
Applied Biosystems, can be obtained in the Documents on
Demand section (http://docs.appliedbiosystems.com/search.taf)
of the Applied Biosystems web site: www.appliedbiosystems.com
Collaborating Institutions
The bulk of the Fugu genome sequencing project was carried
out at the United States Department of Energy’s Joint Genome
Institute (JGI). In addition to the JGI, Celera Genomics,
and the IMCB, other institutions collaborating on the Fugu
genome sequencing project included the Singapore Biomedical
Research Council, the Medical Research Council (UK),
the Cambridge University Department of Oncology, the Institute
for Systems Biology, and Myriad Genetics.
Annotated Version of Fugu Article Available in
Applied Biosystems’ Online Magazine
A web version of this article, with numerous links to
related information, is available in BioBeat Online Magazine
from Applied Biosystems. This online magazine can be accessed
at www.biobeat.com. BioBeat Online Magazine covers life
science research advances made around the globe and
presently includes over 120 richly annotated articles.
Free subscriptions to BioBeat Online Magazine can be obtained
by completing the brief online subscription form at
www.appliedbiosystems.com/biobeat/subscribe.cfm. In addition
to email updates of new story postings, BioBeat subscribers
receive periodic emailings of BioBeat’s popular Journal Watch
and Conference Calendar features.
*The original genus name for Fugu was Takifugu. The shorter name is now commonly used.
For more information on:
ABI PRISM family of DNA Analyzers enter: No. 405
9

10
technical communications
The InteraX System
Monitoring Protein-Protein Interactions
in Different Compartments of the Mammalian Cell
A
better understanding of the specific interactions of
proteins with each other, under physiologically
relevant conditions, is extremely helpful in elucidating
new drug targets and the discovery of new cell
signalling pathways. This in turn may reveal new action
points for new drugs correcting molecular disfunctions.
The InteraX system allows the direct detection of in vivo proteinprotein
interactions in a wide range of cell types and species.
This technology employs the intracistronic complementation
or alpha-complementation of two mutant forms (∆α and ∆ω)
of ß-galactosidase. These two specific mutant forms have
low affinity for each other and do not form an active enzyme
complex. They can be expressed as fusions with the
target proteins under investigation. Upon interaction of the
two target proteins with each other, the two mutant forms of
ß-galactosidase restore an active ß-galactosidase complex.
Consequently, the ß-galactosidase activity measured after
ß-galactosidase complementation is a direct function of the
interaction of the two proteins under investigation (See figure 1).
Figure 1.
Probing Protein-Protein Interaction ß-Galactosidase
Mutant Complementation
A and B
do not interact
A
A
add substrate
No
Light
A and B
do interact
B A B
B
A
B
add substrate
The InteraX system is capable of detecting protein-protein
interactions in different cell lines of different organisms.
The system can be applied to mammalian cell lines
with intact regulatory machinery, which not only allows a
functional readout for orphan and known G-Protein coupled
receptors (GPCRs), but extend to a more general use of the
technology as a tool to map signalling pathways.
Blau et al1 used the above principle of intracistronic
complementation underlying the InteraX system to monitor
EGF receptor dimerisation in live cells. Their experimental
set-up is illustrated in figure 2. Blau et al expressed
chimeric EGF - ß-gal receptors in cell culture. Treatment with
EGF and EGF-like compounds resulted in ligand specific,
reversible dose responses. The dimerisation of the chimeric
EGF-receptor in the cell membrane was inhibited by
antibodies blocking the ligand binding. In addition the
kinetics of EGF receptor dimerisation was investigated.
Low levels of receptor chimeras can be detected avoiding
over-expression of protein.
Figure 2.
Functional Receptor Binding Assay:
Monitoring EGFR Dimerization
FRAP
rapamycin
rapamycin rapa apa yc
FRAP FKBP12
FKBP12
Cytosolic
interaction
Signal / Noise
40
30
20
10
0
Parental
∆ω alone
∆α/∆ω
clone A
∆α/∆ω
clone B
Media
For the cytosolic protein-protein interaction of ∆αFRAP
and ∆ωFKBP12 (See figure 3) high S/N ratios have been
achieved. This documents not only the quantitative nature
of the technology, but also the ability to use it to develop
functional readouts for protein-protein interactions in
different cell compartments.
Figure 3.
Protein Induced Interaction Measured Using Gal-Screen ® Assay.
ß-Gal complementation is demonstrated upon induction with
rapamycin (10 ng/mL) in two clones of transfected cells
(10,000 cells/well) co-expressing the fusion proteins
∆αFRAP and ∆ωFKBP12.
The InteraX system can be applied as well to develop assays
amenable for high-throughput screening. Rees et al2 developed
a homogeneous 384-well assay protocol based on the
Your feedback on Biosystems Solutions
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ecently, with Biosystems Solutions, we included
some market research questions on the content of
the magazine and how you like to be kept informed
with up-to-date information from Applied Biosystems.
Encouragingly most of you found the articles we chose to
include, informative, well balanced and of general interest.
We would welcome your proposals and suggestions for
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If you want to comment on the content of previous issues of
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technical communications
ß-galactosidase complementation technology and studied
pharmacological characteristics of a chimeric EGF-receptor
towards EGF antagonists. The DMSO tolerance of this assay
was observed as up to 2%. In a 1280 compound screen,
retest hit rates of 0.4% were observed, which is far better than
that achieved with reporter gene assays, wherein hit rates of
greater than 5% were obtained. Also, an average mean Z-factor
of 0.55 was obtained throughout the screen. Therefore, one of
the benefits of the technology was determined to be the low
false-positive hit rate compared to functional antagonist assays
and the fact that the readout is detection platform independent.
In summary, the InteraX system employing ß-galactosidase
complementation represents a functional readout for
protein-protein interactions in a variety of different cells and in
different compartments of a cell under physiological conditions.
It is a promising new system for assay development and
high-throughput screening with platform independent detection.
References
1. Bruce T. Blakely, Fabio M.V. Rossi, Bonnie Tillotson, Michelle Palmer, Angeles Estelles,
and Helen Blau; Epidermal Growth Factor Receptor Dimerization monitored in live cells;
Nature Biotechnology, Volume 18, Number 2, p.218-222, February 2000
2. Debbie L.Graham, Nicola Bevan, Peter N. Lowe, Michelle Palmer, and Stephen Rees;
Application of ß-Galactosidase Enzyme Complementation Technology as a High-
Throughput Screening Format for Antagonists of the Epidermal Growth Factor Receptor;
Journal of Biomolecular Screening, Volume 6, Number 6, p.401- 411, 2001
For more information on:
InteraX system datasheet and reprints enter: No. 406
included in future issues then please contact us by emailing
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12
technical communications
Choosing the Right Target!
T
he first challenge for today’s pharmaceutical
companies is not so much hitting the target,
but choosing the right target in the first place.
Over the last hundred years drugs have been designed
against 400-500 disease targets.
The post genome era presents the industry an interesting
dilemma – with the completion of the human genome the
number of ‘druggable’ targets is expected to increase
dramatically with estimated numbers between 3,000 to 10,000.
So how can a company choose which targets to aim at!
Technologies and platforms from Applied Biosystems are being
used by pharmaceutical companies across the globe to more
fully understand the molecular cause of disease. By studying
disease mechanisms researchers are able to identify important
genes and proteins, understanding how they influence and
control biological processes.
At Applied Biosystems we transformed gene discovery research
with Automated DNA Analyzers that decipher entire genomes in
months instead of years. These systems of choice for identifying
disease-related mutations and correlating genetic markers
with disease, are also the primary technology for revealing genes
with altered expression levels in disease. Our Gene Expression
Analysis Systems help to assign function to potential target
genes and also provide novel assays for lead discovery and
biomarkers for clinical trials.
Proteomics offers distinct opportunities for target discovery
and validation, novel assays for lead discovery, and research
to discover biomarkers for clinical trials. Applied Biosystems
is advancing the science of proteomics with Automated
Protein Sequencers, Time-of-Flight (TOF) Mass Spectrometers,
and differential protein expression analysis using ICAT reagents
and software. Applied Biosystems Proteomics Research Center
and Applied Biosystems/MDS SCIEX, work with key leaders in
the field to speed the development of emerging technologies
and novel R & D applications.
Even with the high quality data generated with these
technologies it is still necessary to make sense of this
information before deciding which targets to take forward into
the drug development process. Applied Biosystems has
developed and refined informatics systems that allow the
automation and integration of genomic and proteomic systems
allowing researchers to make informed choices on the targets
for tomorrow’s drugs.
See articles on pages 20, 29 & 40
For more information on:
Systems for DNA Analysis and Gene Expression enter:
Solutions for Proteomics and LC/MS enter:
Informatics Solutions enter:
No. 407
No. 408
No. 409
technical communications
Revolution in 5' Nuclease Assay
TaqMan ® MGB Probes Deliver Simple and Robust SNP Genotyping!
D
etection of single nucleotide polymorphisms (SNPs)
is now central to modern molecular genetics.
Large-scale population scoring of known SNPs requires
a technology with minimal steps and an ability
to automate the assay process. Applied Biosystems vision:
to create a single-step SNP assay making it easy to
unambiguously assign SNP genotypes in a flexible and
scaleable format. Here’s how we’ve delivered on that vision…
Figure 1. SNP scoring (also called allelic discrimination)
assay using 5' nuclease chemistry and TaqMan MGB probes.
Tm=Tm of perfectly matched probe -Tm of mismatched probe.
Discrimination of the two SNP alleles is achieved by using
an annealing/extension temperature within Tm window.
A substantial increase in VIC ® fluorescence only indicates
homozygosity for Allele 1, while a substantial increase in
FAM ® fluorescence only indicates homozgosity for Allele 2.
Both fluorescent signals increase substantially when sample
is heterozygous.
NFQ = Non fluorescent quencher
Three factors contribute to allelic discrimination based
on a single mismatch (Figure 1).
1 A mismatched probe has a lower Tm than a perfectly
matched probe. Shorter probes display greater mismatch
discrimination because the single mismatch has a higher
disruptive effect on the hybridisation kinetics of the
shorter probe.
2 The assay is performed with both probes present in the
reaction tube. The mismatched probes are virtually
prevented from binding to the target due to the stable
binding of the perfectly matched probes.
3 For efficient probe cleavage, the 5' end of the probe must
start to be displaced. Once a probe starts to be displaced,
complete dissociation occurs faster with a mismatch than
with an exact match. Thus, the presence of a mismatch
promotes dissociation rather than cleavage of the probe.
The minor groove binder (MGB) contributes a major
enhancement to the 5' Nuclease Assay. The addition of an
MGB molecule to an oligonucleotide has been shown to
stabilise nucleic acid duplexes, causing a dramatic increase
in the T m of the oligo. Employing the MGB attachment in
a TaqMan probe facilitates the use of shorter probes,
thus resulting in improved mismatch discrimination for
SNP assays and increased design flexibility for both allelic
discrimination assays and gene expression assays.
With a more robust assay, a new generation of products are
being created using 5' Nuclease assay with TaqMan MGB
probes. Building on the efforts of both Celera Genomics and
public sequencing programs, Applied Biosystems is generating
validated SNP assays that can easily be applied for genetic
studies. Using our bioinformatics pipeline for ‘genome-aided’
assay design and our industrial scale production genotyping
lab, as many as 200,000 validated, ready-for-use
Human SNP Assays-on-Demand products and nearly
30,000 Human gene expression assays will be fully released
by Summer 2002. These Assays-on-Demand products
together with the Assays-by-Design SM
service represent
Applied Biosystems Genomic Assays product line (Figure 2),
a set of unique enabling tools that provide the most rapid
and productive path to disease-gene discovery.
Figure 2. Genomic Assays from Applied Biosystems.
Genomic assays are based upon 5' Nuclease assay using TaqMan
MGB probes. Assays-on-Demand products are ready-to-use
Human SNP and Gene expression assays.
Assays-by-Design Service provides assays for customer-specified
SNPs or genes of interest for any species.
For more information on:
Assays-on-Demand Products enter:
No. 410
Assays-by-Design Service enter: No. 411
13

14
technical communications
15 Years in a Thin Glass Tube
The Development of Capillary Electrophoresis Technology
for Genetic Analysis Applications
A
n early publication on automated DNA sequencing
raised an intriguing prospect: the move away from
denaturing polyacrylamide slab gels for sequence ladder
separation. The primary impact of this publication (L. Smith
et al, 1986) was the use of fluorescent dye-labelled
sequencing primers, together with a laser excitation and
photomultiplier detection system, to create a workable
prototype for the automated, real-time reading of DNA
sequences via Sanger dideoxy terminator chemistry.
However, the electrophoretic separation apparatus was also
unusual. A 50cm tube gel, of less than 2mm diameter,
was chosen to maximise the sensitivity of that prototype
sequence reader.
When it came to the production of a usable commercial
instrument, the throughput of a single tube gel was clearly
inadequate so designers turned to a scanning system utilising
a conventional, denaturing polyacrylamide slab gel (C. Conell
et al, 1987). Development of automated DNA sequencing and
DNA fragment analysis continued with slab gel-based systems
for several years, but the attractions of capillary electrophoresis
(CE) remained for separation.
Established as a rapid and sensitive technique for the analysis
of small molecules such as peptides and oligonucleotides,
CE offered fast run times, high resolution and very efficient heat
dissipation from ultra-high voltage separations (up to 20 kV).
Applied Biosystems developed expertise in the field of analytical
CE with the design and production of the 270 instrument in the
late 1980’s. Flowable polymer solutions such as hydroxy-methyl
cellulose could be used to fill narrow bore capillaries for
separating analytes. Ultra-violet absorption detection determined
quantities and retention times of materials separated by
high voltage electrophoresis. For the analysis of synthetic
oligonucleotides, polymerised denaturing gel-filled capillaries
were used as the separation medium. These gave excellent
resolution but offered relatively short lifetimes.
Thus, the major challenge in developing a functional CE-based
DNA sequencing or fragment sizing system lay in the
polymer chemistry. By 1994 a capillary reagent kit had
been produced for the ABI 270HT analytical CE instrument,
for sizing of DNA fragments from restriction enzyme digests,
still using UV detection.
Improvements in polymer design and the application of
the laser excitation technique from Applied Biosystems,
allied to a charge-coupled device camera for fluorescence
detection (as first used in the 377 system), resulted in the
1995 release of the ABI PRISM ® 310 Genetic Analyzer, the first
commercial CE genetic analyzer. The initial model could
sequence over 400 base pairs using a 75µM internal diameter,
Teflon ® lined, fused silica capillary. It also had GeneScan ®
Analysis Software for DNA fragment sizing applications.
The 310 system became a mainstay for low-to-medium
throughput laboratories. Within 2 years of its release,
improvements came with new POP (Performance Optimised
Polymers) and new 50µM i.d. capillaries, allowing better
resolution and heat dissipation. These capillaries were unlined,
utilising dynamic coating of the walls by the improved POP
polymers to achieve superior performance and greatly increased
service lifetimes. Sequencing specification increased to reads
over 600 base pairs with POP-6 polymer, and reliable,
rapid throughput typing of microsatellite markers was
achieved with POP-4 polymer, and a 96-well sample plate to
supplement the earlier 48 tube arrangement.
In the medium-to-high capacity field, slab gel systems
continued as the workhorses of the human genome sequencing
project, and major medical and forensic microsatellite based
typing programmes. The 377 system finally reached 96 lane
capacity, but the demand for ultra high-throughput, rapid turnaround
systems continued, and the goal of complete,
walk away, 24-hour plus operation (as offered by the
310 system) could not be addressed by slab gel instruments.
Applied Biosystems collaborated with Hitachi Ltd, holders of
key patents for analytical CE, and by late 1998 released the
technical communications
96 capillary ABI PRISM ® 3700 DNA Analyzer for Genome scale
projects. Capable of fully automated loading and running of
samples from a series of 96- or 384-well microtitre plates,
the 3700 system was a key factor driving the rapid sequencing
of the human genome. Development of CE polymer chemistry
continued with the release of POP-5 polymer in 2000,
for enhanced sequencing capability on the 3700 system,
and very recently the creation of POP-37 polymer for this
instrument. This offered further increases in sequencing and
fragment analysis throughput.
Another joint project with Hitachi Ltd., produced the ABI PRISM ®
3100 Genetic Analyzer, a 16-capillary system designed for
medium-to-high throughput laboratories and core facilities that
were not so ‘production oriented’ that did not require the
dedicated capacity of the 3700 system. Based on proven 50µM
capillaries and POP polymers, the 3100 system has further
increased the performance of CE based DNA analysis,
with sophisticated laser optics giving increased sensitivity and
signal to noise ratio. A range of capillary array lengths for this
instrument has enabled extremely rapid Single Nucleotide
Polymorphism (SNP) genotyping runs on 22cm capillaries,
and 1,000 base plus sequence reads on 80cm arrays.
This is in addition to more conventional applications run on
36cm and 50cm capillaries.
The standard 3100 system is now joined by the new ABI PRISM ®
3100-Avant Genetic Analyzer, a 4-capillary introductory
version of the full instrument. It offers the low-to-medium
throughput laboratory all the versatility of the 3100 system at
lower initial cost. As the needs of the laboratory grow,
this instrument can be upgraded to full 16-channel capacity
at a time appropriate to the workload.
Applied Biosystems continues to advance the entire line of
CE-based instrumentation through enhanced performance and
the development of new applications.
The newest capillary electrophoresis instruments to be
introduced by Applied Biosystems, in collaboration with
Hitachi High-Technologies Corporation, are the 3730xl and
3730 DNA Analyzers. These instruments have been specifically
designed to deliver improved production capacity and lower
running costs for the high-throughput production laboratory.
References:
1. Smith, L.M., J.Z. Sanders, R.J. Kaiser, P. Hughes, C. Dodd, C.R. Conell, C. Heiner,
S.B.H. Kent and L.E. Hood. (1986). Fluorescence detection in automated DNA
sequence analysis. Nature 321: 674 - 679.
2. C.R. Conell, S. Fung, C. Heiner, J. Bridgham, V. Chakerian, E. Heron, B. Jones,
S. Menchen, W. Mordan, M. Raff, M. Recknor, L. Smith, J. Springer, S. Woo and
M. Hunkapiller. (1987). Automated DNA sequence analysis. BioTechniques 5: 342 - 348.
For more information on:
ABI PRISM family of CE Genetic Analyzers enter: No. 412
15

16
technical communications
Real-Time PCR Analysis of Gene
Expression Pattern in Asthma Research
T
he research of allergic reactions like asthma shows
that not one but many parameters are altered in this
disease. So far, analytical methods for quantitative RNA
analysis of many samples and the required screeningmethods
have not been available. With the help of
Real-Time PCR even complex gene expression patterns
including genes that are expressed only weakly can be
analysed quantitatively and automated. This technique
can be used in research of allergic diseases.
Current methods for the analysis of gene expression are
only detecting the formation of gene products indirectly.
With these immunological methods just a couple of parameters
can be detected in parallel. Furthermore, they are rather low
in sensitivity and assay development is time consuming.
Here, Real-Time PCR-technology leads to new ways.
This very sensitive method only requires very low amounts of
sample material. It even allows a direct measurement
of transcripts if combined with a reverse transcription step.
Thus, it gives a real view into the status of genes expressed in
cells. This enables the detection of differential transcription
patterns in disease related cells if compared with healthy ones.
The pharmaceutical industry already uses results
produced with Real-Time PCR for the development of
new drugs. The goal is to identify substances that interfere
selectively and specifically with altered activation or
deactivation-processes in diseased cells. With Real-Time PCR,
monitoring of pharmacological targets is less time-consuming,
more convenient, cheaper and more sensitive – e.g. using
the automated ABI PRISM ® 7900HT Sequence Detection
System. Real-Time PCR not only reduces the so-called
The ABI PRISM ® 7900HT Sequence Detection System
random screening of big numbers of synthetic and/or
natural substances. It even simplifies the validation of results
in animal testing and in humans too, making this research
ethically more acceptable.
Dr. Andreas Pahl from the Department of Pharmacology at
the University of Erlangen, Germany has already used this
screening approach successfully for many years. The goal of
Dr. Pahls´ research is the identification of candidate substances
for the development of new drugs (leads) that selectively inhibit
a sub-population of T-helper cells, the Th2-cells. The basis of
this research is the so-called Th1/Th2-paradigm (see figure 1).
The two sub-populations of T-helper cells produce different
cytokines. While Th1-cells express the cytokines IL-2, IFN-g
and TNF-ß, Th2-cells produce the cytokines IL4, -5 and -13.
Figure 1.
The Th1/Th2-paradigm shows how Th2-cells
are involved in the development of allergic reactions.
Both subpopulations of T-helper cells produce different
cytokines as a result of an antigen-stimulus.
Th1/Th2-Paradigm
In comparison with Th1-cells, Th2-cells do not only react
against typical antigenes like nematodes or protozoa’s.
Th2-cells are the starting point of a reaction-cascade resulting
in the release of histamines because they react on allergens,
too. Thus, Dr. Pahl tries to identify substances that selectively
inhibit the expression of one or more cytokines expressed
by Th2-cells, but that do not interfere with the expression
of Th1-cytokines.
With the help of Real-Time PCR many parameters that are
altered in a disease can be monitored on the same
reaction plate. In his highly automated laboratory Dr. Pahl
technical communications
incubates cell-cultures in 384-well format with drug libraries.
RNA is isolated from these cells and following reverse
transcription the expression of the relevant cytokines is
analysed. Real-Time PCR is performed on the 7900HT system
with 24-hour unattended operation. This allows the quantitative
analysis of up to 5,300 reactions per day. Finally, the results
are stored in a database and analysed for a certain expression
pattern (see figure 2) in order to identify potential leads for
drug development.
Figure 2.
The results of the drug library screening are collected in a
database. A pre-defined pattern is used to search for
lead-substances that specifically inhibit cytokines expressed
by Th2-cells. Hits are highlighted and will be studied further.
Drug screening results: Pattern Searching
Pattern definition : IL-2 > 0 IL-4,5,13 < -1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Conclusion
Plate Well IL-2 IL-4 lL-5 lL-13 PatternMatch
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
B1
B2
B3
B4
0,00
0,82
1,49
-0,51
1,78
-0,08
-0,58
0,27
0,00
-0,59
-1,07
0,00
0,00
-1,27
-1,81
-0,82
Real-Time PCR allows a higher throughput than conventional
immunologically-based methods. Its very high sensitivity
and large dynamic range allows the analysis of genes
expressed at low levels. The basic PCR chemistry has been
developed further into a very robust, easy to optimise and
standardised method. Assay development is not only fast,
but the assay format itself makes the analysis of many
parameters on the same reaction plate very convenient too.
Therefore, Real-Time PCR offers a new strategy for the
quantitative analysis of gene-expression patterns.
For more information on:
0,00
0,47
0,97
-0,69
1,07
-1,10
-0,63
-2,34
0,00
0,06
0,50
0,00
0,00
-1,59
-1,71
-0,38
0,00
1,39
-0,18
0,09
-0,82
1,10
1,20
-1,4
0,00
-1,40
-0,79
0,00
0,00
-1,09
-0,49
-1,33
0,00
0,73
-0,35
0,30
-0,38
-0,85
-1,19
-3,1
0,00
1,39
-0,18
0,00
0,00
1,10
1,20
-0,96
Real-Time PCR information pack enter: No. 413
X
17

18
new product review
Pro ICAT Software Application
For Automatic Interpretation of Proteomic Data
P
ro ICAT is a software application that works in
conjunction with BioAnalyst software. Its function
is to process LC/MS and LC/MS/MS Information
Dependent Acquisition (IDA) data that has been acquired
from ICAT reagent-labelled proteomics samples using
an API QSTAR ® Pulsar LC/MS/MS System.
The software identifies and quantifies proteins present in
complex samples following labelling with ICAT reagents.
Identification is performed with the new Interrogator search algorithm, which can perform extremely fast
database searches. Quantitation is performed using the
unique three-dimensional LC/MS Reconstruct algorithm.
The system stores all results in a back-end relational database
for easy, flexible access, with the capability to create a second,
expression-dependent MS/MS analysis from the quantitation
results. The software provides fast, accurate interpretation of
data and lets the user directly compare results to raw data in
BioAnalyst software.
Key Features
➜ Automated processing of proteomics data for protein
identification and quantitation
➜ Designed to enhance the ICAT reagent technique
from Applied Biosystems, for quantitative protein
expression analysis
➜ Performs extremely fast database searches for protein
identification using the exclusive Interrogator search
algorithm, which accommodates as many as five variable
modifications and uses a powerful ‘zone modification’ feature
➜ Unique LC/MS Reconstruct quantitation algorithm
performs accurate three-dimensional determination
of experimental:control ratios (D8:D0)
➜ Uses a back-end relational database for storing results
➜ Lets the user create expression dependent LC/MS/MS
methods from quantitation results
➜ One click allows visualisation of quantitative evidence,
protein sequences, and identification evidence with
familiar BioAnalyst software tools
Overview
Traditional mass spectrometry approaches for protein
identification involve separating protein mixtures using 1-D or
2-D gels, isolating the protein spots of interest, enzymatically
digesting the protein into peptides, and analysing the peptides
by mass spectrometry. The recent development of the isotopecoded
affinity tag technique 1 has allowed the investigation of
large numbers of proteins, using a multi-dimensional liquid
chromatography approach (MDLC) in a fraction of the time
typically necessary for traditional 2-D gel techniques.
The ICAT reagent technique is based upon the use of a
specific reagent that selectively modifies cysteine residues.
Once modified, the proteins from two samples are combined
and digested. By taking advantage of a biotin affinity tag on
the reagent, the peptides containing the modified cysteine
residues are selectively purified 2 .
Once purified, ICAT reagent-labelled cysteine-containing
peptides are separated by on-line capillary LC and analysed
directly by mass spectrometry.
Pro ICAT software is designed to identify and quantify
ICAT reagent labelled peptides from LC/MS/MS IDA
data acquired using an API QSTAR Pulsar Hybrid
LC/MS/MS System. The instrument automatically performs
MS and MS/MS on the eluting peptides using IDA3.
The Pro ICAT software application processes the IDA data
to identify proteins from the MS/MS spectra and quantify
ICAT reagent-labelled expression pairs from the MS data.
If desired, the IDA method can be generated from the
quantitation results from an LC/MS run. Thus, expression
dependent MS/MS analysis can be performed on only those
peptides with ratios that meet user-defined limits.
Conclusions
A new software application is now available for processing
proteomics data acquired from an API QSTAR Pulsar
LC/MS/MS System, from samples prepared using the
ICAT reagent technique. Pro ICAT software uses the Interrogator
search algorithm for identifying proteins from un-interpreted
MS/MS data and the LC/MS Reconstruct algorithm for
quantitating expression pairs from the MS data.
Users can define as many as five modifications in the protein
identification search. Additionally, they can search for arbitrary
modifications up to a user-specified mass difference at
run-time (zone modification feature). The LC/MS Reconstruct
algorithm quantifies ICAT reagent expression pairs from
MS data by clustering the data based upon ICAT reagent
fragments and optimally collapsing adjacent spectra for a
maximum signal-to-noise ratio. Users may also run expression
dependent LC/MS/MS IDA experiments based upon the
quantitation results.
new product review
When the goal is to quickly process proteomic data from
ICAT reagent labelled samples from the QSTAR system data,
Pro ICAT software is the answer.
Pro ICAT software identifies and quantifies proteins with
high confidence and offers the additional flexibility of
performing expression dependent analysis from interesting
ratios found in the data.
In summary, Pro ICAT software delivers the most
comprehensive information in the shortest time and with
the highest confidence to find the proteins that matter.
References
1. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold,
R. 1999. Quantitative analysis of complex protein mixtures using isotopecoded
affinity tags. Nature Biotech., 17:994-999.
2. For more information about the ICAT reagent technique, see the ICAT
reagent product bulletin “ICAT Reagents for Quantitative Protein Expression
Analysis Studies” (literature code: 125PB01-01).
3. For more information about IDA, see the LC/MS product bulletin
“Information Dependent Acquisition – The Next Generation of Data Dependent
Experiments for LC/MS/MS Analysis” (literature code: 114PB07-01).
For more information on:
Pro ICAT Software enter:
No. 414
ICAT Reagents enter: No. 415
19

20
new product review
Curtain
Gas
Interface
The New Q TRAP LC/MS/MS System
for Protein Analysis
A
Skimmer
Orifice
Q0
High-
pressure Cell ST Q1
t the recent ASMS conference held in Orlando,
Florida, Applied Biosystems/MDS SCIEX launched
a new linear hybrid ion trap mass spectrometer,
the Q TRAP LC/MS/MS System.
This new Q TRAP LC/MS/MS technology combines the
specificity and robustness of triple quadrupole systems with the
full scan MS/MS sensitivity of ion trap systems into a single
instrument. By combining these capabilities, improvements
in both sensitivity and information content are realised.
Additionally, the instrument has several unique scan modes,
such as the ability to enhance the detection of multiply charged
peptide ions over singly charged ions, mainly due to
chemical noise introduced with a nanospray or LC interface.
These unique features, in combination with acquisition
tools such as Information Dependant Acquisition (IDA),
make the Q TRAP LC/MS/MS System ideal for rapid and
efficient identification of protein digests.
Enhanced Sensitivity
The Q TRAP LC/MS/MS System provides superior sensitivity
in MS and MS/MS modes over that of traditional triple
quadrupole and ion trap mass spectrometers while maintaining
triple quadrupole-like fragmentation. With signal to noise
improvements greater than 100X, identification of protein
digests at femtomole levels can routinely be achieved with
maximum sequence coverage.
Enhanced Resolution
The Q TRAP LC/MS/MS System provides superior resolution
across the entire mass range, making charge state identification
much more reliable up to charge state 5. This capability
improves the ion selection process for complex mixtures and
ensures the appropriate determination of collision energy in
Q2
LINAC
Collision Cell
IQ2 IQ3
Q3 EXB
C2B
DF
DET
IDA mode. The Q TRAP LC/MS/MS System provides constant peak
widths (0.12amu FWHH) across the mass range, thus resulting
in resolution in excess of 4000 and mass accuracies of 50ppm
for tryptic peptides selected by IDA for MS/MS.
Enhancing Multiply Charged Ion Selection
When maximum sample information is desired, it is extremely
important to optimise the data acquisition for ions that will
generate the most useful information. For protein digests at low
levels, detection of multiply charged ions is normally hindered
by the presence of background ions that are predominantly
singly charged ions and usually present at high concentration.
The Q TRAP LC/MS/MS System offers a unique operating mode
where multiply charged ions are selectively detected over singly
charged ions. This mode of operation ensures easy and
efficient detection of peptide ions in both nanospray and
capillary LC/MS mode, thus maximising the collection of useful
information in a minimum amount of time.
Information Dependent Acquisition (IDA)
IDA enables the user to combine the unique modes of
operation of the Q TRAP LC/MS/MS System to acquire MS and
MS/MS data in an automated fashion within a single injection,
thus maximising both instrument usage and information
gathering. This can be achieved by combining modes of
operation that yield very specific MS information such as
precursor ion scans or multiply charged scans for proteolytic
digests, with the enhanced full scan MS/MS mode of the
Q TRAP LC/MS/MS System.
new product review
IDA provides enough flexibility to the user to combine scan
types that will yield the most specific information at the
highest quality level.
Automated identification with ProID
ProID is an application that works in conjunction with
BioAnalyst software, and is designed to identify proteins
from LC/MS/MS data acquired using Applied Biosystems/MDS
SCIEX systems and information dependent acquisition (IDA).
Identification is performed with the Interrogator search
algorithm capable of performing extremely fast database
searches with up to five variable modifications plus a powerful
‘zone’ modification feature for detection of unanticipated
modifications. All results are stored in a back-end relational
database with the ability to directly compare results to raw
data in BioAnalyst software.
The Q TRAP LC/MS/MS System is a cost efficient and reliable
instrument for the identification of proteins. Its productivity,
selectivity and sensitivity make it the instrument of choice for
any proteomic laboratory that is looking for versatility.
For more information on:
Q TRAP LC/MS/MS System enter: No. 416
21

22
new product review
New SQL*LIMS 4.1 Software
The ‘Safe Choice’ for Major Pharmaceutical Companies
C
ontinuing a decade long history of laboratory management
system experience, in 17 of the top 20 pharmaceutical
companies worldwide, Applied Biosystems announces
the release of SQL*LIMS 4.1 Software. The award winning
SQL*LIMS software is installed in over 1000 laboratories world
wide and is available with a full range of modules for enhanced
functionality to meet the specific needs of your laboratory.
As a leader in the LIMS market across a variety of industries,
Applied Biosystems has lead LIMS innovation with features
to assist customers in complying with FDA 21 CFR Part 11
ERES/ESIG and integration with MRP systems, such as SAP’s
R/3 QM. SQL*LIMS software has also lead the way with its
reporting tool, desktop program and instrument data exchange
through its open, industry standards-based Oracle ® and
MS Windows ® architecture.
Furthermore, SQL*LIMS software’s flexible architectural
support allows you to easily scale a system from a single
workgroup to an enterprise wide global implementation. You can
customise the system’s graphical user interface to meet your
requirements and allow access to the system via a web browser.
For more information on:
SQL*LIMS 4.1 Software enter:
No. 417
or email InformaticsInfo@appliedbiosystems.com
The new SQL*LIMS 4.1 software includes many new features
for improved efficiency and performance.
➜ Streamlined user account, audit and electronic
record/signature configuration and monitoring
Support for Oracle account profile functionality
➜ Enhancements to the audit trail and electronic
signature capabilities allowing you to more precisely
map to your workflow
➜ Expanded reporting tool integration that allows more
choice options for direct application integration to
external programs and 3rd-party reporting tools
➜ Enhancements to optimise performance, reliability and
distributed computing that allow you to scale a solution
to meet your exact requirements
The SQL*LIMS system provides you with immediate
access to drug and raw material data, across products,
suppliers and sites. You also benefit from a single
comprehensive audit record that pinpoints data by lot,
supplier, assay and other key criteria, all while meeting
regulatory compliance. SQL*LIMS software is fully auditable
and is the only comprehensive system for laboratory
automation backed by the proven LIMS leader.
New PrepExpress NT Software
A Solution for High-throughput Purification Using the
API 150EX Prep LC/MS System
T
he API 150EX Prep LC/MS System is a useful tool
for high-throughput compound characterisation
and purification. The Prep LC/MS System is easily
configured for both preparative and analytical modes.
Several new enhancements to this product are now
available. The new PrepExpress NT software was introduced
by Applied Biosystems/MDS SCIEX at Pittcon 2002.
It runs on top of Analyst ® software, the Windows NT ®
operating software for LC/MS and LC/MS/MS instruments from
Applied Biosystems/MDS SCIEX.
Overview
The technique of Prep LC/MS combines the separation power of
large scale HPLC with the specificity of mass spectrometry.
This allows for the purification of targeted compounds of known
mass, obviating the need to collect all to the peaks in the
LC run. Automation of the process allows the purification of
hundreds of compounds weekly. Prep LC/MS is gaining
widespread distinction as the tool of choice for high-throughput
characterisation and purification of combinatorial libraries.
Flexible Hardware Setup
As seen in figure 1, the workstation can provide two
separate HPLC flow paths. This accommodates both
analytical and preparative configurations. The analytical flow
path may be used for analytical scale sample characterisation,
including purity assessment, prior to purification.
Figure 1.
API 150EX Prep LC/MS System schematic
new product review
Alternatively, the analytical flow path may be replaced by either
a semi-preparative flow path, or a second preparative flow path
for increased throughput.
Central to the Prep LC/MS System is the API 150EX single
quadrupole mass spectrometer with a TurboIonSpray source
and the Gilson ® 215 liquid handler that may be used as both
sample injector and fraction collector. This provides enhanced
sample capacity and a wider range of collection vessels,
including 48- and 24-well microtitre plates. Also, the Gilson
204 fraction collector may be used. Multiple collectors of either
type may be employed to further increase sample capacity.
PrepExpress NT Software
The new flexible PrepExpress NT Software provides one-stop
control of all fraction collection parameters.
Important features of the software are:
➜ High-throughput unattended batch purification
➜ MS (both mass-specific or non mass-specific) or UV
directed fraction collection
➜ Embedded, automatic purity calculations, batch creation
and submission
➜ Easy ‘wizard aided’ sample submission
➜ Walk up sample introduction
➜ Automated report generation and easy sample tracking
For more information on:
PrepExpress NT Software for prep LC/MS enter: No. 418
23

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new product review
Introducing the ABI PRISM ®
3100-Avant Genetic Analyzer
Growing with Your Throughput Needs!
O
ver a period of several years, Applied Biosystems
has steadily introduced and developed a range of
genetic analyzers based on capillary electrophoresis
technology. This family has been built up to provide
projects of all scales with a genetic analyzer of an
appropriate capacity.
To address the needs of low-to-medium throughput
laboratories, with requirements falling between the capacity
of either the ABI PRISM ® 310 Genetic Analyzer or the
ABI PRISM 3100 Genetic Analyzer, we have launched the
ABI PRISM 3100-Avant Genetic Analyzer.
This new multi-capillary instrument is capable of processing
a considerable number of samples in a fully automated
24-hour run, producing up to 48,000 bases of sequence
data with the rapid sequencing protocol. A 24-hour, 5 dye
fragment analysis‚ run can produce up to 5,760 genotypes with
microsatellite samples or 3,840 genotypes with the multiplex
SNaPshot ® kit (single nucleotide polymorphism) assay.
This level of performance is achieved by coupling an array
of four capillaries to the proven 3100 system optics,
polymer pump system and autosampler (accepting either a
96- or a 384-well microtitre plate, which is sufficient for
loading samples for a full 24-hour run). The repeated,
robotic loading of samples for successive injections allows
true, walk-away automation.
4-capillary array
The 3100-Avant system, like all CE genetic analyzers from
Applied Biosystems, will benefit from our ceaseless research
and development programme that has, over a period of several
years, consistently produced sequential and significant
improvements in polymer performance, sequencing chemistry
and downstream data analysis for existing members of the
CE family. For example, the 3100-Avant system is capable of
using the new 22cm and 80cm capillary lengths that were
recently developed for the 3100. The short 22cm array enables
higher throughput for genotyping applications with very fast
runs, whereas the long 80cm array can be used for enhanced
length-of-read in sequencing applications. Having a choice of
four available arrays (22, 36, 50 and 80cm), one can achieve
the best possible balance between throughput and resolution
based upon your specific application and needs.
Internal view of the ABI PRISM ® 3100-Avant Genetic Analyzer
Futhermore, the 3100-Avant system can be upgraded to full
3100 system specification, so that its throughput can be
extended in parallel with the demands of an expanding research
programme. This option provides an effective way of spreading
the capital investment for a major project through its initial
(method development and establishment), and expansion
(main data production), phases.
With the release of the 3100-Avant system, Applied Biosystems
now offers a range of genetic analyzers to suit all scales of
laboratory requirements, from occasional rapid turn-around
tests on individual samples, right through to the factory scale
24-hour a day operations of the major genome projects.
De novo
Sequencing
new product review
Sequence Analysis
Software
Comparative
Sequencing
BigDye ®
Chemistry
SNP Discovery
and Validation
Microsatellite
Analysis
Linkage Mapping Set
Chemistry
ABI PRISM ® 3100-Avant Genetic Analyzer
SeqScape TM
Software
GeneScan ®
Software
Genotyper ®
Software
SNP Validation
and Screening
SNaPshot ® Multiplex
Chemistry
GeneMapper TM
Software
Finally, proven sequencing and fragment analysis chemistries,
along with new application-specific downstream software,
make the 3100-Avant system an integral part of our
complete solution for all your research needs.
See article on page 14
For more information on:
ABI PRISM 3100-Avant Genetic Analyzer enter: No. 419
25

26
new product review
Affinity Depletion Cartridges
For Removal of Abundant Proteins from Human Serum
Purpose
Removal of abundant proteins from human serum using a highly
specific immunoaffinity chromatography support.
Overview
One of the major difficulties in analysing the proteome of
human serum is the dynamic range of the concentrations
of the proteins present in the sample. Human serum albumin
(HSA) constitutes 57-71% of total serum protein and
gamma-immunoglobulin (IgG) ranges from 8-26%. Removal of
these two proteins alone clears about 75% of the total
protein present in serum, therefore allowing the detection
of the remaining proteins that are present in far lower
concentration. Here we describe the characterisation of
anti-HSA and Protein G affinity chromatography cartridges,
which remove HSA and IgG from human serum, with respect
to their percentage depletion, non-specific binding and
cross-reactivity.
Key features
➜ Highly specific, immunoaffinity ligand, exhibiting very low
to zero non-specific binding
➜ Proven POROS ® support for high throughput processing
and cleanability
➜ Long lifetime and reusability
➜ Process samples by automated (VISION workstation) and
manual modes using unique cartridge format
Chromatography Media Development
The new POROS anti-HSA support was developed using antibody
ligand that has been optimised to specifically bind human
albumin. Immobilisation of this immunoaffinity ligand was to
POROS Perfusion chromatography media using polyethylene
glycol spacers. Using the 0.2ml cartridge device, we have shown
that the media will completely bind the albumin in a 10-70µl
sample of human serum that has been diluted 1:10 (albumin
concentration of 3.5mgs/ml). In combination with the Protein G
cartridge, both albumin and IgG can be effectively removed in
one step.
Technology Application
Experimental Goal
In this work, a sample of human serum was passed over
both a Protein G and Anti-HSA cartridge. The flow through
(serum proteins) and eluted fractions (albumin and IgG) were
analysed by 1 dimensional and 2 dimensional SDS PAGE gel,
as well as a commercially available ELISA assay. This is in order
to quantitate the removal of HSA and IgG from the sample,
and examine the level of non specific binding.
Bands from the 1D Gel were further analysed using peptide
mass fingerprinting analysis on the Voyager workstation in order
to identify visualised bands that did not correspond to the intact
molecular weight of HSA.
Comparisons of binding capacities of albumin from different
species were also compared to determine cross reactivity.
Experimental Conditions
70µL of human serum diluted 1:10 with Phosphate buffered
saline (PBS) was passed through first to a 4mmDx15mmL
(0.2ml) Protein G cartridge and then the flow-through FT
fraction was diluted to 400µL. Then 100µL of the diluted
protein G FT fraction was applied to a 4mmDx15mmL (0.2ml)
anti-HSA cartridge at a flow rate of 0.5mls/min. Elution of each
cartridge was done using 3 mls of 12mM HCL at 1ml/min and
the cartridge was then cleaned with a 5ml step of 1M NaCl.
All chromatographic steps were carried out using the VISION
Workstation and the peak FL and eluted EL fractions were
collected for further analysis.
Protein concentration for each fraction was determined
using Bradford assay. Equal amount of protein (6.5µg)
from each fraction was analysed by SDS-PAGE. 2-D gel analysis
was done using 200µg human serum, before and after
affinity depletion by both cartridges and the proteins visualised
by silver stain. MALDI-TOF peptide mass fingerprinting (PMF)
was performed on bands from 1D Gel that did not correspond
to the intact molecular weight of human Albumin using
Voyager-DE STR Biospectrometry Workstation from in-gel
digested peptides using 10µg/mL modified bovine trypsin in
25mM ammonium bicarbonate.
Anti-HSA and anti-human IgG ELISA kits (Bethyl Laboratory)
was performed according to the manufacture protocol.
The FT fractions used for the ELISA were pooled from multiple
runs (six runs from two anti-HSA cartridges from the same lot
and four runs from two Protein G cartridges from the same lot).
new product review
Results and Discussion
Figure 1 shows the chromatograph of affinity depletion of HSA
from human serum using POROS and anti-HSA antibody affinity
cartridge operated on the VISION Workstation. The sample was
previously run over a POROS protein G cartridge in order to
remove IgG from the solution.
Figure 1. Chromatograph of affinity subtraction using POROS
Anti-HSA cartridge
Absorbance (A280)
Figure 2 shows SDS-PAGE analysis of serum sample (lane 2)
the FT and EL fractions and the enrichment of low intensity
bands in the protein G and anti-HSA FT fraction (lane 5).
The predominant proteins in the eluted fractions (lanes 4 and 6)
are IgG heavy chain (55kDa) and light chain (25kDa) and HSA
(66kDa) respectively. To further characterise the proteins eluting
from the anti-HSA cartridge, peptide mass fingerprinting was
performed on low intensity bands. That did not correspond to the
intact molecular weight of Albumin. Most of these bands were
identified as proteolytic fragments of Albumin.
Figure 2. 4-20% SDS PAGE gel stained with coomassie blue.
All lanes loaded with 6.5µg total protein. Bands A,B,D were
identified as Albumin fragments, C was identified as Albumin
fragments and Keratin.
Lane 1 Molecular weight markers
Lane 2 Serum sample diluted 1:10 with Phosphate buffered saline
Lane 3 Flow through of POROS Protein G cartridge
Lane 4 Eluted fraction of POROS Protein G cartridge
Lane 5 Flow through of POROS Protein G and POROS Anti-HSA cartridges
Lane 6 Eluted fraction of POROS Anti-HSA cartridge
page 28
27

28
new product review
Figure 3 shows an expanded region of a 2 dimensional gel the
enrichment of low intensity protein spots before and after the
affinity depletion using 2-D gel. In many cases, protein spots
that were barely visible in a normal sample became much more
intense upon depletion of the albumin and IgG.
Figure 3. Expanded section of 2-Dimensional Silver stained gels.
Top gel is serum sample before removal of IgG and HSA.
Bottom gel is serum sample after processing on POROS Protein
G and Anti-Hsa cartridges. Circled spots represent increase in
concentration of some low abundant proteins
Figure 4 shows results of measuring the removal of IgG and
albumin from the flow through fraction of both cartridges.
Removal of both IgG and HSA was shown to be greater
than 99%.
Figure 4. Results from measurement of IgG and HSA in the
serum sample using an ELISA assay, before and after affinity
subtraction using the POROS Protein and Anti-HSA cartridges.
The cross reactivity of the Anti-HSA antibody was also
examined to determine binding capacity for various species of
Albumin. Table 1 shows the capacity measurements made on
the media using purified forms of albumin from various
species. Although albumin from other species do bind with
some specificity, this antibody demonstrates the highest
specificity for Human Serum Albumin.
Table 1. Cross reactivity of Anti-HSA media for albumin
from various species. Capacity measurements are based
on 10% breakthrough capacity measured on a
2.1mmDx100mmL column.
Species mg Bound mMoles Bound
per ml media per ml media
Bovine 0.24 0.004
Goat 0.01 0.000
Human 2.04 0.031
Mouse 0.70 0.011
Porcine 0.52 0.008
Rabbit 0.31 0.005
Rat 0.75 0.011
Sheep 0.01 0.000
Conclusions
POROS Protein G and Anti-HSA cartridges can quickly and
efficiently remove both IgG and HSA, which are the two most
abundant proteins in human serum. Using this immunoaffinity
technique greater than 99% of both proteins can be removed
from serum with very low to zero non-specific binding of other
proteins in the sample. Using a convenient cartridge format,
samples can be processed manually or using an automated
LC system to increase throughput. Capacity for human serum
Albumin was measured at around 2.5mgs/ml.
For more information on:
Affinity Depletion Cartridges enter: No. 420
Moving forward in Genomic Research
Assays-by-Design service
Assays-by-Design service is a custom assay design service
dedicated to providing quality controlled assay products for
gene expression and SNP genotyping needs. You simply submit
your target DNA sequence, and Applied Biosystems returns a
QC-verified, 5' nuclease MGB assay that is ready to use with
TaqMan ® Universal PCR Master Mix and to load on your
sequence detection system platform of choice. Assays for any
DNA sequence of any species can be generated and delivered
in a convenient single tube format.
Assays-by-Design service;
➜ shortens your path to results
➜ lets you focus on research questions, not on designing assays
➜ eliminates the manual task of designing primers and probes
➜ eliminates assay optimisation, saves time and labour
➜ is designed to ensure a successful experiment
See also pages 13 & 46 for related articles
For more information on:
Assays-by-Design Service and
Assays-on-Demand Products enter:
No. 421
new product review
Applied Biosystems Introduces the New Assays-by-Design SM Service
and Assays-on-Demand Products
Assays-on-Demand products
The introduction of the Assays-on-Demand products for SNP
genotyping and gene expression studies for all human genes,
signals Applied Biosystems’ next major innovation to move
genome analysis to the next level by changing the point of
departure for genetic research from gene sequence to gene
information content. Off-the-shelf products for 30,000 genes
respectively 200,000 SNPs; researchers will be able to order
these validated assays by gene name or attribute without the
requirement of knowing the gene’s sequence or oligonucleotide
design techniques. Just add your sample and go!
Assays-on-Demand products;
➜ provide rapid, quantitative gene expression or
SNP genotyping results
➜ eliminate manual design, optimisation and functional testing
➜ allow direct and accurate comparison of data between labs
➜ ensure high quality, high performance and reliability
For more information on Assays-on-Demand please visit our
web store at https://store.appliedbiosystems.com or contact
your local Sales Office. Registered customers can order
assays on-line from this website*. Feel free to re-visit this
site periodically as new assays are continually being added,
fully released by summer 2002.
* Please contact your local Applied Biosystems office if you don’t find your
country in the drop down menu on the registration page
29

30
new product review
Improved Software Tools for
DNA Polymorphism Data Analysis
D
NA sequencing and polymorphic marker analysis
could have been viewed as two disparate
investigative techniques in the past. Direct sequencing
provided a ‘gold standard’ for absolute typing of
polymorphisms, allowing both detection and definition
of mutations in one assay. In comparison, DNA fragment
analysis provided a high-throughput, marker typing technique
for analysing large numbers of samples such as the data sets
needed for linkage mapping projects, or human identification
databases. More recently, single nucleotide polymorphism
(SNP) genotyping has bridged the gap between those original
techniques, being a direct sequence interrogation assay that is
applicable to mass screening projects.
All these techniques face a similar challenge as genotyping
projects grow in scope and complexity: the need to handle
increasing volumes of data reliably and efficiently.
Comparative sequencing and DNA fragment-sizing analyses,
though originally undertaken by entirely separate software
programmes, followed the same basic workflow, in three stages
– data collection, sample file analysis and overall data review.
For sequencing projects, this meant sequence ladder tracking
and data collection, sequence analysis and processing
by Factura and Sequence Navigator ® software. For fragment
analysis the route was data collection, GeneScan ® analysis and
Genotyper ® software review of the data set.
One drawback of this organisation was the separation of primary
data analysis and data review. All the sample data, good and
bad, would have to be reviewed (and possibly edited) at the
initial analysis stage before proceeding to compilation and
review by the downstream software (Sequence Navigator or
Genotyper software). Alternatively, analysed sample data could
be loaded directly into the downstream programme and
processed, but this carried the risk that poor or anomalous data
from some samples would require a return to the basic analysis
software for editing or re-analysis.
Recently, new software products were released by
Applied Biosystems which increased the efficiency of such
data analysis and review, enabling much larger data sets to
be handled conveniently and accurately by one operator.
For comparative sequencing, SeqScape software replaces
Sequence Analysis, Factura and Sequence Navigator software for
reviewing data in pursuit of SNP discovery or mutation profiling.
In fragment analysis and SNP projects, GeneMapper carries
out all the functions formerly handled separately by the
GeneScan, Genotyper and GenBase software packages.
Earlier this year, updated versions of both these software
programmes were released: SeqScape software v1.1 and
GeneMapper software v2.0 , both with significant improvements.
Both of them feature data quality indicators which enable the
operator to identify immediately any sample data that needs to
be reviewed, edited or possibly rejected. By eliminating the
need to scan all primary data visually for acceptance,
data throughput is greatly increased. Also, because review and
re-analysis or rejection of a minority part of the sample data can
be done within the same programme, efficiency of operation and
centralising of operator intervention is much improved.
As an example, an operator using GeneMapper software for a
linkage project can process up to 48,000 genotypes in a six hour
period, an increase of six-fold over the data-handling capacity of
the previous GeneScan/Genotyper system.
SeqScape software v1.1 provides Quality Values (QV) for every
base call, derived from the Trace Tuner software (developed by
Paracel Inc.) now embedded in the new version (see figure 1).
The QV figure bears an inverse logarithmic relationship to the
calculated probability of error in the base call. QV’s are displayed
as scaled bars above each base call, which can be customised
for colour and range by the user to allow instant identification
of good, moderate and unacceptable data reliability in
sequence displays.
Figure 1.
Quality Values
This facility dramatically improves ease and speed of editing a
data set to complete a project. The performance of SeqScape
software v1.1 was illustrated in a poster published at the
American Society for Human Genetics meeting 2001 (ref. 1).
GeneMapper software v2.0 offers Process component-based
Quality Values (PQV) to assist data evaluation and substantially
reduce examination time (see figure 2). The PQV’s can be drawn
from the many stages of sample data processing, and specific
sub-sets can be selected as appropriate for certain kinds of data
analysis: SNP’s or microsatellite markers, and for the latter,
for dinucleotide repeats or tri- and tetra-nucleotides. Allele calls
are assigned to one of three categories, pass, check and low
quality, on the basis of PQV scores. A concordance control
PQV allows allele calling from a known internal control sample
to confirm the validity of the chosen analysis parameters for a
body of related sample data.
Figure 2.
GeneMapper software v2.0 also features an Automatic
Bin-Builder algorithm, which optimises bin centres and
boundaries on the basis of recorded and processed data,
to enhance reliable allele calling for micro-satellite analysis.
The GeneMapper Database is pre-loaded with size standard,
marker and panel information to complement our Linkage
Mapping Set of human genomic markers, to help provide a
complete genotyping solution. To maximise the potential of SNP
based genotyping assays, GeneMapper software v2.0 has an
Auto-Paneliser algorithm to allow the highest effective level of
marker multiplexing when used in conjunction with the newly
released SNaPshot ® Primer Focus kit. The enhanced multiplex
SNP analysis capabilities of GeneMapper software v2.0 were the
subject of a poster publication at ASHG 2001 (ref. 2).
new product review
In conclusion, ongoing and intensive research and development
programmes have yielded similar benefits in throughput,
accuracy and ease-of-use for both DNA sequencing and
fragment analysis applications.
References
1. “Enhanced performance of SeqScape software, a sequence comparison
tool for variant identification” C. Kosman et al, 51st American Society for
Human Genetics meeting, October 2001.
2. “Automated scoring of SNaPshot kit reactions with GeneMapper
software” D. Bishop et al, 51st American Society for Human Genetics
meeting, October 2001.
For more information on:
Improved Software Tools enter: No. 422
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new product review
PhotoSpray Source
A New Ionisation Technique for LC/MS
T
he PhotoSpray source is an alternative ionisation
source to TurboIonSpray ® or APCI/Heated Nebuliser
for API 150EX, API 3000 and QSTAR ® Pulsar LC/MS
and LC/MS/MS systems.
The PhotoSpray source provides:
➜ A better sensitivity for low polar compounds
➜ A better flexibility to the ionisation range of the
API 150EX, API 3000 and QSTAR Pulsar systems
The availability of PhotoSpray, TurboIonSpray and APCI
sources allows researchers to ionise a wider range of
compounds than in the past.
Principles of Photoionisation for LC/MS
Photoionisation at atmospheric pressure may be used to
generate ions from the vaporised LC eluent 1 . A Krypton
discharge lamp (hν=10eV), can selectively ionise most
analytes in the presence of the common LC solvents.
Provided hν> Ionisation Potential, single photon ionisation
may occur M + hν →M+ + e-.
However, the efficiency of direct photoionisation is
relatively poor, partly because solvent molecules and other
species absorb the limited photon flux without generating ions.
By adding large quantities of an ionisable dopant to the
ionisation region, dopant photoions can be created in great
abundance. At atmospheric pressure, in the presence of
solvent and analyte, the dopant ions initiate a rapid series of
ion-molecule reactions. Provided that the thermodynamics
are favourable, the end result is that the charge from the
dopant photoions ultimately resides with analyte molecules.
M+ and/or MH+ ions are generated with high efficiency,
the predominant ion type being determined by the IP and
proton affinity of the analyte 2 . The PhotoSpray source in
negative mode gives M-H ions with the same efficiency
as APCI.
Description
Where the Ionspray source produces ions by the process of ion
evaporation from liquid phase, the PhotoSpray source uses a
Heated Nebuliser to vaporise the sample prior to inducing
ionisation by atmospheric pressure photionisation. The source
typically operates at temperatures between 300 and 450°C.
This vaporisation process leaves the molecular constituents of
the sample intact. Molecules are ionised via the process
of photoionisation, induced by a beam of ultraviolet radiation
in the presence of a dopant molecule, as they pass through
the ion source block and into the interface region.
The source block is positioned off-axis to the orifice and the
optimum position is not compound dependent. Proven Curtain
Gas interface technology protects the mass analyser from
contamination and provides ruggedness to the system.
Principles of Photoionisation for LC/MS
Charge Transfer Process is suitable for non-polar compounds.
This process rarely occurs with APCI.
Proton Transfer Process is suitable for compounds that exhibit
higher polarity. This is the dominating process.
Dopant Selection
The dopant is selected for its ability to undergo photoionisation,
because of favourable ionisation energy – normally just below
UV photon energy – and for the ease with which it can
be available in high purity grade – preferably HPLC grade.
Ideally it should exhibit low toxicity. Toluene, (ionisation
potential of 8.83eV) meets all these requirements and
is the preferred dopant compound for the PhotoSpray
applications. The dopant infusion rate is 5–15% of the
mobile phase flow rate (no split).
Flow Rate
The PhotoSpray source operates, in principle, with flow rates
up to 2.0mL/min, with an optimum flow rate between 200
and 500µL/min, making the source well adapted to 2mm
I.D. LC columns. Most applications have been demonstrated
under this regime. The source operates under reversed or normal
phase chromatographic conditions:
➜ MeOH/Water or Acetonitrile
➜ Isooctane/Isopropanol/Methylene Chloride and other
LC solvents
new product review
PhotoSpray Applications
The PhotoSpray source can ionise low polar compounds,
with better sensitivity than the APCI source. PhotoSpray showed
important sensitivity improvements for Steroid analysis,
PolyAromatic Hydrocarbons, Vitamins, Quinones, Antioxidants,
Pesticides, Pharmaceuticals and Nutraceuticals and several
other classes of compounds. So the PhotoSpray source is a
complementary source to the TurboIonSpray and APCI sources.
TurboIonSpray
Shown here is a direct plot of the relationship of PhotoIonisation
to both the APCI and TurboIonSpray techniques as compound
polarity increases against molecular weight.
API 3000 LC/MS/MS Steroids Analysis
Performance comparisons of the PhotoSpray source versus an
APCI source have been performed under normal-phase
chromatographic conditions for steroids 3 . Testosterone and
Ethynyl Estradiol were tested with the photoionisation
source and compared with the conventional APCI source.
Using APCI steroids, compounds normally exhibit different
sensitivity levels and fragmentation depending on their
hydroxylation numbers. Ethynyl Estradiol is a steroid compound
having significant economical importance for its estrogenic
effect. This compound exhibits in-source decomposition and
the fragment at 279amu is followed for the MRM transition.
We compared the sensitivity of these steroids for the two sources
in MS scan to evaluate relative ionisation efficiency and to
measure LOD and LOQ using MRM transitions 4 . Toluene was
used as dopant for the source at a typical flow rate of 20µL/min.
A Keystone Betasil Diol 5µm, 100Å, 2x100mm has been used
for the LC separation in normal phase conditions. Mobile phase
composition was isocratic isooctane (94%)/isopropyl alcohol
(6%). Reversed phase separations have been achieved
isocratically on a BDS Hypersil Cyano 5µm, 120Å, 2 x 250mm
using a mobile phase composition of MeOH (50%)/HOH (50%).
Mobile phase flow rate was 200µL/min in both normal and
reversed phase conditions.
page 34
33

34
new product review
In MRM mode the signal gain, when combined with a reduction
in background noise, translates into between 5 to 20 times
better signal-to-noise ratios. The response was linear with an
upper limit of determination of 2000 pg on column for
Testosterone, and 4000 pg on column for Ethynyl Estradiol.
Relative response for Testosterone and Ethynyl Estradiol with
PhotoSpray (PS) and APCI, as recorded in MRM mode.
The same respective amount of compounds were injected
on column using Normal phase chromatographic conditions.
Estimated LODs and improvement Factor of S/N versus APCI
Normal Reversed
Phase Phase
Steroid LODs PS Ratio PS/APCI Steroid LODs PS Ratio PI/APCI
Testosterone 0.16 pg 7.5 Testosterone 0.08 pg 22.2
Ethynyl Estradiol 1.0 pg 5.0 Ethynyl Estradiol 1.1 pg 8.1
Polycyclic Aromatic Hydrocarbons (PAHs)
The PhotoSpray source was used to develop an analytical
method for polycyclic aromatic hydrocarbons (PAHs),
comparing the difference under reverse and normal phase
chromatographic conditions. A total of 16 PAHs commonly
found in atmospheric samples from both rural and urban
areas were analysed. Airborne PAHs, usually found in
aerosol particles, are implicated in heterogeneous atmospheric
processing and as important indicators of gas-phase
atmospheric processes. Certain carcinogenic PAHs may
be carried by ultra fine particles in ambient air into the
lungs and are often the targets of rigorous environmental
monitoring. A 20ppm stock standard solution containing
16 common PAHs was used for this experiment 5 .
Reverse Phase PAH separation showing ten
MRM transistions for 16 common PAHs
Conclusion
The PhotoSpray source provides a better detection limit to the
API 150EX , API 3000 and QSTAR Pulsar systems for low
polar class of compounds. The API 3000 and QSTAR Pulsar
LC/MS/MS systems are the only tandem MS systems with three
different ionisation sources, which allows researchers to have a
high ionisation range flexibility.
References
1. Bruins, A. P., Robb, D.B.: A New Ionisation Technique for LC/MS, 16th
Montreux LC/MS Symposium, Nov. 3rd, 1999 (WO3)
2. Robb, D.B., Covey, T.R., Bruins, A.P., Anal. Chem. 2000, 72, 3653-3659.
3. Robb, D.B., Bruins, A.P., Peters, H.A.M., Jacobs, P.L.; Atmospheric Pressure
Photoionisation (APPI) for High Sensitivity LC/MS in Bioanalysis,
ASMS 2000 poster MPA-015
4. Alary, J.F., Comparative Study: LC-MS/MS Analysis of Four Steroid
Compounds Using a New Photoionisation Source and a Conventional APCI,
ASMS 2001 poster TPA-009
5. Impey, G., Kieser, B., Alary, J.F., The Analysis of Polycyclic Aromatic
Hydrocarbons (PAHs) by LC/MS/MS using a New Atmospheric Pressure
Photoionisation Source, ASMS 2001 poster TPH-187
For more information on:
PhotoSpray Ionisation Source enter: No. 423
new product review
TaqMan ® Assays for Food Testing
and Food Pathogen Detection
F
ood quality and food safety is a subject of growing
public concern. Public food control institutions,
private service laboratories and commercial food
manufacturers see a growing need to test for genetically
modified organisms (GMO) and pathogens in food.
In July 1999, Switzerland implemented legal regulations
which require food containing more than 1% GMO to be
labelled. The European Community and many other countries
around the world followed this example.
Assays for detecting GMO must be quantitative, sensitive and
reproducible. Immunoassays are fast and inexpensive, but not
quantitative. Common PCR and its gel-based analysis is only
semi-quantitative and bares a high risk of cross-contamination.
Real-time quantitative PCR produces quantitative results in
a closed tube assay, which avoids crossover contamination.
The use of fluorescent TaqMan probes adds an extra level of
specificity to the PCR. An internal positive control labelled with
a different fluorescent dye controls for false-negative results.
TaqMan GMO Detection Kits for soy and maize from
Applied Biosystems use a universal target to detect the
presence of the transcriptional regulatory elements common
to most GMOs, which express the desired traits such as
resistance to insects and herbicides. This transcriptional
regulator is the 35S promoter of the cauliflower mosaic virus.
Figure 1. Amplification plot of processed foods purchased
from a local supermarket and tested with the TaqMan GMO
Soy 35S Detection Kit run on an ABI PRISM ® 7700
Sequence Detection System.
A) Soy-based infant formula, 50% GMO content (yellow)
B) Energy bar, 8% GMO content (green)
C) Soy-based drink, GMO-negative (red)
A plant-specific endogenous control has two advantages:
it allows a relative quantitation of GMO soy or GMO maize in
the sample, and no amplification of the control indicates the
presence of PCR inhibitors. The probe for the endogenous
control is labelled with VIC ® dye, so the 35S target and the
control can be detected simultaneously in a multiplex reaction.
Figure 2. Amplification plot of processed foods purchased
from a local supermarket and tested with the TaqMan GMO
Maize 35S Detection Kit run on an ABI PRISM ® 7000
Sequence Detection System.
A) Puppy food, 50% GMO content (green)
B) Corn snack, 5% GMO content (red)
C) Blue corn chips, GMO-negative (blue)
A different source of public concern is contamination by
food-borne pathogens. Salmonella is the second most
frequent cause of food-borne illness in Europe and the
United States. According to the World Health Organisation
(WHO), diseases caused by the major pathogens alone are
estimated to cost up to US $35 billion annually (1997) in
medical costs and lost productivity in the USA alone.
Applied Biosystems recently released the new TaqMan ®
Salmonella Gold Detection Kit. Now a complete line of
assays for food testing is available, including kits for detection
of Salmonella, E. coli O157:H7 and E. coli STX-1/2 (Shiga-like
toxin; VT1 and VT2).
The TaqMan Salmonella Gold Kit detects all serotypes
of Salmonella while closely related non-Salmonella bacteria
test negatively. Hot-start with AmpliTaq Gold ® DNA Polymerase
and the TaqMan probe contribute to the high level of
specificity. After pre-enrichment, 10-100 cfu per sample can be
detected reproducibly. Salmonella-specific and internal positive
control assays use FAM ® and VIC reporter dyes. They are run
as duplex 5' nuclease assays, which can be analysed
and detected on the ABI PRISM ® 7000, 7700, and 7900HT
Sequence Detection Systems.
Like the GMO Kits, the Salmonella Detection Kit provides positive
and negative controls. In addition, the GMO and Food Pathogen
Kits include a label licence, which conveys certain PCR service
rights when used with an authorised thermal cycler.
For more information on:
TaqMan assays for food testing enter:
No. 424
TaqMan assays for food pathogen detection enter: No. 425
35

36
promotions
Free Subscription!
To the Award-Winning Online Magazine from Applied Biosystems
B
ioBeat ® Online Magazine (www.biobeat.com) covers life
science research around the world that is enabled by
technology from Applied Biosystems. This award-winning
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please contact BioBeat editor Mike O’Neill at:
oneillmd@appliedbiosystems.com
T
he Gene Expression package from Applied Biosystems takes
your experiments from RNA sample preparation to successful
real-time quantitative PCR with TaqMan ® MGB probes.
The Gene Expression package includes:
ABI PRISM 6100 Nucleic Acid PrepStation
➜ For highly reproducible preparation of high yield,
high purity RNA from cultured cells, plant and
animal tissue and whole blood.
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detection for multiplex quantitation
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Expression studies, including
• TaqMan ® Universal PCR Master Mix
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components ensures robust real-time PCR reactions
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For more information on:
ABI PRISM 6100 Nucleic Acid PrepStation enter: No. 426 Gene Expression Package enter: No. 428
ABI PRISM 7000 Sequence Detection System enter: No. 427 Real-Time PCR Information Pack enter: No. 429
37

38
promotions
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Europe online @
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isit our new European website and gain access to
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39

40
customer relations
SQL*LIMS Installed to
Streamline Product Release
Baxter BioScience, Neuchatel, Switzerland
T
he demands on pharmaceutical companies for
quality assurance based on accurate information,
from both the public and the regulators, are increasing.
At the same time, market competitiveness is becoming
ever more cut-throat, so it is more vital than ever that
products of verifiable quality be brought to market in
the shortest practicable time. This means that product
development and its associated information handling must
be as streamlined as possible. A drug under development
spends its life in the laboratory and therefore increasing
the productivity of laboratories is a high priority for the
pharmaceutical industry. The ability to capture, process and
disseminate information electronically has radically changed
the efficiency with which laboratories operate.
A key step towards improving productivity is the
implementation of a laboratory information management
system (LIMS). Deciding which system to implement is a
crucial decision for any manufacturing site, especially one
being built from scratch – a fact not lost on Alexandre Boillat,
the IS manager of Baxter BioScience’s new multi-product
biosciences facility in Neuchatel, Switzerland. The objective of
the Neuchatel site is to significantly reduce time-to-market for
Baxter BioScience’s next-generation therapies.
The Baxter BioScience division is responsible for developing
and producing therapeutic proteins, from plasma and
through recombinant methods, to treat haemophilia, immune
deficiencies and other blood-related disorders. Its portfolio of
therapies includes coagulation factors, immunoglobulins,
albumin, wound management products and vaccines.
Construction work at the $131 million Neuchatel facility
began in 1998 and production began on the site’s first product
- a recombinant protein-free manufactured Factor VIII therapy
for haemophilia - in late 2000. More products are scheduled
to come on line over the next few years, with the workforce
growing to around 400.
“We took great care in deciding our strategy,” Alexandre Boillat
explained. “We were looking around to see what systems were
available to help and support us. It was important that we got
the right one.”
From Left: Alexandre Boillat, IS Manager and Pascal Schneider,
project leader of the LIMS implementation, Baxter BioScience,
Neuchatel, Switzerland
Pascal Schneider, project leader of the LIMS implementation
at Neuchatel, knew that Baxter was keen to achieve economies
of scale and minimise costs by standardising systems across
the corporation. “Baxter chose the SQL*LIMS software from
Applied Biosystems as its corporate standard and we stayed
with that choice,” he said. The Neuchatel site was one of the
first in the world to implement SQL*LIMS v4.0.
At Neuchatel, the LIMS implementation team began
by focussing on a key utility – water. “Starting from this
point we developed a clear project vision for our LIMS,”
said Alexandre Boillat. Emphasising that a manufacturing
site’s water supply must be under tight control, he explained:
“In the pharmaceutical industry you have a lot of things
to do for the validation of products and there is a lot of
data. For example, water is a critical supply for us and
monitoring water treatment is a part of quality assurance.
The quality assurance people need accurate information on
this, among other things, to take the decision whether or not to
release a product. In fact, obtaining good information is a
critical part of the product release process.”
“We also asked ourselves how it would fit with other systems,”
he went on, “and how many stages would be necessary
to implement it. Finally, we also had to be sure that a
computerised system would be better than a paper-based one.”
Baxter BioScience, Neuchatel, Switzerland
Rule 21 CFR part 11
As LIMS begin to offer all the advantages of the latest
computer systems’ capabilities, so the paperless laboratory gets
closer and closer. Under SQL*LIMS, laboratory methodology is
built up using easy-to-use forms. From a single screen,
the operator can define test requirements such as which
components to assay, which calculations to perform on results,
whether to calculate the cost of testing, or which analyst or
instrument to assign.
Part of the FDA’s Rule 21 CFR part 11 rule requires the
implementation of a number of security features to control and
monitor an individual’s access to data. LIMS can meet these
requirements through password verification and by providing a
modular electronic signature option that works with any form of
signature, including biometrices.
At Neuchatel, the LIMS implementation team persuaded Baxter
to let them purchase the latest version of SQL*LIMS, v4.0.
Pascal Schneider said: “It’s a big improvement over previous
versions, compliant with the FDA’s regulations and that
was what we needed.”
Implementation
Having decided which system to purchase, the next step was
to set up a team to run the project. According to Pascal
Schneider, there were three very important considerations.
“Firstly, identifying the resources needed and then defining
roles and responsibilities. The third consideration, training,
was highly important. We wanted to transfer as much
as possible SQL*LIMS knowledge to the project team.
All the team members were involved in a week’s training.
At first, some of the laboratory people had difficulty in
grasping the unfamiliar jargon. It was very important for us to
have everybody involved in training, so they could pick up
customer relations
the terminology, as well as learn how to use the system and
how it was configured. It meant that everybody had the
same understanding of the product from the beginning.
Following a short period of time where we put the system
through its paces, we started to define the specifications
we needed. Significant efforts were made at this point
to ensure that all validation was performed within the
regulated environment requirements.” Alexandre Boillat added:
“From the local point of view, the day-to-day implementation of
the system and the support were very good. We were very happy
with the support from Applied Biosystems in Switzerland.”
Strong systems, strong support
With the system having been successfully implemented
for Neuchatel’s plant, attention is now being turned
to environmental monitoring. Over the next few years,
LIMS will also be used to coordinate in-process testing and
final product testing. “We now have to roll out the other phases.
Our policy for IS projects is always to involve end users.
They strongly participate along the way and then, when the
development of a system is complete, there are no surprises.”
Alexandre Boillat said. “Our project vision should be
implemented with the LIMS within three or four years.”
The procedure should be quicker next time around.
“We had limited knowledge of LIMS for phase one,
although our project organisation and team structure were
good. Now that we have some experience, it will be easier to
conduct the next phase.” Recognising the need to have a
flexible informatics solution to cope with an ever-increasing
volume of data, Alexandre Boillat added: “One day we will
have to see how SQL*LIMS will be interfaced with our
laboratory instrumentation.”
To help support customers like Alexandre Boillat,
Applied Biosystems has a professional services organisation
that provides Rapid Integration Solutions. “In the software
industry it is hard to find an application that is bug-free,”
explained Alexandre. “For us another challenge was to
receive appropriate support from the supplier to make sure
that the bugs are recognised and that they will be corrected.
We did not want to customise our system and have to deal
with these problems alone.”
For more information on:
SQL*LIMS Software enter: No. 430
41

42
customer relations
GENUINE
SERVICE CONTRACTS
Servicing the Global Oligonucleotide
Operations (GOO)
Kingsland Grange, Warrington, UK
T
he occasional clanking and hissing of the waste
valves, and the constant whining of the vacuum
pumps drowning out the faint clicking of delivery valves
is a familiar sound in the high-throughput laboratory at
the GOO facility in Warrington, where rows of DNA
Synthesisers fill the room.
The GOO Facility
Over the last twelve months worldwide demand for customoligonucleotides
has increased, causing a three-fold growth of
the facility at Kingsland Grange and this site has become a
key account for the UK Service Department. GOO has taken
delivery of twenty-nine ABI 3900 High-throughput DNA
Synthesizers since February 2001. They already had fourteen
synthesizers, which placed significant demand on Field Service
resources in the UK.
In April 2001 we reviewed the provision of service for this site;
GOO placed service calls, like any other customer, through the
service administration department. Although the service
provided was very good, and compared favourably with industry
standards, it still could not meet the demands of this busy
manufacturing site. The service department made a guarantee
to have a Service Engineer on site all day from Monday to
Friday, providing service that is second to none, but having a
Field Engineer based on site permanently took its toll on the
rest of the field based resource in the UK.
In June 2001 it was decided to recruit an In-house Service
Engineer whose role it is to ensure the smooth running of the
DNA Synthesizers in all three of the Oligo Labs at the
Kingsland Grange site. This solution enabled us to provide a
quick response to breakdowns and ensure that all planned
maintenance checks are carried out promptly, and at a
time convenient to the customer. We are now providing an
instrument up-time of about 95% and most breakdowns are
repaired within one day, which is vital for the production
requirements of this site. This is achieved with support from
the members of the Field Service and Application teams who
provide assistance when necessary.
This key site also has its own Call Tracking Database,
which provides the users with the ability to log a call as soon
as a breakdown occurs. Once a call has been logged, an email
is sent to a Service Administrator at Applied Biosystems,
Lingley House in Warrington, who is dedicated to co-ordinating
all service calls for the UK key service accounts. Service calls
show up on the UK Oligo Engineers’ PC within seconds of
being logged so the engineer can then react immediately.
Paul A Johnson, Service Engineer at work
The Call Tracking Database provides a comprehensive
maintenance history for all the instruments, which is vital for
visiting engineers, and is also a good source of information for
Production Team Leaders and Operators at GOO, who have
access to the database at all times. This information is vital for
checking compliance with ISO standards and analysis of
production records.
You can see what has been achieved in a relatively short space
of time to enhance the level of service delivery to a Key
Account site. As the demand for oligos steadily increases,
the importance of optimum instrument performance will
always be a major priority. As the site expands so will
the demand on the Service Department. This is something
Applied Biosystems constantly reviews to ensure we meet
our customer’s expectations, both now and in the future!
Paul A Johnson, Service Engineer, GOO, Warrington, UK
Proteomics Research Center
Embraces RIS Implementation
European Proteomics Support Center, Langen (Frankfurt, Germany)
R
ecently, a vast number of proteomics projects
have been funded in various European countries.
The success of most of these projects relies on
the integration of traditional techniques like twodimensional
gel electrophoresis, chromatography
and Edman sequencing with new technologies to
enable high-throughput protein identification as
well as protein quantitation and characterisation.
A Rapid Integration Solutions (RIS) program that combines
Laboratory Information Management Software (LIMS),
and services to automate laboratory procedures integrates
proteomics tools and workflows into a center for proteomics
innovations and discoveries.
Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF optics
Resources of the Proteomics Support Center
Many of the new techniques and tools that ushered in the
proteomics era – ICAT Reagent Technology, MALDI-TOF mass
spectrometry systems, and the human genome database – are
resources of the Proteomics Support Center demonstration lab.
Volker Kruft Manager at the German Mass Spectrometry
Proteomics Support Center in Langen, Germany explains
that “the full suite of Applied Biosystems' proteomics
instrumentation can be tested during our customer
demonstrations.” The state-of-the-art instruments, which are
installed in the demonstration facility, include – the 4700
Proteomics Analyzer, the Voyager-DE PRO workstation,
Voyager-DE STR MALDI-TOF mass spectrometers, ESI and
oMALDI QSTAR ® Pulsar Systems, as well as biochromatography,
Procise ® sequencer and peptide synthesizers.
customer relations
Managing Proteomics Process Information
In Proteomics labs today, researchers need an automated
information management system, not only because they must
process large numbers of samples, but because they must
be able to interpret and organise the vast volumes of data
that can be generated from a single protein sample.
According to Dr. Armin Graber, Bioinformatics Manager at the
Proteomics Research Center (PRC) in USA, “Even a simple
proteomics experiment can yield up to 10,000 data
points that need to be tracked, analysed, and stored in a
retrievable database.”
The LIMS components of a RIS implementation track
and manage basic lab operations. However, for an RIS
implementation, an Applied Biosystems professional services
team configures the system to suit the internal processes of
each customer. The team then integrates any third-party
systems used in the lab with Applied Biosystems tools and
technologies to provide a seamless system tailored to the
research needs of the customer. “The way RIS works depends
on the questions researchers ask and how they approach a
project,” says Dr. Graber. “For example, someone may be
comparing spots from 2-D gels to study protein expression
levels under different conditions. In that case, the RIS team
can tailor the system to recognise similar spots on each
gel that differ in size, so that it uses mass spectra data to
identify proteins represented by the spots and annotates the
2-D gels accordingly.”
Come and Visit the Proteomics Demonstration Centers
Together, the Proteomics Demonstration Centers and RIS
represent a cornerstone of Applied Biosystems' investment in
proteomics. A tour of the PRC can be arranged by contacting
an Applied Biosystems' Marketing Representative, or by taking
a virtual tour of the lab on the Applied Biosystems' Website
http://www.appliedbiosystems.com/apps/proteomics/prc/nav.swf
For more information on:
The Proteomics Research Center enter: No. 431
43

44
customer relations
German Society of Mass Spectrometry
Grants ‘Life Science Award’
to Prof. Jasna Peter-Katalinic, for Scientific Excellence
in Mass Spectrometry
F
or the first time, the German Society of
Mass Spectrometry (Deutsche Gesellschaft für
Massenspektrometrie, DGMS) has granted the newly
established ‘Life Science Award’ for Scientific
Excellence in Mass Spectrometry to a researcher.
The first awardee is Prof. Jasna Peter-Katalinic,
University of Muenster, where she heads the
Biomedical Analysis Group at the Institute of
Medical Physics and Biophysics.
A 5000 Euro prize was donated by Applied Biosystems
Germany, which was open to researchers all over Europe.
The award honours scientific work from all areas of
methodology development and applications of mass
spectrometry within the life sciences. Prof. Peter-Katalinic
was honoured in appreciation of her outstanding work in
the field of the structural analysis of glycoconjugates.
left to right: Prof. Dr. Michael Przybylski, Mrs Prof. Jasna Peter-Katalinic
and Dr. Holm Sommer
The prize was awarded with a certificate at a special award
presentation during the conference of the German Society of
Mass Spectrometry (DGMS) on 5 March 2002 in Heidelberg.
The chairman of the DGMS, Prof. Dr. Jürgen Grotemeyer,
said he was proud to award this prize for the first time.
The prize is awarded exclusively through nomination by a
scientific jury appointed by the Board of the German Society of
Mass Spectrometry. Prof. Dr. Michael Przybylski, chairman of
the nominating committee, gave the awarding speech.
In his speech, he honoured Prof. Peter-Katalinic for having
built up one of the best known international groups working in
the field of biological mass spectrometry for glycoconjugate
analysis and glycomics. She has published more than
150 articles in peer-reviewed, high impact factor journals,
and 200 presentations at scientific meetings. She is serving
as a reviewer for several scientific journals in her field.
Her research projects have been funded by the Deutsche
Forschungsgemeinschaft (DFG) and the German Federal
Ministry of Research and Education (BMBF).
In the main areas of her research she is focussing on:
➜ New instrumentation methods of mass spectrometry
to tackle the most challenging analytical questions,
e.g. with quadrupole time-of-flight and with highresolution
Fourier-transform ion cyclotron resonance
mass spectrometry
➜ Novel strategies for structure elucidation of complex
N- and O-glycosylation in proteins, which have proven
difficult to analyse with common methods
➜ Applying these strategies for elucidation of the role of
glycoconjugates in the field of molecular medicine,
e.g. in the etiology of genetic diseases.
Following this introduction, Prof. Peter-Katalinic herself
presented ‘Glycomics in the Post-Genome Era and the way
to get it: 20 Years Glycoanalysis’. She described her early
work on oligosaccharide determinants of blood groups,
and on gangliosides in the development of the human brain,
both of high biological relevance. In her more recent work,
applying a broad range of glycoanalytical methods,
she both contributed to clinical research, where the differential
expression of glycoconjugates in normal and diseased
specimens may be used as a diagnostic tool, and to basic
customer relations
Kiel University Hospital Collaborates
with Applied Biosystems
A
research where she investigates protein – carbohydrate
interactions. Amazingly enough, there are still novel
glycosylation types in proteins to be detected.
Apart from her position as a professor in Muenster,
Germany, she is involved in establishing a new laboratory for
biological mass spectrometry and proteomics at the Institute of
Molecular Medicine and Genetics, Medical College of Georgia,
Augusta, USA. At both locations, she applies a broad range of
analytical methods using different instrumentation.
When asked for her first response when she was informed about
the prize, Prof. Peter-Katalinic said that she was surprised,
pplied Biosystems Group, an Applera Corporation
business, and the University Hospital in Kiel in
Germany, have recently announced a collaborative
study on high-throughput genotyping led by Dr. Stefan
Schreiber, Professor of Medicine and Gastroenterology.
Up to 2,000 single nucleotide polymorphisms (SNPs) in
approximately 5,000 individuals will be investigated in the
course of the study using the local high-throughput SNP
genotyping infrastructure to identify if any have associations
with chronic intestinal inflammation, in particular Crohn’s
disease and ulcerative colitis.
In the past, Professor Schreiber’s research group has
examined a large number of genes for disease associations on
various chromosomes and was among those who described
the first Crohn’s disease susceptibility gene, NOD2,
on chromosome 16. This new study, which will use an approach
called high-density association mapping, will attempt to define
further interacting disease genes in this polygenetic disorder.
The study has received considerable support from
the Federal Ministry for Education and Research (BMBF),
the German National Genome Network, and the German Crohns
and Colitis Foundation, who helped assemble the patient
database for the project.
but highly honoured. She admitted that she hadn’t yet had the
time to celebrate, but has promised to take her students out to
mark the occasion. At that time she spent another research
period at the Medical College of Georgia. Young scientist’s
education in the field of mass spectrometry in life sciences,
a dynamic and truly interdisciplinary field for physicists,
chemists and biologists is one of her major concerns.
Applied Biosystems would like to congratulate
Prof. Peter-Katalinic and wish her well with her future
career. We hope that the prize will contribute to her
future research success.
Professor Schreiber is one of the first users of
the recently introduced Assays-by-Design SM service from
Applied Biosystems, where individual assay kits for known
SNPs are designed and tested to order. This approach can save
time, money and manual labour and when used in combination
with the ABI PRISM ® 7900HT Sequence Detection System,
can help researchers analyse up to 250,000 genotypes
a day. As part of the Applera-wide discovery program,
Applied Biosystems is dedicated to providing scientists
with the tools they need for high-throughput, low-cost SNP
genotyping and gene expression research.
For more information on:
The Assays-by-Design service enter:
No. 432
or see: http://home.appliedbiosystems.com/press
(Press release dated October 25, 2001)
or visit: www.appliedbiosystems.com/assaysbydesign
45

46
customer relations
Enabling Discovery:
Genotyping in the Genomic Era
European Seminar Series
Enabling adj. Rendering possible by supplying the means
Discovery n. Information learned for the first time
Genotyping Solutions for linkage & association studies
Applied Biosystems’ Enabling Discovery European Seminar
Series took place on nine dates between 25th February and 8th
March, 2002. The series of one-day events began in Munich
and subsequently visited Berlin, Rotterdam, Madrid, London,
Copenhagen, Stockholm, Paris and Naples. Experts from
Applied Biosystems presented our innovative genotyping
solutions for linkage mapping and association studies.
From microsatellite analysis to SNP discovery, validation
& scoring, Applied Biosystems provides a range of high quality
tools and expertise for the diverse customer needs.
Distinguished scientists from across Europe were invited to
present their research and discuss how genotyping Solutions
from Applied Biosystems are employed in their laboratories.
Delivering the genome
Scientific discovery continues to require a scientist with a
hypothesis to conduct experiments. At Applied Biosystems,
we are rising to the challenge of mining the vast amounts
of human genome data to bring our customers useful
information and robust tools to enable the next phase of
discovery. The Genome Initiative was first announced by
Applera Corporation in July 2001. This initiative combines
data from Celera Genomics and public programs with
Applied Biosystems' long history of technology innovation and
expertise. The Enabling Discovery seminar series provided the
forum to present the goals of this ambitious initiative to our
European customers for the very first time. Through this
initiative, the resulting Genomic Assay will enable our
customers to engage in genotyping studies at a previously
undreamed of ease, scale, speed and value.
Highlights from our customers
It would be impossible to capture in detail each of the
interesting customer presentations. Here, we briefly summarise
three presentations given by our invited speakers.
Dr. Andreas Koch, a post-doctoral fellow in the laboratory of
Dr. Stefan Schreiber at Christian-Albrecht’s University, Kiel,
Germany presented data from candidate gene studies and fine
mapping of genes related to Inflammatory Bowel Disease.
As one of the three National Genome Research Network
(NGFN) laboratories, they have chosen the ABI PRISM ® 7900HT
Sequence Detection System and Assays-by-Design sm Service
as the technology solution to provide high-throughput
genotyping services to the research community in Germany.
In January, using these new tools, they completed a study of
130 SNPs on a cohort of samples from 2,500 patients.
The Rotterdam Study was described by Dr. Andre Uitterlinden,
Head of the Genomic Laboratory, Department of Internal
Medicine at Erasmus University, Rotterdam, Netherlands.
Started in 1991, this prospective population-based cohort
study consists of samples from >7,000 patients selected for
the research of the determinants of chronic and disabling
diseases in the elderly. Dr. Uitterlinden’s interests are in
endocrine diseases, and more specifically in the genetics of
osteoporosis. He presented methods for SNP genotyping
of candidate genes, showing data from their application of
SNaPshot ® Multiplex Kit technology on the ABI PRISM ® 3100
Genetic Analyzer.
For more than 6 years, the MRC HGMP Resource Center
in Cambridge, United Kingdom has carried out whole
genome microsatellite-based mapping for the UK academic
community. Dr. Tom Weaver, Head of Biology Services,
outlined a collaborative project between HGMP and a
research group at Hammersmith Hospital in London,
which resulted in the elucidation and publication of a
disease gene for Dyskeratosis Congenita (DC).
He went on to describe a series of technology evaluations and
their testing of virtually all SNP genotyping products on the
market. He concluded by describing the reasons that they have
selected 5' Nuclease assay with TaqMan ® MGB probes as
their method of choice for high-throughput SNP genotyping.
He cited five critical factors which gave TaqMan reagents
the ‘winning’ score:
1) Homogeneous single tube assay
2) High-throughput
3) 100% accuracy across their evaluation sample set
4) Low cost when employed for association studies
5) Simple implementation
Enabling the next phases
The characterisation of genetic predisposition to complex
diseases is one of the greatest challenges of the genomic
era. Genetic susceptibility is a composite of several genes,
each contributing only a small risk, thus rendering the
identification of susceptibility genes a time-consuming and
expensive task. Association studies will involve populations much
larger than traditional Applied Biosystems' technologies could
deliver. The Genomic Assays products will enable the next
phase in genomic discovery. Now, it is possible to eliminate the
risks and reduce the costs and time that are associated with
design, optimisation and assay failure. By using our validated
Human SNP Assays-on-Demand products and supplementing,
Customer Training Courses
Call Eve Lightfoot 01925 282551 or Josephine Kinsey 01925 282475
Basic PCR - Theory Optimisation & Applications
customer relations
if necessary, with our Assays-by-Design service, you can
implement the same, simple, single-step SNP genotyping
workflow in any laboratory.
Enabling Discovery, we deliver the tools, so that you can
deliver the science.
See related articles on pages 13 & 29
For more information on:
Assays-on-Demand Products enter:
No. 433
Assays-by-Design Service enter: No. 434
DNA Sequencing - Practical Troubleshooting 12-13 September
ABI PRISM ® 3100 Genetic Analyzer 01-02 & 29-30 August 24-25 September 22-23 October
ABI PRISM ® 310 Genetic Analyzer User Training 21-22 August 09-10 October
ABI PRISM ® 7700 Sequence Detection System User Training 01-02 August 03-04 September 01-02 October
ABI PRISM ® 7000 Sequence Detection System User Training 06-07 August 10-11 September 15-16 October
ABI PRISM ® 7900 Sequence Detection System User Training 08-09 August 19-20 September 24-25 October
(Training Courses held in The Netherlands, in English)
Informatics v4 System Manager
Informatics Transition Training 07-10 October
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Informatics v4 Key Personnel 09-13 September No. 444
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