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Q TRAP™ LC/MS/MS SystemMore metabolites, more proteins,more confidence in your results

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Biosystems SolutionsE d i t o r i a lApplied Biosystems, Lingley House, 120 Birchwood Boulevard, Warrington, Cheshire WA3 7QH, UK.Tel: +44 (0)1925 825650 Fax: +44 (0)1925 282502 email: [email protected]

E d i t o rKay 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 sPeter 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 sApplera 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, SNaPshot, API QSTAR, QSTAR, GeneScan, Analyst, Genotyper, BioBeat, FAM, VIC, POROS, Sequence Navigator and Gal-Screen are registered trademarks and Assays-by-Design is a Service mark of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

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 partnershipbetween 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. 6,342,345Certain aspects of the technology described herein are covered under current or pending US and/or international patents.

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Information subject to change without notice.No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any other means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the copyright holder. Copyright rests with the publisher.

©2002 Applied Biosystems. All rights reserved. Printed in the UK 06/02

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3

contentscover story - 05

Q TRAP™ LC/MS/MS System More metabolites, more proteins,

more confidence in your results

customer relations

40-48SQL*LIMS Installed to Streamline Product ReleaseGlobal Oligonucleotide OperationsProteomics Research Center Embraces RIS ImplementationGerman Society Grants ‘Life Science Award’Collaborative Study on High-throughput GenotypingGenotyping in the Genomic EraTraining Dates

18Pro ICAT SoftwareAutomatic Interpretation of Proteomic Data

243100-Avant SystemIntroducing a New Genetic Analyzer

38Europe OnlineVisit the New European Website

technical communications

07-17 The BlastMachine™ System The InteraX™ SystemChoosing the Right Target!Revolution in 5' Nuclease Assay15 Years in a Thin Glass TubeAnalysis of Gene Expression Pattern in Asthma Research

prot

eom

ics

Maybe you do it by unlocking an insight about your research when you least expect it.

Toward a discovery.

A new drug target or pathway. Even a cure.

You’ve just got to find the order in the chaos.> If you work in proteomics,

you know complexity firsthand.

So your work starts to take on a new direction.

Your next proteomics discovery awaits.Applied Biosystems, the company that enabled the sequencing of the humangenome, 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 breakthroughslike 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

The Discovery Momentnew product review

18-35Q TRAP LC/MS/MS System for Protein AnalysisSQL*LIMS™ 4.1 SoftwarePrepExpress NT SoftwareAffinity Depletion CartridgesMoving Forward in Genomic Research...Improved Software Tools for Data AnalysisPhotoSpray™ SourceTaqMan® Assays for Food Testing

promotions

36-39Free Subscription to BioBeat!Start off your Gene Expression Studies!

08A Sequence to Die For!Fugu Genome DraftAnnounced

ne of the first steps in the investigation of thebiotransformation 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

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.

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page 6

Q TRAP™ LC/MS/MS SystemMore Metabolites, More Proteins, More Confidence in Your ResultsProf. Gérard Hopfgartner, University of Geneva, Switzerland

54

n April 23, 2002 Applied Biosystems announcedthe introduction of the Applied Biosystems 3730

and 3730xl DNA Analyzers. These next generationproduction scale systems are expected to improvedata quality and increase productivity by a factor of two or more compared to current technologyplatforms. They bring powerful new tools for rapid,accurate, and cost-effective DNA analysis todiscovery researchers worldwide studying human and other genomes.

The addition of the 3730 and 3730xl DNA Analyzersto the Applied Biosystems existing portfolio ofindustry-leading DNA analyzers provides researcherswith a wider range of platforms to address theirthroughput and application needs. The 3730analyzer, a 48-capillary electrophoresis system,combines the core technology of the highly successfulABI PRISM® 3100 Genetic Analyzer with new advancesin automation and optics, to meet the needs of higherthroughput individual researchers and core facilities.The 3730 platform can also be upgraded to the3730xl DNA Analyzer, the 96-capillary successor tothe ABI PRISM® 3700 DNA Analyzer.

While researchers and production facilities areexpected to primarily use the 3730xl DNA Analyzerfor production sequencing – both de novo andresequencing – the 3730 DNA Analyzer is expectedto be used for a wider range of sequencing andfragment analysis applications including de novo andcomparative sequencing and genotyping. The newsystems run both ABI PRISM® BigDye® Terminatorchemistries for sequencing and five-dye chemistriesfor fragment analysis, as do all Applied BiosystemsDNA analyzers.

Throughput, Automation, Efficiency ImprovementsThe 3730 and 3730xl DNA Analyzers can reduce sequencing project costs and increaseefficiency by providing longer read lengths, enhanced instrument sensitivity and efficiency and the highest 24-hour unattended capacity of anyDNA analyzer. Sequence read lengths of greater than 1,000 base pairs and improved basecallingcould reduce the number of samples needed to complete de novo genome sequencing andresequencing projects by 20-40% respectively.

Applied Biosystems: Pioneers in Genetic Analysis SystemsBoth new systems are part of the pioneering line ofcapillary electrophoresis instruments for DNAanalysis from Applied Biosystems. The ABI PRISM®

310 Genetic Analyzer, the first commerciallyavailable capillary sequencer, was introduced in1995. That was followed in 1998 by the introductionof the 96-capillary ABI PRISM® 3700 DNA Analyzer.Government and commercial researchers in theUnited States, Europe and Asia used the 3700 system to sequence the human genome.Researchers worldwide currently use the 3700system for the industrial-scale analysis of genomicinformation. The 16-capillary ABI PRISM® 3100Genetic Analyzer, introduced in 2000 was designed to provide the flexibility of the 310 system withadded throughput for medium-to-high throughputlaboratories. The ABI PRISM® 3100-Avant GeneticAnalyzer, introduced in April 2002, is a four-capillaryelectrophoresis system that can be upgraded to the16-capillary 3100 system (see page 14).

Applera Announces New Knowledge Business forCelera Discovery System (CDS)Applera Corporation announced earlier this year thatits Applied Biosystems Group will become theexclusive distributor of the Celera Discovery System™

(CDS) operated by its Celera Genomics Group andthat Applied Biosystems will integrate CDS and othergenomic and biological information into a newKnowledge Business.

For the future, Applied Biosystems has plans for its Knowledge Business to include genomic assaysand related content (see page 29), as well as otherinformation-rich products, services, and analyticaltools to meet the needs of its life sciences customers. The Knowledge Business products andservices will be marketed, in part, on an expandedinformation portal.

For more information on 3730/3730xl DNA Analyzers enter: No. 401

O

Introducing the Next Generation of Production Level DNA Analysis Systems

Corporate review

cover story

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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

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.

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

The BlastMachine™ Systemaracel’s BlastMachine system is a turnkey softwareand hardware solution running a Paracel-optimised

version of the NCBI BLAST algorithm on a pre-packagedLinux 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 multi-

processor 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 sequence-

search 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:Paracel products enter: No. 404

No. 403

POptimised BLAST System for Sequence Similarity Analysis

Figure 1.

Figure 2.

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technical communicationstechnical communications

8

A Sequence to Die For!

n October 26, 2001, at the Genome Sequencing andAnalysis Conference (GSAC) in San Diego, California,

an international team of investigators, includinglegendary molecular biologist Dr. Sydney Brenner,announced completion of a draft sequence of the Fugurubripes genome.*

Fugu, a pufferfish, is a delicacy in Japan, but it can be a riskymeal, as it contains a deadly neurotoxin, and if not properlyprepared, can prove fatal to the diner. The Fugu genome isparticularly interesting to scientists because it contains very littleof the so-called ‘junk’ DNA that lies between gene segments inother 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 apotentially more direct route to the identification of genes andthe analysis of gene function, by allowing them to avoid thecomplications of sifting through all the non-coding sequencethat 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 containapproximately the same number of genes as the much largerhuman genome (3,000 million DNA base pairs) i.e., the Fugugenome is more than eight times as gene-dense as the humangenome. In addition, the Fugu and the human genomes arethought to share very similar sets of genes.

O

Fugu Genome Draft Announcedby Michael D. O'Neill, BioBeat® Online Magazine (www.biobeat.com)

Dr. Sydney Brenner at the Gordon Conference on Human MolecularGenetics in Newport, Rhode Island, August 1999. (Photo by Michael D. O'Neill, BioBeat Online Magazine).

For these reasons, it is believed that sequence comparisonsbetween the Fugu genome and the human genome mayaccelerate the identification of human genes, because genes that can be relatively quickly identified in the gene-dense Fugugenome can be used to find the corresponding genes in the muchless gene-dense human genome. Looking for a gene in the humangenome can be likened to looking for a needle in a haystack.Looking for the corresponding gene in the Fugu genome mayallow 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 outon ABI PRISM® 3700 DNA Analyzers at Celera Genomics. In addition, sequencing of cosmids from Fugu genomic librarieswas carried out at the Institute of Molecular and Cell Biology(IMCB) in Singapore, which presently has two ABI PRISM 3700DNA Analyzers and two ABI PRISM 377 DNA Sequencers.

IMCB Sequencing Leader Lauds Performance of Applied Biosystems SequencersDr. Alice Tay, leader of the DNA Sequencing & Analysis Facility at the IMCB, said, “We are very pleased with theperformance 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 obtainat 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 thissequencing project, as well as on other products from Applied Biosystems, can be obtained in the Documents onDemand section (http://docs.appliedbiosystems.com/search.taf)of the Applied Biosystems web site: www.appliedbiosystems.com

Collaborating InstitutionsThe bulk of the Fugu genome sequencing project was carried out at the United States Department of Energy’s Joint GenomeInstitute (JGI). In addition to the JGI, Celera Genomics, and the IMCB, other institutions collaborating on the Fugugenome sequencing project included the Singapore BiomedicalResearch Council, the Medical Research Council (UK), the Cambridge University Department of Oncology, the Institutefor Systems Biology, and Myriad Genetics.

Annotated Version of Fugu Article Available in Applied Biosystems’ Online MagazineA 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 accessedat www.biobeat.com. BioBeat Online Magazine covers lifescience research advances made around the globe and presently includes over 120 richly annotated articles.

Free subscriptions to BioBeat Online Magazine can be obtainedby completing the brief online subscription form atwww.appliedbiosystems.com/biobeat/subscribe.cfm. In additionto email updates of new story postings, BioBeat subscribersreceive periodic emailings of BioBeat’s popular Journal Watchand 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

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)

Dr. Alice Tay, leader of the DNA Sequencing & Analysis Facility at theInstitute of Molecular and Cell Biology (IMCB) in Singapore. (Image courtesy of Dr. Tay).

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 toinclude, informative, well balanced and of general interest.We would welcome your proposals and suggestions forfuture articles on the advancement of life science researchthroughout Europe.

If you want to comment on the content of previous issues ofBiosystems Solutions or request to see specific topics

included in future issues then please contact us by [email protected] using ‘BS comment’as your email subject.

Finally, to continue to keep you informed it is important that our records of your contact details are as up-to-date as possible. So if you have recently changed your address,or are about to, please complete the postage free reply cardin this magazine and send it back to us. Or alternativelyemail us at the above address.

RYour feedback on Biosystems Solutions

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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.

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

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.

References1. 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 20002. 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

The InteraX™ SystemMonitoring Protein-Protein Interactions in Different Compartments of the Mammalian Cell

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better understanding of the specific interactions ofproteins with each other, under physiologically

relevant conditions, is extremely helpful in elucidatingnew drug targets and the discovery of new cell signalling pathways. This in turn may reveal new actionpoints for new drugs correcting molecular disfunctions.

The InteraX system allows the direct detection of in vivo protein-

protein 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).

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.

A

Figure 1.

Probing Protein-Protein Interaction ß-Galactosidase

Mutant Complementation

add substrate

A and Bdo not interact

A and Bdo interact

A

A

A

AB

B B

B

add substrate

NoLight

Figure 2.

Functional Receptor Binding Assay: Monitoring EGFR Dimerization

rapamycinrapamycin

rapamycinrapaa ycapa

FRAP

FRAP

FKBP12

FKBP12

Cytosolicinteraction

Sign

al /

Noi

se

Par

enta

l

∆ωal

one

∆α/∆

ω

clon

e A

∆α/∆

ωcl

one

B

Med

ia

40

30

20

10

0

Figure 3.

Protein Induced Interaction Measured Using Gal-Screen® Assay.

ß-Gal complementation is demonstrated upon induction withrapamycin (10 ng/mL) in two clones of transfected cells (10,000 cells/well) co-expressing the fusion proteins ∆αFRAP and ∆ωFKBP12.

Figure 2. Genomic Assays from Applied Biosystems.Genomic assays are based upon 5' Nuclease assay using TaqManMGB probes. Assays-on-Demand products are ready-to-useHuman SNP and Gene expression assays. Assays-by-Design Service provides assays for customer-specifiedSNPs or genes of interest for any species.

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Choosing the Right Target!he first challenge for today’s pharmaceuticalcompanies is not so much hitting the target,

but choosing the right target in the first place. Over the last hundred years drugs have been designedagainst 400-500 disease targets.

The post genome era presents the industry an interestingdilemma – with the completion of the human genome thenumber of ‘druggable’ targets is expected to increasedramatically 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 beingused by pharmaceutical companies across the globe to morefully understand the molecular cause of disease. By studyingdisease mechanisms researchers are able to identify importantgenes and proteins, understanding how they influence andcontrol biological processes.

At Applied Biosystems we transformed gene discovery researchwith Automated DNA Analyzers that decipher entire genomes inmonths instead of years. These systems of choice for identifyingdisease-related mutations and correlating genetic markers with disease, are also the primary technology for revealing geneswith altered expression levels in disease. Our Gene ExpressionAnalysis Systems help to assign function to potential target genes and also provide novel assays for lead discovery andbiomarkers 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™ reagentsand software. Applied Biosystems Proteomics Research Centerand 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 thesetechnologies it is still necessary to make sense of thisinformation before deciding which targets to take forward intothe drug development process. Applied Biosystems hasdeveloped and refined informatics systems that allow theautomation and integration of genomic and proteomic systemsallowing 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. 409No. 408No. 407

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Revolution in 5' Nuclease Assay TaqMan® MGB Probes Deliver Simple and Robust SNP Genotyping!

etection of single nucleotide polymorphisms (SNPs)is now central to modern molecular genetics.

Large-scale population scoring of known SNPs requiresa 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 tounambiguously assign SNP genotypes in a flexible andscaleable format. Here’s how we’ve delivered on that vision…

Three factors contribute to allelic discrimination basedon a single mismatch (Figure 1).1 A mismatched probe has a lower Tm than a perfectly

matched probe. Shorter probes display greater mismatchdiscrimination because the single mismatch has a higherdisruptive effect on the hybridisation kinetics of the shorter probe.

2 The assay is performed with both probes present in thereaction tube. The mismatched probes are virtuallyprevented from binding to the target due to the stablebinding of the perfectly matched probes.

3 For efficient probe cleavage, the 5' end of the probe muststart to be displaced. Once a probe starts to be displaced,complete dissociation occurs faster with a mismatch thanwith an exact match. Thus, the presence of a mismatchpromotes dissociation rather than cleavage of the probe.

The minor groove binder (MGB) contributes a majorenhancement to the 5' Nuclease Assay. The addition of anMGB molecule to an oligonucleotide has been shown to

stabilise nucleic acid duplexes, causing a dramatic increase in the Tm 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 allelicdiscrimination assays and gene expression assays.

With a more robust assay, a new generation of products arebeing created using 5' Nuclease assay with TaqMan MGBprobes. Building on the efforts of both Celera Genomics andpublic sequencing programs, Applied Biosystems is generatingvalidated SNP assays that can easily be applied for geneticstudies. Using our bioinformatics pipeline for ‘genome-aided’assay design and our industrial scale production genotypinglab, 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-DesignSM 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.

For more information on:Assays-on-Demand Products enter:Assays-by-Design Service enter: No. 411

No. 410

D

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 indicateshomozygosity 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


15

Improvements in polymer design and the application of the laser excitation technique from Applied Biosystems, allied to a charge-coupled device camera for fluorescencedetection (as first used in the 377 system), resulted in the1995 release of the ABI PRISM® 310 Genetic Analyzer, the firstcommercial CE genetic analyzer. The initial model couldsequence 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-mediumthroughput laboratories. Within 2 years of its release,improvements came with new POP™ (Performance OptimisedPolymers) and new 50µM i.d. capillaries, allowing betterresolution and heat dissipation. These capillaries were unlined,utilising dynamic coating of the walls by the improved POPpolymers to achieve superior performance and greatly increasedservice lifetimes. Sequencing specification increased to readsover 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 tosupplement the earlier 48 tube arrangement.

In the medium-to-high capacity field, slab gel systemscontinued as the workhorses of the human genome sequencingproject, and major medical and forensic microsatellite basedtyping programmes. The 377 system finally reached 96 lanecapacity, but the demand for ultra high-throughput, rapid turn-around 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 ofkey patents for analytical CE, and by late 1998 released the

96 capillary ABI PRISM® 3700 DNA Analyzer for Genome scaleprojects. Capable of fully automated loading and running ofsamples from a series of 96- or 384-well microtitre plates, the 3700 system was a key factor driving the rapid sequencingof the human genome. Development of CE polymer chemistrycontinued 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 thisinstrument. This offered further increases in sequencing andfragment analysis throughput.

Another joint project with Hitachi Ltd., produced the ABI PRISM®

3100 Genetic Analyzer, a 16-capillary system designed formedium-to-high throughput laboratories and core facilities thatwere not so ‘production oriented’ that did not require thededicated capacity of the 3700 system. Based on proven 50µMcapillaries and POP polymers, the 3100 system has furtherincreased the performance of CE based DNA analysis, with sophisticated laser optics giving increased sensitivity andsignal to noise ratio. A range of capillary array lengths for thisinstrument has enabled extremely rapid Single NucleotidePolymorphism (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 on36cm 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-mediumthroughput laboratory all the versatility of the 3100 system atlower 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 andthe development of new applications.

The newest capillary electrophoresis instruments to beintroduced by Applied Biosystems, in collaboration with Hitachi High-Technologies Corporation, are the 3730xl and3730 DNA Analyzers. These instruments have been specificallydesigned to deliver improved production capacity and lowerrunning 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

14


n early publication on automated DNA sequencingraised an intriguing prospect: the move away from

denaturing polyacrylamide slab gels for sequence ladderseparation. The primary impact of this publication (L. Smithet al, 1986) was the use of fluorescent dye-labelledsequencing primers, together with a laser excitation andphotomultiplier detection system, to create a workableprototype for the automated, real-time reading of DNAsequences via Sanger dideoxy terminator chemistry.

However, the electrophoretic separation apparatus was alsounusual. A 50cm tube gel, of less than 2mm diameter, was chosen to maximise the sensitivity of that prototypesequence reader.

When it came to the production of a usable commercialinstrument, the throughput of a single tube gel was clearlyinadequate so designers turned to a scanning system utilisinga conventional, denaturing polyacrylamide slab gel (C. Conellet al, 1987). Development of automated DNA sequencing andDNA fragment analysis continued with slab gel-based systemsfor several years, but the attractions of capillary electrophoresis(CE) remained for separation.

Established as a rapid and sensitive technique for the analysisof small molecules such as peptides and oligonucleotides, CE offered fast run times, high resolution and very efficient heatdissipation from ultra-high voltage separations (up to 20 kV).

Applied Biosystems developed expertise in the field of analyticalCE with the design and production of the 270 instrument in thelate 1980’s. Flowable polymer solutions such as hydroxy-methylcellulose could be used to fill narrow bore capillaries forseparating analytes. Ultra-violet absorption detection determinedquantities and retention times of materials separated by high voltage electrophoresis. For the analysis of syntheticoligonucleotides, polymerised denaturing gel-filled capillarieswere used as the separation medium. These gave excellentresolution but offered relatively short lifetimes.

Thus, the major challenge in developing a functional CE-basedDNA 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.

A

15 Years in a Thin Glass TubeThe Development of Capillary Electrophoresis Technology for Genetic Analysis Applications

17

technical communicationstechnical communications

16

Real-Time PCR Analysis of GeneExpression Pattern in Asthma Research

he research of allergic reactions like asthma showsthat not one but many parameters are altered in this

disease. So far, analytical methods for quantitative RNAanalysis of many samples and the required screening-methods have not been available. With the help of Real-Time PCR even complex gene expression patternsincluding genes that are expressed only weakly can beanalysed quantitatively and automated. This techniquecan 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

T

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.

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

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.

ConclusionReal-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:Real-Time PCR information pack enter: No. 413

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.

Figure 2.

The results of the drug library screening are collected in adatabase. 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.

1111111111111111

A1A2A3A4A5A6A7A8A9

A10A11A12B1B2B3B4

0,000,821,49-0,511,78-0,08-0,580,270,00-0,59-1,070,000,00-1,27-1,81-0,82

0,000,470,97-0,691,07-1,10-0,63-2,340,000,060,500,000,00-1,59-1,71-0,38

0,001,39-0,180,09-0,821,101,20-1,40,00-1,40-0,790,000,00-1,09-0,49-1,33

0,000,73-0,350,30-0,38-0,85-1,19-3,10,001,39-0,180,000,001,101,20-0,96

Plate Well IL-2 IL-4 lL-5 lL-13 PatternMatch

Drug screening results: Pattern SearchingPattern definition : IL-2 > 0 IL-4,5,13 < -1

X

Th1/Th2-Paradigm

The ABI PRISM® 7900HT Sequence Detection System

OverviewTraditional mass spectrometry approaches for proteinidentification involve separating protein mixtures using 1-D or2-D gels, isolating the protein spots of interest, enzymaticallydigesting the protein into peptides, and analysing the peptidesby mass spectrometry. The recent development of the isotope-coded affinity tag technique1 has allowed the investigation oflarge numbers of proteins, using a multi-dimensional liquidchromatography approach (MDLC) in a fraction of the timetypically 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 combinedand digested. By taking advantage of a biotin affinity tag on the reagent, the peptides containing the modified cysteineresidues are selectively purified2.

Once purified, ICAT reagent-labelled cysteine-containingpeptides are separated by on-line capillary LC and analyseddirectly 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 thequantitation results from an LC/MS run. Thus, expressiondependent MS/MS analysis can be performed on only thosepeptides with ratios that meet user-defined limits.

ConclusionsA new software application is now available for processingproteomics data acquired from an API QSTAR Pulsar LC/MS/MS System, from samples prepared using the ICAT reagent technique. Pro ICAT software uses the Interrogatorsearch algorithm for identifying proteins from un-interpretedMS/MS data and the LC/MS Reconstruct algorithm forquantitating expression pairs from the MS data.

Users can define as many as five modifications in the proteinidentification search. Additionally, they can search for arbitrarymodifications up to a user-specified mass difference at run-time (zone modification feature). The LC/MS Reconstructalgorithm quantifies ICAT reagent expression pairs from MS data by clustering the data based upon ICAT reagentfragments and optimally collapsing adjacent spectra for amaximum signal-to-noise ratio. Users may also run expressiondependent LC/MS/MS IDA experiments based upon thequantitation results.

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.

References1. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. 1999. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotech., 17:994-999.2. For more information about the ICAT reagent technique, see the ICATreagent product bulletin “ICAT Reagents for Quantitative Protein ExpressionAnalysis 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 DependentExperiments for LC/MS/MS Analysis” (literature code: 114PB07-01).

For more information on:Pro ICAT Software enter:ICAT Reagents enter: No. 415

No. 414

ro ICAT is a software application that works inconjunction with BioAnalyst™ software. Its function

is to process LC/MS and LC/MS/MS InformationDependent Acquisition (IDA) data that has been acquiredfrom 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 algorithmperforms 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

For Automatic Interpretation of Proteomic Data

Pro ICAT Software Application

P

19

new product review

18

new product review

The New Q TRAP™ LC/MS/MS Systemfor Protein Analysis

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 thespecificity and robustness of triple quadrupole systems with thefull scan MS/MS sensitivity of ion trap systems into a singleinstrument. 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 chargedpeptide 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 andefficient 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 triplequadrupole and ion trap mass spectrometers while maintainingtriple quadrupole-like fragmentation. With signal to noiseimprovements greater than 100X, identification of proteindigests at femtomole levels can routinely be achieved withmaximum sequence coverage.

Enhanced ResolutionThe Q TRAP LC/MS/MS System provides superior resolutionacross the entire mass range, making charge state identificationmuch more reliable up to charge state 5. This capabilityimproves the ion selection process for complex mixtures andensures the appropriate determination of collision energy in

IDA mode. The Q TRAP LC/MS/MS System provides constant peakwidths (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 SelectionWhen maximum sample information is desired, it is extremelyimportant to optimise the data acquisition for ions that willgenerate the most useful information. For protein digests at lowlevels, detection of multiply charged ions is normally hinderedby the presence of background ions that are predominantlysingly charged ions and usually present at high concentration.The Q TRAP LC/MS/MS System offers a unique operating modewhere multiply charged ions are selectively detected over singlycharged ions. This mode of operation ensures easy andefficient detection of peptide ions in both nanospray andcapillary LC/MS mode, thus maximising the collection of usefulinformation in a minimum amount of time.

A

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 andMS/MS data in an automated fashion within a single injection,thus maximising both instrument usage and informationgathering. This can be achieved by combining modes ofoperation that yield very specific MS information such asprecursor ion scans or multiply charged scans for proteolyticdigests, with the enhanced full scan MS/MS mode of the Q TRAP LC/MS/MS System.

IDA provides enough flexibility to the user to combine scantypes that will yield the most specific information at the highest quality level.

Automated identification with ProIDProID is an application that works in conjunction withBioAnalyst™ software, and is designed to identify proteins from LC/MS/MS data acquired using Applied Biosystems/MDSSCIEX systems and information dependent acquisition (IDA).Identification is performed with the Interrogator™ searchalgorithm capable of performing extremely fast databasesearches with up to five variable modifications plus a powerful‘zone’ modification feature for detection of unanticipatedmodifications. All results are stored in a back-end relationaldatabase 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 reliableinstrument for the identification of proteins. Its productivity,selectivity and sensitivity make it the instrument of choice forany proteomic laboratory that is looking for versatility.

For more information on:Q TRAP LC/MS/MS System enter: No. 416

21

new product review

20

new product review

Skimmer

DET

Q0 High-

pressure Cell ST Q1

IQ2 IQ3

Q3 EXB

Q2LINAC

Collision CellDF

Orifice

Curtain Gas

Interface

C2B

New SQL*LIMS™ 4.1 Software

ontinuing a decade long history of laboratory managementsystem experience, in 17 of the top 20 pharmaceutical

companies worldwide, Applied Biosystems announces the release of SQL*LIMS 4.1 Software. The award winningSQL*LIMS software is installed in over 1000 laboratories worldwide and is available with a full range of modules for enhancedfunctionality 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:

or email [email protected]

The new SQL*LIMS 4.1 software includes many new featuresfor 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.

No. 417

C

he API 150EX Prep LC/MS System is a useful toolfor high-throughput compound characterisation

and purification. The Prep LC/MS System is easilyconfigured 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.

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


PrepExpress NT Software for prep LC/MS enter: No. 418

T

New PrepExpress NT SoftwareA Solution for High-throughput Purification Using the API 150EX™ Prep LC/MS System

23

new product review

22

new product review

Figure 1.

API 150EX™ Prep LC/MS System schematic

The ‘Safe Choice’ for Major Pharmaceutical Companies

De novoSequencing

ComparativeSequencing

SNP Discoveryand Validation

MicrosatelliteAnalysis

SNP Validationand Screening

BigDye®

ChemistryLinkage Mapping Set

ChemistrySNaPshot® Multiplex

Chemistry

ABI PRISM® 3100-Avant Genetic Analyzer

Sequence AnalysisSoftware

SeqScapeTM

SoftwareGenotyper®

Software

GeneScan®

Software

GeneMapperTM

Software

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.

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.

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


ABI PRISM 3100-Avant Genetic Analyzer enter: No. 419

ver a period of several years, Applied Biosystemshas steadily introduced and developed a range of

genetic analyzers based on capillary electrophoresistechnology. This family has been built up to provideprojects of all scales with a genetic analyzer of anappropriate 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.

O

Introducing the ABI PRISM®

3100-Avant Genetic AnalyzerGrowing with Your Throughput Needs!

25

new product review

24

new product review

4-capillary array

Internal view of the ABI PRISM® 3100-Avant Genetic Analyzer

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 ApplicationExperimental 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).

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 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.

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Figure 1. Chromatograph of affinity subtraction using POROSAnti-HSA cartridge

Abs

orba

nce

(A2

80

)

Figure 2. 4-20% SDS PAGE gel stained with coomassie blue. All lanes loaded with 6.5µg total protein. Bands A,B,D wereidentified as Albumin fragments, C was identified as Albuminfragments and Keratin.

Lane 1 Molecular weight markersLane 2 Serum sample diluted 1:10 with Phosphate buffered salineLane 3 Flow through of POROS Protein G cartridgeLane 4 Eluted fraction of POROS Protein G cartridgeLane 5 Flow through of POROS Protein G and POROS Anti-HSA cartridgesLane 6 Eluted fraction of POROS Anti-HSA cartridge

Affinity Depletion Cartridges

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

page 28

For Removal of Abundant Proteins from Human Serum

Moving forward in Genomic Research

Assays-by-Design serviceAssays-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:

Assays-on-Demand productsThe 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 yourcountry in the drop down menu on the registration page

No. 421

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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 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%.

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.

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.


Affinity Depletion Cartridges enter: No. 420

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 ProteinG and Anti-Hsa cartridges. Circled spots represent increase inconcentration of some low abundant proteins

Figure 4. Results from measurement of IgG and HSA in theserum sample using an ELISA assay, before and after affinitysubtraction using the POROS Protein and Anti-HSA cartridges.

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 Boundper ml media per ml media

Bovine 0.24 0.004Goat 0.01 0.000Human 2.04 0.031Mouse 0.70 0.011Porcine 0.52 0.008Rabbit 0.31 0.005Rat 0.75 0.011Sheep 0.01 0.000

Applied Biosystems Introduces the New Assays-by-DesignSM Serviceand Assays-on-Demand™ Products

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new product review

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.

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).

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.

References1. “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

Improved Software Tools for DNA Polymorphism Data Analysis

NA sequencing and polymorphic marker analysiscould have been viewed as two disparate

investigative techniques in the past. Direct sequencingprovided a ‘gold standard’ for absolute typing ofpolymorphisms, allowing both detection and definition of mutations in one assay. In comparison, DNA fragmentanalysis provided a high-throughput, marker typing techniquefor analysing large numbers of samples such as the data setsneeded for linkage mapping projects, or human identificationdatabases. More recently, single nucleotide polymorphism(SNP) genotyping has bridged the gap between those originaltechniques, being a direct sequence interrogation assay that isapplicable 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.

D

Figure 1.

Figure 2.

Quality Values

PhotoSpray™ SourceA New Ionisation Technique for LC/MS

he PhotoSpray source is an alternative ionisationsource to TurboIonSpray® or APCI/Heated Nebuliser

for API 150EX, API 3000™ and QSTAR® Pulsar LC/MSand 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 APCIsources allows researchers to ionise a wider range of compounds than in the past.

Principles of Photoionisation for LC/MSPhotoionisation at atmospheric pressure may be used togenerate ions from the vaporised LC eluent1. A Kryptondischarge 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 otherspecies absorb the limited photon flux without generating ions.

By adding large quantities of an ionisable dopant to theionisation region, dopant photoions can be created in greatabundance. At atmospheric pressure, in the presence of solvent and analyte, the dopant ions initiate a rapid series ofion-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 andproton affinity of the analyte2. The PhotoSpray source innegative mode gives M-H ions with the same efficiency as APCI.

DescriptionWhere the Ionspray™ source produces ions by the process of ionevaporation from liquid phase, the PhotoSpray source uses aHeated Nebuliser to vaporise the sample prior to inducingionisation by atmospheric pressure photionisation. The sourcetypically operates at temperatures between 300 and 450°C.This vaporisation process leaves the molecular constituents ofthe 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.

T

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new product review

The source block is positioned off-axis to the orifice and theoptimum position is not compound dependent. Proven CurtainGas™ interface technology protects the mass analyser fromcontamination and provides ruggedness to the system.

Dopant SelectionThe 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, (ionisationpotential of 8.83eV) meets all these requirements and is the preferred dopant compound for the PhotoSprayapplications. The dopant infusion rate is 5–15% of the mobile phase flow rate (no split).

Flow RateThe 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 demonstratedunder this regime. The source operates under reversed or normalphase chromatographic conditions:

➜ MeOH/Water or Acetonitrile

➜ Isooctane/Isopropanol/Methylene Chloride and other

LC solvents

PhotoSpray ApplicationsThe PhotoSpray source can ionise low polar compounds, with better sensitivity than the APCI source. PhotoSpray showedimportant sensitivity improvements for Steroid analysis,PolyAromatic Hydrocarbons, Vitamins, Quinones, Antioxidants,Pesticides, Pharmaceuticals and Nutraceuticals and severalother classes of compounds. So the PhotoSpray source is acomplementary source to the TurboIonSpray and APCI sources.

API 3000 LC/MS/MS Steroids AnalysisPerformance comparisons of the PhotoSpray source versus anAPCI source have been performed under normal-phasechromatographic conditions for steroids3. Testosterone andEthynyl Estradiol were tested with the photoionisation source and compared with the conventional APCI source. Using APCI steroids, compounds normally exhibit differentsensitivity levels and fragmentation depending on theirhydroxylation numbers. Ethynyl Estradiol is a steroid compoundhaving significant economical importance for its estrogeniceffect. 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 sourcesin MS scan to evaluate relative ionisation efficiency and tomeasure LOD and LOQ using MRM transitions4. Toluene wasused as dopant for the source at a typical flow rate of 20µL/min.A Keystone Betasil Diol 5µm, 100Å, 2x100mm has been usedfor the LC separation in normal phase conditions. Mobile phasecomposition was isocratic isooctane (94%)/isopropyl alcohol(6%). Reversed phase separations have been achievedisocratically on a BDS Hypersil Cyano 5µm, 120Å, 2 x 250mmusing a mobile phase composition of MeOH (50%)/HOH (50%).Mobile phase flow rate was 200µL/min in both normal andreversed phase conditions.

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 exhibithigher polarity. This is the dominating process.

TurboIonSpray

Shown here is a direct plot of the relationship of PhotoIonisationto both the APCI and TurboIonSpray techniques as compoundpolarity increases against molecular weight.

page 34

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TaqMan® Assays for Food Testingand Food Pathogen Detection

ood quality and food safety is a subject of growingpublic concern. Public food control institutions,

private service laboratories and commercial foodmanufacturers see a growing need to test for geneticallymodified organisms (GMO) and pathogens in food.

In July 1999, Switzerland implemented legal regulations which require food containing more than 1% GMO to belabelled. The European Community and many other countriesaround the world followed this example.

Assays for detecting GMO must be quantitative, sensitive andreproducible. Immunoassays are fast and inexpensive, but notquantitative. Common PCR and its gel-based analysis is onlysemi-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 ofspecificity to the PCR. An internal positive control labelled witha 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 thepresence of the transcriptional regulatory elements common to most GMOs, which express the desired traits such asresistance to insects and herbicides. This transcriptionalregulator is the 35S promoter of the cauliflower mosaic virus.

A plant-specific endogenous control has two advantages:it allows a relative quantitation of GMO soy or GMO maize inthe sample, and no amplification of the control indicates thepresence of PCR inhibitors. The probe for the endogenouscontrol is labelled with VIC® dye, so the 35S target and thecontrol can be detected simultaneously in a multiplex reaction.

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 areestimated to cost up to US $35 billion annually (1997) inmedical 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-liketoxin; 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 Polymeraseand 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 positivecontrol 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 positiveand negative controls. In addition, the GMO and Food PathogenKits include a label licence, which conveys certain PCR servicerights when used with an authorised thermal cycler.

For more information on:TaqMan assays for food testing enter:

TaqMan assays for food pathogen detection enter: No. 425

No. 424

F

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)

Figure 2. Amplification plot of processed foods purchased from a local supermarket and tested with the TaqMan GMOMaize 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)

Relative response for Testosterone and Ethynyl Estradiol withPhotoSpray (PS) and APCI, as recorded in MRM mode.The same respective amount of compounds were injected on column using Normal phase chromatographic conditions.

In MRM mode the signal gain, when combined with a reductionin background noise, translates into between 5 to 20 timesbetter signal-to-noise ratios. The response was linear with anupper limit of determination of 2000 pg on column forTestosterone, and 4000 pg on column for Ethynyl Estradiol.

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 analyticalmethod for polycyclic aromatic hydrocarbons (PAHs), comparing the difference under reverse and normal phase

chromatographic conditions. A total of 16 PAHs commonlyfound in atmospheric samples from both rural and urban areas were analysed. Airborne PAHs, usually found in aerosol particles, are implicated in heterogeneous atmosphericprocessing and as important indicators of gas-phaseatmospheric processes. Certain carcinogenic PAHs may be carried by ultra fine particles in ambient air into the lungs and are often the targets of rigorous environmentalmonitoring. A 20ppm stock standard solution containing 16 common PAHs was used for this experiment5.

ConclusionThe PhotoSpray source provides a better detection limit to theAPI 150EX™, API 3000 and QSTAR Pulsar systems for low polar class of compounds. The API 3000 and QSTAR PulsarLC/MS/MS systems are the only tandem MS systems with threedifferent ionisation sources, which allows researchers to have ahigh ionisation range flexibility.

References1. 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-0154. 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-0095. 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

Reverse Phase PAH separation showing tenMRM transistions for 16 common PAHs

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he Gene Expression package from Applied Biosystems takesyour experiments from RNA sample preparation to successful

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orders, check product availability, view your pricing and more...

**please contact your local Applied Biosystems office if you don't findyour country in the drop down menu on the registration page.

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isit our new European website and gain access to Applied Biosystems Europe online. As a visitor to the

new website you will be able to take full advantage of the site’s features:

➜ Current special offers*

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➜ Online event registration

➜ European training course listing*

➜ Biosystems Solutions online*

➜ The new European eCommerce store*

*features specific to the European website

Special Offers - Keep up-to-date with current special offers

from Applied Biosystems. You can view full details of each

featured offer and request a quote by simply submitting

your details online.

New Product Features - Review our latest technology and

services via the web and request further information or prices

online for any of the products featured.

Online Event Registration - Register online for European events,

seminars and user meetings hosted by Applied Biosystems.

Each listing provides an in-depth profile of the event including

agenda topics, guest speakers and product focus.

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Visit Applied Biosystems Europe online @europe.appliedbiosystems.com

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SQL*LIMS™ Installed toStreamline Product Release

he demands on pharmaceutical companies forquality assurance based on accurate information,

from both the public and the regulators, are increasing.At the same time, market competitiveness is becomingever more cut-throat, so it is more vital than ever thatproducts of verifiable quality be brought to market inthe 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.”

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.”

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

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 supportWith 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

Baxter BioScience, Neuchatel, Switzerland

T

From Left: Alexandre Boillat, IS Manager and Pascal Schneider,project leader of the LIMS implementation, Baxter BioScience,Neuchatel, Switzerland

Baxter BioScience, Neuchatel, Switzerland

Servicing the Global OligonucleotideOperations (GOO)Kingsland Grange, Warrington, UK

he occasional clanking and hissing of the wastevalves, and the constant whining of the vacuum

pumps drowning out the faint clicking of delivery valvesis a familiar sound in the high-throughput laboratory atthe GOO facility in Warrington, where rows of DNASynthesisers fill the room.

Over the last twelve months worldwide demand for custom-oligonucleotides has increased, causing a three-fold growth ofthe facility at Kingsland Grange and this site has become a key account for the UK Service Department. GOO has takendelivery of twenty-nine ABI 3900 High-throughput DNASynthesizers since February 2001. They already had fourteensynthesizers, which placed significant demand on Field Serviceresources in the UK.

In April 2001 we reviewed the provision of service for this site;GOO placed service calls, like any other customer, through theservice administration department. Although the serviceprovided was very good, and compared favourably with industrystandards, it still could not meet the demands of this busymanufacturing site. The service department made a guaranteeto have a Service Engineer on site all day from Monday toFriday, providing service that is second to none, but having aField Engineer based on site permanently took its toll on therest of the field based resource in the UK.

In June 2001 it was decided to recruit an In-house ServiceEngineer whose role it is to ensure the smooth running of theDNA Synthesizers in all three of the Oligo Labs at theKingsland Grange site. This solution enabled us to provide aquick response to breakdowns and ensure that all plannedmaintenance 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 arerepaired within one day, which is vital for the productionrequirements of this site. This is achieved with support fromthe members of the Field Service and Application teams whoprovide 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 soonas a breakdown occurs. Once a call has been logged, an emailis sent to a Service Administrator at Applied Biosystems,Lingley House in Warrington, who is dedicated to co-ordinatingall service calls for the UK key service accounts. Service callsshow up on the UK Oligo Engineers’ PC within seconds ofbeing logged so the engineer can then react immediately.

The Call Tracking Database provides a comprehensivemaintenance history for all the instruments, which is vital forvisiting engineers, and is also a good source of information forProduction Team Leaders and Operators at GOO, who haveaccess to the database at all times. This information is vital forchecking compliance with ISO standards and analysis ofproduction records.

You can see what has been achieved in a relatively short spaceof time to enhance the level of service delivery to a KeyAccount 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 somethingApplied 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

T

The GOO Facility

Paul A Johnson, Service Engineer at work

Proteomics Research Center Embraces RIS ImplementationEuropean Proteomics Support Center, Langen (Frankfurt, Germany)

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 two-dimensional 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.

Resources of the Proteomics Support CenterMany 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 bio-

chromatography, Procise® sequencer and peptide synthesizers.

Managing Proteomics Process InformationIn 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 CentersTogether, 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


The Proteomics Research Center enter: No. 431

R

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Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF™ optics

SERVICE CONTRACTS

GENUINE

or the first time, the German Society of Mass Spectrometry (Deutsche Gesellschaft für

Massenspektrometrie, DGMS) has granted the newlyestablished ‘Life Science Award’ for ScientificExcellence 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.

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 high-

resolution 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

F

to Prof. Jasna Peter-Katalinic, for Scientific Excellence in Mass Spectrometry

German Society of Mass SpectrometryGrants ‘Life Science Award’

left to right: Prof. Dr. Michael Przybylski, Mrs Prof. Jasna Peter-Katalinic

and Dr. Holm Sommer

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Kiel University Hospital Collaborateswith Applied Biosystems

pplied Biosystems Group, an Applera Corporationbusiness, and the University Hospital in Kiel in

Germany, have recently announced a collaborative study on high-throughput genotyping led by Dr. StefanSchreiber, Professor of Medicine and Gastroenterology.Up to 2,000 single nucleotide polymorphisms (SNPs) inapproximately 5,000 individuals will be investigated in thecourse of the study using the local high-throughput SNPgenotyping infrastructure to identify if any have associationswith chronic intestinal inflammation, in particular Crohn’sdisease and ulcerative colitis.

In the past, Professor Schreiber’s research group has examined a large number of genes for disease associations onvarious 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 approachcalled high-density association mapping, will attempt to definefurther 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 Crohnsand Colitis Foundation, who helped assemble the patientdatabase for the project.

Professor Schreiber is one of the first users of the recently introduced Assays-by-DesignSM service fromApplied Biosystems, where individual assay kits for knownSNPs are designed and tested to order. This approach can savetime, 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 SNPgenotyping and gene expression research.

For more information on:The Assays-by-Design service enter:or see: http://home.appliedbiosystems.com/press(Press release dated October 25, 2001)or visit: www.appliedbiosystems.com/assaysbydesign

No. 432

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,

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.

Enabling the next phasesThe characterisation of genetic predisposition to complexdiseases 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 theidentification of susceptibility genes a time-consuming andexpensive task. Association studies will involve populations muchlarger than traditional Applied Biosystems' technologies coulddeliver. The Genomic Assays products will enable the next phase in genomic discovery. Now, it is possible to eliminate therisks and reduce the costs and time that are associated withdesign, optimisation and assay failure. By using our validatedHuman SNP Assays-on-Demand™ products and supplementing,

if necessary, with our Assays-by-Design service, you canimplement the same, simple, single-step SNP genotypingworkflow 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:

Assays-by-Design Service enter: No. 434

No. 433

Genotyping Solutions for linkage & association studiesApplied Biosystems’ Enabling Discovery European SeminarSeries took place on nine dates between 25th February and 8thMarch, 2002. The series of one-day events began in Munichand subsequently visited Berlin, Rotterdam, Madrid, London,Copenhagen, Stockholm, Paris and Naples. Experts fromApplied Biosystems presented our innovative genotypingsolutions for linkage mapping and association studies.

From microsatellite analysis to SNP discovery, validation & scoring, Applied Biosystems provides a range of high qualitytools and expertise for the diverse customer needs.

Distinguished scientists from across Europe were invited topresent their research and discuss how genotyping Solutionsfrom Applied Biosystems are employed in their laboratories.

Delivering the genomeScientific discovery continues to require a scientist with ahypothesis 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 usefulinformation and robust tools to enable the next phase ofdiscovery. The Genome Initiative was first announced byApplera Corporation in July 2001. This initiative combines data from Celera Genomics and public programs with Applied Biosystems' long history of technology innovation andexpertise. The Enabling Discovery seminar series provided theforum to present the goals of this ambitious initiative to ourEuropean customers for the very first time. Through thisinitiative, the resulting Genomic Assay will enable ourcustomers to engage in genotyping studies at a previouslyundreamed of ease, scale, speed and value.

Highlights from our customersIt would be impossible to capture in detail each of theinteresting customer presentations. Here, we briefly summarisethree 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 finemapping of genes related to Inflammatory Bowel Disease.

As one of the three National Genome Research Network(NGFN) laboratories, they have chosen the ABI PRISM® 7900HTSequence Detection System and Assays-by-Designsm Service as the technology solution to provide high-throughputgenotyping services to the research community in Germany.In January, using these new tools, they completed a study of130 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 InternalMedicine at Erasmus University, Rotterdam, Netherlands.Started in 1991, this prospective population-based cohortstudy consists of samples from >7,000 patients selected for the research of the determinants of chronic and disablingdiseases in the elderly. Dr. Uitterlinden’s interests are inendocrine diseases, and more specifically in the genetics ofosteoporosis. He presented methods for SNP genotyping of candidate genes, showing data from their application ofSNaPshot® Multiplex Kit technology on the ABI PRISM® 3100Genetic 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 academiccommunity. 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 andtheir testing of virtually all SNP genotyping products on themarket. 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

European Seminar SeriesEnabling adj. Rendering possible by supplying the meansDiscovery n. Information learned for the first time

Enabling Discovery:Genotyping in the Genomic Era

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Customer Training CoursesCall Eve Lightfoot 01925 282551 or Josephine Kinsey 01925 282475

Basic PCR - Theory Optimisation & Applications

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



(Training Courses held in The Netherlands, in English)Informatics v4 System Manager

Informatics Transition Training 07-10 October

Informatics v4 Key Personnel 09-13 September No. 444

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