Biosystems
Applied Biosystems
Lingley House
120 Birchwood Boulevard
Warrington, Cheshire
WA3 7QH
TEL: 01925 825650
FAX: 01925 282502
Solutions
European Edition, Issue 5 - Autumn 2002
European
Sales Offices
Austria
Tel: +43 (0)1 867 35 75 0
Belgium
Tel: +32 (0)2 532 44 84
Denmark
Tel: +45 45 58 60 00
Finland
Tel: +358 (0)9 693 794 27
France
Tel: +33 (0)1 69 59 85 85
Germany
Tel: +49 (0)6151 96 700
Italy
Biosystems Solutions
Editorial
Applied Biosystems, Lingley House, 120 Birchwood Boulevard, Warrington, Cheshire WA3 7QH, UK.
Tel: +44 (0)1925 825650 Fax: +44 (0)1925 282502 email: Kay_L_Hill@eur.appliedbiosystems.com
Editor
Kay L Hill
Contributors - Applied Biosystems
Peter Boogaard, Tony Hardware, Martin Heinrich, Karsten Lueno, Barbara Maniglia, Friederike Gerdes, Wolfgang Mayser, Sue Ann Molero, Michael O'Neill,
Pierre Paroutaud, Nico Stom, Jean-Luc Gy, Fabienne LeFloch, Rupa Ark, Brigitte Fortnagel, Silvana Biraghi, Raimo Tanzi, and Dave Watts
Tel: +39 039 83891
The Netherlands
Tel: +31 (0)180 392 400
Norway
Tel: +47 23 16 25 75
Portugal
Tel: +351 22 605 33 14
Design/Production
MacRae Communications Ltd, 3 Belgreen House, Green Street, Macclesfield, Cheshire SK10 1JQ, UK.
Tel: +44 (0)1625 869689 Fax: +44 (0)1625 511678 email: info@macraemarketing.com Web: www.macraemarketing.com
Spain
Trademarks
Applera Corporation is committed to providing the world's leading technology and information for life scientists.
Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses.
AB (Design), Applera, GeneMapper, Assays-on-Demand, Assays-by-Design, VISION, oMALDI, TurboIonSpray, SQL*LIMS, BioMaintenance,
BioRepair, BioParts, BioAssurance, cAMP-Screen Direct, TR717, TOF/TOF are trademarks and Applied Biosystems, ABI PRISM, BigDye, SNaPshot, QSTAR,
GeneScan, Genotyper, BioBeat, Primer Focus, SymBiot are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
Sweden
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.
Paracel is a registered trademark of Paracel Inc.
The ABI PRISM 3100 and the 3100-Avant Genetic Analyzers include patented technology licensed from Hitachi, Ltd. as part of a strategic partnership between Applied
Biosystems and Hitachi, Ltd., as well as patented technology of Applied Biosystems.
The Applied Biosystems 3730 and 3730xl DNA Analyzers include patented technology licensed from Hitachi, Ltd. as part of a strategic partnership
between Applied Biosystems and Hitachi, Ltd., as well as patented technology of Applied Biosystems.
The PCR process and the 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.
HitHunter and the DiscoveRx name are trademarks of DiscoveRx.
Q TRAP, NanoSpray and PhotoSpray are trademarks of Applied Biosystems/MDS SCIEX instruments, which is a joint venture between Applera Corporation and MDS Inc.
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.
Certain aspects of the technology described herein are covered under current or pending US and/or international patents.
Microsoft is a registered trademark and Xbox is a trademark of Microsoft Corporation.
Oracle is trademark of Oracle Corpoation.
All other names are the property of their respective owners.
Tel: +34 91 806 1210
Tel: +46 (0)8 619 4400
Switzerland
Tel: +41 (0)41 799 77 77
United Kingdom
Tel: +44 (0)1925 825650
European
Managed Territories
Africa
Tel: +27 11 478 0411
Czechia
Tel: +420 2 3536 5189
Hungary
Tel: +36 1 270 83 98
Poland
Russia
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.
S.E. Europe, Middle East,
West Asia
europe.appliedbiosystems.com
see p
ages
5-6
Tel: +48 22 866 4010
For Research Use Only. Not for use in diagnostic procedures.
©2002 Applied Biosystems. All rights reserved. Printed in the UK 10/02
Gen
o
Assamic
DEMO ys
AVAIL CD
ABLE
Tel: +7 095 935 8898
Tel: +44 (0)1925 282481
The Whole Genome...
Ready-to-Use
Science that changes lives
contents
Three revolutionary scientific inventions.
technical communications
07-12
cover story - 05
Accurate High-Throughput Protein Identification
HitHunter™ EFC Non-Selective Inhibitor Probe Assay System
Pancreatic Cancer
The Whole Genome... Ready-to-Use
Edison´s light bulb (1879)
new product review
Applied Biosystems Q TRAP™ System (2002):
A unique system combining an API triple quadrupole with a linear ion trap
13-25
SNaPshot® Primer Focus® Kit Released
AmpliTaq Gold® DNA Polymerase, LD
Life Science LIMS Software
New Quadrupole Time-Of-Flight LC/MS/MS System
Advanced Informatics Solutions for Genomics & Proteomics
Paracel™ BLAST Software
Diving Deeper into the Proteome
promotions
26-36
New Cleavable ICAT™ Reagents
50th ASMS Conference – Posters & Presentations Overview
Genetics of Complex Diseases and Isolated Populations
cAMP-Screen™ Direct Immunoassay System
Assays-on-Demand™ Products Promotion
World Drug Discovery and Development Summit
The European In-House Sales Team
BioBeat® Wins Publishing Excellence Awards
customer relations
37-43
ABI PRISM® 3100 Data Collection Software v1.1 Released
The 34th European Conference of Human Genetics
New Service Agreement Plans
The Q TRAP™ LC/MS/MS System sets a new standard in
Mass Spectrometry technology.
In 1879, Thomas A. Edison invented the electric light bulb. In 2002, Applied Biosystems /
MDS SCIEX introduced the Q TRAP LC/MS/MS system: a unique, patented linear ion
trap technology, that combines the specificity and robustness of a triple quad mass
spectrometer with the full scan MS/MS sensitivity of ion trap instruments into a single
instrument. So for proteomics, you get fast full scans at unmatched sensitivity, real-time
charge state determination, plus superior mass accuracy. For metabolites, you get all
of the above, plus accurate quantitation. Advanced mixed scan modes and application
specific software ties it all together. It adds up to more value, more metabolites,
more proteins and more confidence in your results.
Want more information? Visit: europe.appliedbiosystems.com
14
22
38
40
New Applied Biosystems
3730xl DNA Analyzer
For Proteomics Research
Applied Biosystems
Proteomics Research Center
Expanding in Europe
Faster, Better, Cheaper
Automation Capabilities
3rd Year in Operation
Applied Biosystems
3
cover story
Corporate review
Applied Biosystems/MDS SCIEX – proven supplier
of quality mass spectrometers and services
A
pplied Biosystems has had a joint venture
The strong relationship with Applied Biosystems
with MDS SCIEX for almost three decades.
over
Its major customers are pharmaceutical and
biotechnology companies throughout the world.
In this partnership, MDS SCIEX is responsible
for the research, development and production
many
years
has
helped
shape
mass
spectroscopy technology products. This includes:
1981 - MDS SCIEX created the first commercial,
triple quadrupole mass spectrometer.
of instrumentation, with Applied Biosystems
1983 - MDS SCIEX pioneered the inductively
directing worldwide sales, marketing and customer
coupled plasma mass spectrometer.
service. Applied Biosystems/MDS SCIEX instruments
are designed primarily for use by researchers
and scientists in the rapidly growing biotechnology,
biomedical and pharmaceutical fields, and are
widely used for high-throughput analyses in
1989 - MDS SCIEX introduced the first viable
mass spectrometry system linked with liquid
chromatography - a breakthrough that revolutionised
the pharmaceutical industry.
support of proteomics, clinical trials and drug
1998 - AB/MDS SCIEX introduced the API QSTAR®
metabolism studies.
Pulsar LC/MS/MS system and the oMALDI™ ion
source in collaboration with the University of
Manitoba. This technology promises to revolutionise
the field of proteomics.
2001 - AB/MDS SCIEX introduced the revolutionary
API 4000
™
system, a triple quadrupole system
that sets a new standard of performance in the
pharmaceutical industry.
2002 - AB/MDS SCIEX produced the first combined
systems for triple quadrupole and ion trap
technology with the Q TRAP LC/MS/MS system.
The joint venture partnership recently introduced
the Q TRAP™ LC/MS/MS System, a high performance
qualitative and quantitative analysis tool for use in
both proteomics and early stage small molecule
Over the last 25 years, MDS SCIEX has established
drug development. The Q TRAP System combines
a worldwide reputation for innovation and a
two important mass spectrometry technologies for
record of market leadership in pioneering the
the first time – triple quadrupole and ion trap – to
pharmaceutical,
and
identify proteins and peptides in proteomics
technological applications of mass spectrometry.
research and identify and quantify small molecule
MDS SCIEX was born from a relationship formed
drug
between academia and government, and today
development (see further details on page 7).
environmental,
clinical
MDS SCIEX maintains strong relationships with
universities across North America and Europe.
The company employs approximately 450 people.
4
metabolites
important
in
The Whole Genome...Ready-to-Use
T
he historic sequencing of the human genome
marked the beginning of the next phase in the
journey to discovery. Obtaining the human genome draft
sequence was a major scientific accomplishment, but the
application of this genomic information to the understanding of
biology and disease will have the greatest potential to impact
medicine. In this phase of discovery, life science researchers
will need the ability to transform the 3.2 Gb of raw sequence
data directly into meaningful biological results. Only then can
we advance our understanding of biology and disease to move
closer to a vision of personalised medicine.
An Initiative of Unprecedented Breadth and Scope
The Applera Genomic Initiative is an unprecedented
project that will transform the human genome sequence
information into novel and tangible products. The mission of
Applied Biosystems is to deliver these powerful, enabling tools
that will accelerate this new phase of discovery. Our aim is to
provide scientists with the ability to navigate simply an
immense amount of data content, and then easily select
ready-to-use, robust SNP and gene expression assays.
These Assays-on-Demand™ SNP and Gene Expression products
represent the most comprehensive set of biological assays
available for making the human genome ‘ready-to-use’ in
laboratories around the globe.
Applied Biosystems’ high-throughput genotyping validation
laboratory by genotyping 90 individual DNA samples from
two distinct populations to determine allele frequency.
These assays are designed to simplify and accelerate
candidate-gene and candidate-region association studies,
linkage mapping, linkage disequilibrium and eventually,
whole genome mapping analyses.
The Assays-on-Demand Gene Expression Products will ultimately
comprise an assay for every transcribed gene in the human
genome. The first set of gene expression assays are designed
to quantify expression of the RefSeq transcripts. These will be
followed by assays for remaining genes and major splice variants
for a total of approximately 30,000 assays.
therapeutic
The Assays-on-Demand SNP Genotyping products, comprise
approximately 200,000 functionally tested and fully
validated assays, for SNPs located in human gene or gene
regulatory regions. Assay validation is accomplished in
page 6
5
cover story
technical communications
Accurate, High-Throughput
Protein Identification
The gene expression assays will be the ideal complement to
high density microarray screening to confirm and validate
putative hits, allowing generation of accurate, and reproducible
gene expression patterns for large numbers of samples.
Future developments will include a custom configurable,
pre-loaded microfluidic card specifically designed for use
on the ABI PRISM® 7900HT Sequence Detection System.
These microfluidic cards will further simplify the use of our
gene expression assays and reduce the required amount of
precious RNA sample.
The Assays-by-DesignSM service completes the Genomic Assays
product line by offering custom-designed assays for both
SNP genotyping and gene expression assays unique to a
customer’s research project. The customer provides the DNA
target sequence; Applied Biosystems designs, tests and
delivers the specified genomic assay in the same,
simple, ready-to-use format.
Fundamental components of Genomic Assays
Technology & Content
TaqMan® probe based chemistry for real-time PCR is the
‘gold standard’ for gene expression, providing unparalleled
reproducibility. The chemistry has the unique features of
being a homogeneous, single-step, PCR-based assay.
These features drastically reduce the manual labour steps
involved in many other technologies available today.
The third generation TaqMan MGB Probes provided the
necessary technological advance for the 5' nuclease assay to
become a robust and reliable standardised technique for
high-throughput SNP genotyping (see Biosystems Solutions
Issue 4, page 13 for more details).
Today, the robustness, ease-of-use and sensitivity of TaqMan
probe based chemistry make it the ideal technology for
performing both gene expression and SNP genotyping.
This ‘third generation’ assay format, with its simplified
implementation on ABI PRISM Sequence Detection Systems,
was chosen as the basis for Genomic Assays and is poised
to become accepted as the worldwide standard for SNP and
gene expression analysis.
Applied Biosystems has recognised that in order to
adopt 5' nuclease chemistry, customers have two fundamental
requirements: elimination of time involved for design and
optimisation of TaqMan assays and reduction in costs.
The Genomic Initiative has responded to both of these
requirements by creating a large portfolio of pre-formulated,
robust assays at a competitive price. These ready-to-use
assays deliver first time success, eliminate optimisation
steps and make failed assays a thing of the past,
thereby reducing overall project costs.
Applied Biosystems and Celera Genomics are working together,
using the best data available, in the development of these
products. All assays are generated through an extensive
bioinformatics design pipeline, which uses a combination of
DNA sequence data from both the Celera and public sequence
databases. The collection of Assays-on-Demand products
can easily be queried on our web-based ordering system.
6
Using the new Q TRAP™ LC/MS/MS System and Pro ID Software
Overview
Powerful search and filtering tools enable customers to quickly
identify the assays relevant to their research.
For SNP selection, comprehensive information is provided,
including NCBI gene name, reference marker, db SNP ID,
Celera ID, chromosome location, allele frequency and more.
RefSeq ID, Celera ID, gene name, and when known, molecular
function or biological process, are provided for each
gene expression assay. The Applied Biosystems web-based
ordering system is open for customers to view* at
https://store.appliedbiosystems.com/ or see the attached
CD-ROM on page 5. Contact your local Sales Engineer
if your magazine does not have the CD.
*Please contact your local Applied Biosystems office if you don’t find
your country in the drop-down menu on the registration page.
A Revolution in Biological Research
The Assays-on-Demand products, complemented by our
Assays-by-Design service, represent the first step of
the Applera Genomic Initiative that will enable scientists
to undertake studies of growing complexity and scope.
The assays provide a more efficient workflow by streamlining
the experimental design process and simplifying analysis.
The savings in precious resources and time will free
researchers and bioinformatics experts for other work.
As a result, we expect this product offering will vastly
accelerate discovery by shortening ‘time-to-results’ by weeks
or months. In the future, the Applera Genome Initiative will
continue to generate valuable information and products to
advance life science through resequencing and gene
prediction efforts. The genome-wide human assay sets from
Applied Biosystems represent only the beginning of a
revolution in biological research.
For details of the Assays-on-Demand products
promotion see pages 30-33 or visit:
europe.appliedbiosytems.com
Researchers have traditionally performed protein identification
by liquid chromatography mass spectrometry (LC/MS) using
three-dimensional (3-D) quadrupole ion traps or hybrid
quadrupole time-of-flight mass spectrometers, such as the
API QSTAR® Pulsar LC/MS/MS system. Instruments such as
the QSTAR system offer premium performance, while 3-D
quadrupole ion traps, because of their lower cost, have gained
widespread acceptance. However, conventional 3-D ion traps
have several limitations for proteomics applications.
No. 501
No. 502
➜ Pro ID software with patented Interrogator™ search
algorithm allows rapid, accurate protein identification,
even from proteins with multiple modifications
➜ Application-specific software provides ease-of-use Yeast
Proteome LC/MS/MS Experiment
➜ Poor mass accuracy, resulting in ambiguous database
search results
➜ Important low mass ions are not observed in typical
MS/MS experiments
➜ Longer cycle times when performing higher-resolution
scans, with the increased resolution providing no increase
in mass accuracy
➜ True precursor ion and neutral loss scans, useful for protein
characterisation, are not available
The Q TRAP system combines superior ion trap capabilities
with the powerful scan modes of a triple quadrupole
mass spectrometer. This versatile instrument combines the
typical ion trap capabilities of high sensitivity without
sacrificing mass accuracy and resolution. In addition,
all normal triple quadrupole features – including true precursor
ion and neutral loss scans – are maintained. In addition,
the intuitive acquisition software and Pro ID software,
for automated processing, ensure that more proteins are
identified with higher confidence in less time.
Key Features
➜ Enhanced resolution and mass accuracy compared to
conventional 3-D ion trap and standard triple
quadrupole mass spectrometers, which provides greater
confidence in your database search results
➜ Superior full-scan sensitivity in MS and MS/MS modes
permits analysis of important low-copy proteins
For more information on:
Genomic Assays Gene Expression Products enter:
Genomic Assays SNP Genotyping Products enter:
➜ Unique scan functions that include MS3, neutral loss,
precursor ion, and multiply charged scans provide more
flexibility for protein identification and characterisation
➜ Standard triple quadrupole-like MS/MS fragmentation with
no low mass cut-off, provides better peptide sequence
coverage, improving database search results
Q TRAP LC/MS/MS System – Plug-and-Play Source
Yeast Proteome LC/MS/MS Experiment
Because of its ease of cultivation, genetic manipulation,
and short generation times, yeast is an ideal system for the
study of biological processes relevant to higher eukaryotes.
The complete genome of yeast is known, and research
efforts continue to characterise its complete proteome.
Using the Q TRAP system the proteome of wild-type yeast
(Saccharomyces cerevisiae) was analysed.
Method
Yeast was grown to mid-log phase (OD600 = 0.7) and
extracts prepared using the liquid nitrogen (LN2) grinding
method. The resultant protein samples were labelled with
iodoacetamide and digested with trypsin. After digestion,
the samples were separated into 25 fractions by ion
exchange chromatography using a VISION™ workstation.
page 8
7
technical communications
The cation exchange fractions were loaded into the
LC Packings autosampler and automatically injected onto
the Q TRAP system for data analysis. We programmed the
instrument with the IDA Method Acquisition Wizard to
automatically perform one Enhanced MS (EMS) survey
scan, two Enhanced Resolution (ER) scans, and two Enhanced
Product Ion (EPI) scans throughout the entire LC/MS/MS
run. Enhanced scan modes use the ‘trap’ capabilities of the
instrument to improve sensitivity, resolution, and mass accuracy.
Upon completion of data acquisition, the Pro ID software
program automatically processed the data for protein
identification. For each ER scan, Pro ID determines the
charge state and accurate mass of the precursor and thus
the peptide molecular weight. Using the corresponding
MS/MS data from those precursors, matches to peptides in a
protein or DNA database were established.
technical communications
search. In comparison, a search of similar data acquired using
a 3-D ion trap required larger MS (1.5 Da) and MS/MS (0.7 Da)
tolerances due to the lower mass accuracy.
Table 1.
Protein Category
%
Ribosomal proteins
28
Dehydrogenases
5
Protein kinases
4
tRNA synthetases
4
Elongation factors
4
Heat shock proteins
4
Transferases
3
Isomerases
3
Translational proteins
3
Transcription factors
2
Helicases
2
Aldolases
2
Endonucleases
2
Other proteins
22
Hypothetical/Unknown
12
For the yeast data, the fragment ion mass accuracy on the
Q TRAP system was ±0.1 Da, while for the 3-D ion trap it
was ±0.75 Da.
Pro ID software processes all cycles in the data and creates a
summary report consisting of a list of identified proteins and
the peptides that were identified with their confidence values.
In addition, all results were saved in a relational database to
allow easy data retrieval and comprehensive queries.
Depending upon the size of the database searched and the
number of modifications included in the search, the processing
of a typical one-hour LC/MS/MS data file by Pro ID software
takes 1–10 minutes.
Results and Discussion
Table 1 categorises the proteins identified in
and the percentages of each, identified from
exchange fractions combined. Proteins typically
being of high abundance as well as some lower
proteins are observed.
this study
the cation
considered
abundance
Yeast contains over 6,000 open reading frames (ORFs).
In one cation exchange fraction, 120 proteins were identified
with a confidence of 90% or higher using an MS tolerance of
0.5 and MS/MS tolerance of 0.3 Daltons for the database
8
The 3-D ion trap identified only 62 proteins with a confidence
of 90% or higher – only 51% as many as the higher mass
accuracy data from the Q TRAP system. For the abundant
protein HSP 70, the Q TRAP system identified 12 peptides that
matched to this protein, while the 3-D ion trap found only 5.
The Q TRAP system obtained better protein coverage,
increasing confidence in the identification.
Conclusions
The Q TRAP LC/MS/MS System is a new linear ion trap hybrid
mass spectrometer that outperforms 3-D ion traps and delivers
high-performance triple quadrupole functionality.
This powerful platform provides high sensitivity for protein
identification for proteomics applications. Coupled with Pro ID
and BioAnalyst™ software, the Q TRAP system lets you identify
more proteins faster and with higher confidence.
Furthermore, the unique scan functions of the Q TRAP system
enable highly specific biomolecule identification and
characterisation. Now you can go straight to the answers!
For more information on:
Q TRAP LC/MS/MS System enter:
Yeast Proteome LC/MS/MS experiment enter:
No. 503
No. 504
™
The HitHunter EFC Non-Selective
Inhibitor Probe (NSIP) Assay System
Probing kinase ATP binding sites without antibodies
or peptide substrates
P
rotein phosphorylation by protein kinases is one of
the major mechanisms by which the cell is
triggering and regulating cellular responses to
extracellular signals. Protein kinases are critical components
Figure 1. HitHunter EFC assay system principle
of the cell signalling process and represent approximately 2%
of the genes encoded by the human genome. The organisation
of the catalytic domain is now well established1. Two domains,
N- and C-terminal, are connected by a single polypeptide
strand and ATP binds in a pocket between these two domains2.
Because of their importance in numerous signalling pathways,
protein kinases have become significant therapeutic targets
in drug discovery.
The majority of traditional kinase activity assays measures
the phosphorylation reaction catalysed by a kinase enzyme
and is dependent on specific antibodies and substrate
for the enzyme under study. The need for specific probing
substrates hampers efforts to rapidly develop screening
assays for novel kinases. The HitHunter EFC NSIP assay
system removes this bottleneck and represents a kinase
ligand-binding assay using a non-selective inhibitor probe.
The NSIP used is an Enzyme Donor (ED) labelled
staurosporine, which probes the ATP binding site of a
target with kinase activity.
The HitHunter EFC assay system uses technology based
on a genetically engineered ß-galactosidase enzyme that
consists of two fragments – Enzyme Acceptor (EA) and
Enzyme Donor (ED). Only when the two fragments are
complementing each other is the ß-galactosidase activity
restored. The restored ß-galactosidase activity is measured
using a chemiluminescent readout. ED conjugated to
staurosporine, a potent ATP inhibitor of many kinases,
competes with kinase inhibitors under study for binding to
the ATP binding pocket of any target with kinase activity.
The higher the affinity of kinase inhibitors, the higher
the concentration of free ED-staurosporine; this leads to an
increase in ß-gal activity (Figure 1). This homogeneous assay
is suited for screening and can be used with 96-, 384- and
1536-well plates.
The ED-staurosporine NSIP assay, using PKC-a as a target, was
compared with traditional kinase assays. The IC-50 values
obtained with the new assay were in good agreement with
staurosporine and several other, low- and high-potency kinase
inhibitors. At the same time, the binding of staurosporine and
ED-staurosporine is competitive (Figure 2).
Figure 2. Comparison of functional kinase assays for STK and
ED-staurosporine NSIP Enzyme Binding Assay.
page 10
9
technical communications
Both potent and weak inhibitors can be detected with
the assay (Figure 3).
Figure 3. Weak inhibitors of serine threonine kinase.
Concentration range up to 10 µM
technical communications
In summary, the HitHunter ED-Staurosporine NSIP assay
system is flexible and easily miniaturised into 96-, 384- and
1536-well microplate formats. The assay is homogeneous,
non-radioactive and uses a highly sensitive chemiluminescent
read-out. To develop screening assays for novel kinases,
no specific antibodies or substrates are needed nor is labelling
of the target required.
Pancreatic Cancer
Researchers Locate First Susceptibility Gene
to a Region of Chromosome 4
Michael D. O'Neill, BioBeat® Online Magazine (www.biobeat.com)
References:
1. Hanks, S.K. and Hunter, T. (1995) Protein kinases 6. The eukaryotic protein kinase
superfamily: kinase (catalytic) domain structure and classification, FASEB J. 9, 576-596
2. Scapin, S. (2002) Structural biology in drug design: selective protein kinase inhibitors;
DDT Vol 7, No.11, p.601-611
3. Data sheet – HitHunter Enzyme Fragment Complementation; ED Staurosporine NSIP
Enzyme Binding Assay Kit ; (2002); DiscoverX Corporation, Fremont, CA; USA
The NSIP Enzyme Binding Assay can be used for screening.
In a mini-screen with 336 kinase enriched library compounds
using ED-staurosporine NSIP Kinase Enzyme Binding
Assay, calibration curves were assayed with 2-7% CV and a
z-factor of 0.7. Interference of compounds with subsequent
EFC complementation was tested as well and found to
be negligible. Only one compound exhibited interference
with complementation3.
For more information on:
No. 505
NSIP Enzyme Binding Assay Kit enter:
Other HitHunter Enzyme Fragment Complementation
No. 506
based screening assays systems enter:
No. 507
c-AMP enter:
No. 508
Kinases enter:
No. 509
Phosphatases enter:
No. 510
Nuclear Receptors enter:
T
he first susceptibility gene specific for pancreatic
cancer has been localised to a region of chromosome
4 by researchers reporting in the American Journal of
Human Genetics1. The scientists said that they hope to move
quickly forward to identifying the susceptibility gene itself,
and suggested that determination of the function of this gene
may prove to be the cornerstone of future efforts to develop
effective chemotherapeutic interventions for this dread disease.
Pancreatic cancer is an especially devastating form of cancer,
with almost everyone who is diagnosed with the disease
going on to die within six months of diagnosis. Localisation of
the susceptibility gene depended critically on genetic linkage
analysis of an unusual, large family (Family X) that displayed
autosomal dominant inheritance of pancreatic cancer,
marked by early age of disease onset. Another key to the
success of this research study was the availability of an
ultrasound-based endoscopic surveillance programme to detect
pre-cancerous changes in the pancreases of family members.
The authors noted that this is the first time a successful linkage
analysis study for pancreatic cancer has ever been reported.
Pancreatic Dysplasia.
Your feedback on Biosystems Solutions
R
ecently, with Biosystems Solutions, we included
some market research questions on the content of
the magazine and how you like to be kept informed
with up-to-date information from Applied Biosystems.
Encouragingly most of you found the articles we chose
to include, informative, well balanced and of interest.
We would welcome your proposals and suggestions for
future articles on the advancement of life science research
throughout Europe.
If you want to comment on the content of any issues of
Biosystems Solutions or request to see specific topics
10
included in future issues then please contact us by emailing
abdirect@eur.appliedbiosystems.com 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 card
in this magazine and send it back to us. Or alternatively
email us at the above address.
This stained tissue section shows a pre-cancerous dysplastic
pancreatic duct dominating the frame of the image. Notable are
the enlarged clumped nature of the pre-cancerous cells and the
abnormally large nuclei of these cells. A normal-appearing
duct is seen at the left, slightly below centre.
(Image courtesy of Dr. Teresa Brentnall).
Pancreatic Cancer - A Silent Killer
Pancreatic cancer is one of the most fearsome of all
cancers, killing almost all its victims within six months of
diagnosis. The target of the cancer, the pancreas, has two chief
functions: to secrete insulin for the control of blood sugar levels
and to produce enzymes that are sent to the intestine to aid in
the digestion of food. Cancer of the pancreas has been referred
to as a ‘silent’ disease because it occurs in a relatively
inaccessible part of the body and remains asymptomatic
until well advanced. This form of cancer is notoriously difficult
to detect, early to metastasise, and resistant to treatment.
When symptoms finally do develop, they include pain,
biliary obstruction, clinical wasting, and early subclinical
metastases. Only a biopsy can yield a certain diagnosis.
The survival rate is poor, with only about 3% of patients still
alive five years after diagnosis. Better survival rates have been
obtained at certain specialised medical centres, for those rare
individuals whose cancers are detected when surgery is still an
option. The median age at diagnosis is 70 and the cancer rarely
occurs before age 40.
Genetics of Pancreatic Cancer
Most cases of pancreatic cancer are sporadic.
Familial clustering of the disease has been observed,
and it is now estimated that up to 10% of the cases of
pancreatic cancer arise from autosomal dominant inheritance
of the disease in the absence of either other cancers or chronic
pancreatitis. The genetic investigation of the inheritance of
pancreatic cancer has been hampered until now by a number
of factors. These include the generally late age of disease onset
and the generally short time between diagnosis and death.
In addition, there tend to be no pre-clinical signs allowing for
early identification of affected individuals. Finally, the degree
of penetrance is unknown.
Unusual Characteristics of Family X Prove Key
The characteristics of Family X varied from this general pattern
in ways that were favourable to genetic analysis: The average
age of disease onset (43 years) was much earlier than
usually seen for pancreatic cancer and the penetrance
was high (over 80%). And very importantly, affected members
of the family displayed pre-cancerous changes that could
be detected by endoscopic ultrasound surveillance,
allowing for early identification of ‘at-risk’ individuals.
page 12
11
technical communications
These unusual characteristics, coupled with the family’s
large size (42 members in a four-generation pedigree) and
willingness to participate in the study, were key to the
success of the linkage analysis. The collection of key data
was aided by the development of a pioneering, pancreatic
cancer-screening program by Dr. Teresa Brentnall’s group at the
University of Washington.
Dr. Teresa Brentnall’s group at the University of Washington
Linkage Analysis Places Gene on Chromosome 4
The effort to locate the susceptibility gene began with a
genome scan of DNA samples from 20 affected individuals
in Family X using 373 microsatellite marker loci from
the ABI PRISM® Linkage Mapping Set (v2). The markers
were amplified by PCR and the products separated by
electrophoresis on ABI PRISM® 377 DNA Sequencers.
Data collection was accomplished with the ABI PRISM®
GeneScan® Analysis Software, while analysis and allele
assignment were done with the ABI PRISM® Genotyper® Software
(v 2.5), or other software. This and related analysis allowed the
scientists to identify a region (4q32-34) on chromosome 4 as
being the most likely location of the susceptibility gene. The
researchers then genotyped DNA samples from the 20 affected
individuals, and additional family members, using an additional
17 DNA markers specific for this region. Analysis of the results
of this genotyping lent further support to the localisation of the
susceptibility gene to this region. The researchers added that
haplotype analysis indicated that a single haplotype was in
general present in all affected individuals and absent from the
unaffected individuals. They said that crossover analysis
indicated that the susceptibility gene lies between markers
D4S413 and D4S2910 on chromosome 4.
Finding the Gene
Having established that the gene is located in a particular
region of chromosome 4 (4q32-34), the researchers believe
that they will be able to identify the gene itself in a relatively
12
new product review
short time. There are about 100 genes in the region.
The researchers noted that the availability of relevant gene
mapping tools, a number of BACs and ESTs that map to the
4q32-34 region, will likely aid their gene identification effort
immensely. “With the use of these tools, we will be able to
identify regions of 4q32-34 that show allelic loss or gain,
as well as changes in gene expression during tumour
development. We have constructed an array of all of the ESTs
that map to this region, we are currently analysing the
expression pattern, and we are screening patient samples,
for allelic loss, with the BAC clones that map to the area.
This information will aid in the identification of the mutant
gene in this family,” the researchers wrote in their American
Journal of Human Genetics article 1.
Possible Gene Function
Elucidation of the normal function(s) of the susceptibility
gene product will likely await gene identification and
characterisation of the protein it codes for. Nevertheless,
clues to this function(s) may already be available.
The pre-cancerous changes observed in Family X (widespread
fibrosis and multifocal dysplasia in the pancreas), coupled with
the late-stage tumour heterogeneity that is also frequently
observed in affected family members, may reflect the
involvement of a common genetic pathway associated with the
susceptibility gene on chromosome 4. One hypothesis is that
the gene may be involved in the pathway associated with the
desmoplastic reaction (the pervasive growth of fibrous tissue
around the tumour) that is seen in pancreatic cancer.
®
®
SNaPshot Primer Focus Kit Released
S
NaPshot® Multiplex kits from Applied Biosystems
offer an efficient way of genotyping samples
with single nucleotide polymorphism (SNP) markers.
The technique uses the advantages of the five dye fluorescent
reporter principle for labelled DNA detection on the range of
genetic analysers from Applied Biosystems.
To optimise the use of these multiplex SNP genotyping kits,
it is necessary to determine which combinations of markers will
work together in effective multiplex reactions. It is also useful
to set up the most efficient means of automated analysis for
these ‘marker panels’.
The new SNaPshot Primer Focus kit allows accurate
measurement of the mobility of extended SNP probes,
without the sacrifice of valuable template DNA. It does this
by means of a transferase, rather than a template-directed
polymerase, extension reaction.
The products obtained from the SNaPshot Primer Focus kit
reaction include all four possible permutations of the single
base polymorphism. The principle of this extension process and
the comparability of the products with those of a conventional
SNP genotyping reaction are shown in Figure 1.
Figure 1. The Primer Focus Reaction produces all allele combinations
Hope for Future
In conclusion, the first susceptibility gene specific for
pancreatic cancer has been located and this offers great hope
that the molecular pathways underlying the development of
this long-mysterious and dreaded disease can be unravelled.
Elucidation of these pathways should provide scientists with a
better understanding of the mechanisms by which this cancer
is generated; ultimately, this might serve as the basis for
the development of rational chemotherapeutic interventions
for this lethal disease.
no marker overlap) are used by the software to create a
validated panel of usable markers with their respective bin
sizes. Any markers that would not give clear results are
rejected for inclusion in the panel, and therefore must be used
in another combination.
The following rules are used for the creation of a validated
SNP multiplex panel:
➜ Bins of the same colour may not overlap anywhere in a panel
➜ A bin is one colour. There are up to four bins or colours per
marker, one for each of the four bases
➜ Markers in a panel may overlap as long as bins of the same
colour do not overlap
➜ Bins in a marker are single continuous ranges; there is only
one bin of each colour in a marker
➜ Bins in a marker must be different colours and may overlap;
for almost all markers there will be some overlap of their
constituent bins
➜ Duplicate bin names are not allowed in a marker
➜ Each marker can have only one bin set
These rules, plus the chosen user preferences, govern the
selection of the SNP marker bins. The bin sets created for a
series of marker panels can be incorporated into a new
GeneMapper software analysis method for SNP data processing.
An example of a SNaPshot data panel, analysed by the
GeneMapper software, is shown in Figure 2.
Figure 2. The Auto Panelising feature of GeneMapper software
defines new SNP markers and bin sets. The software determines
the overlap potential of SNP markers and recommends panels to
achieve accurate SNaPshot multiplexing results.
A more detailed version of this story can found in BioBeat
Online Magazine http://www.biobeat.com at this specific URL
http://www.appliedbiosystems.com/biobeat/pancreatic_cancer/
References:
1. Eberle, M.A. et al., American Journal of Human Genetics 70(4): 1044-1048 (April 2002)
For more information on:
ABI PRISM family of DNA Analyzers enter:
Linkage Mapping Sets enter:
No. 511
No. 512
The data obtained can be used by the new Auto-Paneliser
feature of GeneMapper™ version 2.0 to create groups of SNP
markers which can be multiplexed and run together to give a
clear, unambiguous genotype for each locus. In using the
SNaPshot Primer Focus kit, each extended SNP oligonucleotide
probe is run separately, so that the relative mobility of each of
the four possible extension products can be recorded.
These values, together with user-set preferences (for minimum
and maximum multiplex numbers, and maximum allowable or
In conclusion, the new SNaPshot Primer Focus kit offers a rapid
and convenient method for creating efficient, easily analysed
multiplex SNP marker panels. It also avoids the consumption of
valuable template DNA in preliminary assay optimisation.
For more information on:
SNaPshot Primer Focus Kit enter:
No. 513
13
new product review
new product review
Improve Data Quality
➜ Greater signal uniformity
➜ High raw signal
➜ High signal-to-noise ratio
➜ Run temperature of up to 70°C
➜ Forgiving of variations in template type and concentration
➜ Long length of read
Process fewer samples (20-50% less) to
complete a project
Increase Production Capacity
➜ Highest 24-hour unattended capacity of any
DNA Analyzer
Increased Production Capacity
Sequencing
Run Module
Rapid
Standard
Long Read
(36 cm array)
(36 cm array)
(50 cm array)
Average LOR (Phred Q18)*
Runs per Day
Phred Q20 Bases per Read
550
40
500
700
24
650
>1000
12
>800
Phred Q20 Mb per Day:
3730 system
3730xl system
0.96
1.92
0.75
1.50
>0.46
>0.92
*98.5% base calling accuracy, less than 2% N’s, using pGEM® plasmid as template
➜ Enhanced automation and sample tracking
➜ Multiple run modules
➜ Minimal downtime
Complete a project in 1/2 to 1/4 of the time required
with any other platform
Minimise Reagent Usage
➜ 30x less polymer
➜ 20-50x less buffer
➜ Further optimisation of sequencing chemistry
DNA Analysis Faster, Better, Cheaper
With the next generation Applied Biosystems 3730xl DNA Analyzer
T
he Applied Biosystems 3730xl and 3730 DNA
Analyzers are the newest capillary electrophoresis
instruments introduced by Applied Biosystems in
collaboration with Hitachi High-Technologies Corporation.
Building upon the innovative core technology applied to the
ABI PRISM® 3100 DNA Analyzer, the 3730xl and 3730
instruments have been specifically designed to deliver improved
production capacity with reduced operational complexity.
➜ Automated plate loading from a stacker that
accommodates up to 16 plates (96- or 384-well)
All these needs and more have been met with the introduction
of the Applied Biosystems 3730xl and 3730 DNA Analyzers.
They provide both researchers and production facilities with
the ideal platform for faster, better and cheaper sequencing.
For the development of this new high-throughput sequencing
platform, three main goals have been formulated. The new
platform has been specifically designed and built to:
➜ Automated polymer replenishment
➜ Enhance data quality in sequencing and fragment analysis
➜ Internal barcode reader
➜ Increase production capacity
➜ Benchtop unit
➜ Minimise reagent consumption
Main features of the 3730xl and 3730 DNA Analyzers
➜ 96- or 48-capillary array
➜ Simultaneous injection and analysis of 96 or 48 samples
➜ Walk-away automation
➜ Low operating costs
14
Even in the post-human genome-era, both de novo sequencing
as well as resequencing efforts will continue to require a vast
sequencing capacity. But today, scientists not only need
increased capacity, but also ask for lower cost per kilobase to
enable new projects and new approaches. They also want lower
cost of ownership and require a high-throughput yet flexible,
fully automated and robust DNA sequencer.
➜ 25-60x less waste generated
➜ 1-2x lower sample volumes
Significantly reduce the reagents required to
complete a project
Enhanced data quality in sequencing and fragment analysis
The 3730xl platform consistently delivers high quality data
with long lengths of read as shown in the data displayed below.
Top panel represents the compressed (full view) of the analysed
data, while the bottom panel shows a selected expanded
region of the sequence (855-900bp). Note the distribution of
quality values where red bars equal QV of 0-20, green bars
equal QV of 20-30 and blue bars equal QV of 30-50.
BigDye® Terminator v3 Sequencing Standard run using the
Long Read sequencing protocol.
Long Read Sequencing on the
Applied Biosystems 3730xl DNA Analyzer
Increased production capacity
Sequencing
Microsatellite-Based Genotyping
Applied Biosystems 3730 (36cm array)
Dye Set
Runs per Day
Samples per Day
Genotypes per Day**
G5
48
2,304
46,080
**Assumes 20 genotypes per sample
Minimised Reagent Consumption
A comparison of reagents and waste for 100 Runs
3700 System
3730xl System
~ 42L Water
~ 500ml Water
~ 20L Buffer
~ 500ml Buffer
~ 0.8L Polymer
~ 25ml Polymer
Waste ≈ 63L
Waste ≈ 1.025L
Low sample volumes and
reduced CE reagent
consumption
With the release of the 3730xl and 3730 DNA Analyzers,
Applied Biosystems now offers a new level of automation,
throughput and economy in high-throughput sequencing and
fragment analysis. Both new instruments are ideally suited to
add additional capacity to your laboratory or to save costs by
replacing existing 3700 Systems.
For more information on:
The 3730xl & 3730 DNA Analyzers enter:
No. 514
15
new product review
new product review
®
AmpliTaq Gold DNA Polymerase, LD
Life Science LIMS Software
When purity counts
The core laboratory management component of Rapid Integration Solutions
from Applied Biosystems for laboratory integration and automation
R
esearch applications such as identification
of bacterial DNA in culture-negative samples,
taxonomic classification of bacteria by typing 16S rRNA
genes and certain analyses of eukaryotic genes cloned in
BAC vectors rely on PCR amplification of template DNA.
Such experiments often suffer from contamination with
bacterial DNA which is co-purified with the Taq DNA
polymerase protein from either Thermus aquaticus or E. coli
host bacteria in which cloned Taq DNA polymerase is overexpressed. This contamination can show as false positive
results, signals in no-template controls or false high signal
levels in quantitative PCR. Preparations of native Taq
polymerase from T. aquaticus, which is not over-expressed,
can have a particularly high load of bacterial DNA.
The chemical hot start capability of AmpliTaq Gold DNA
Polymerase, LD, like in the proven AmpliTaq Gold DNA
Polymerase, significantly increases PCR yield and sensitivity,
while reducing primer oligomers and misprimed amplicons.
The gradual time-release of active enzyme matches template
concentration and enhances specificity, even when the
heat-activation step has been omitted. AmpliTaq Gold DNA
Polymerase, LD combines purity with the ‘gold standard’
chemical hot start process. This is particularly useful for low
copy number PCR amplifications and microbiological work.
Solutions (RIS) programme from Applied Biosystems,
addresses a laboratory’s exact needs by combining
state-of-the-art software components and world-class
professional services, to deliver tailored informatics solutions
to integrate and automate a laboratory.
More time for science – let LIMS organise your workflow and data
Central to any RIS implementation is Life Science LIMS 5
Software from Applied Biosystems. The software is the latest
generation of Laboratory Information Management System
(LIMS) software from Applied Biosystems that is specifically
focused on the sample information and workflow needs of the
molecular biology laboratory. This core component helps
ensure that a laboratory’s operations proceed efficiently,
so researchers can focus on the pursuit of useful and
profitable scientific discoveries.
Figure 1.
Detection of contaminating bacterial 16S DNA from PCR
enzyme addition. All reactions were carried out as per
manufacturers suggestions and went through 40 cycles of PCR.
1, 12:
2, 3:
4, 5:
6, 7:
8, 9:
10, 11:
ptimising throughput and improving the overall
quality of results are critical success factors for
today’s rapidly evolving, medium- to high-throughput
laboratories. But every lab is unique. The Rapid Integration
The RIS program allows customers to choose the tools that best
suit their applications – from sample management and tracking
to analysis and database searching. RIS systems can integrate
data from a variety of technology platforms and information
sources, both public and proprietary, into the laboratory's
preferred data analysis software packages. This provides greater
data security and better data management by providing a
unified database, ideal for finding relationships and
correlations that might otherwise be overlooked. The RIS
programme currently provides solutions for sequencing,
gene expression, genotyping and proteomics laboratories.
The new AmpliTaq Gold DNA Polymerase, LD enzyme is purified
in a proprietary separation process to minimise the amount of
co-purified bacterial DNA, thus LD for low DNA. The enzyme is
quality control tested to have less than 10 copies of 16S rRNA
gene sequences in a standard 2.5 Unit aliquot (figure 1).
Lane
Lane
Lane
Lane
Lane
Lane
O
AmpliSize marker, Bio-Rad
AmpliTaq Gold DNA Polymerase, LD
AmpliTaq Gold DNA Polymerase
Native Taq
Antibody hot start enzyme
Competitive hot start enzyme
Complete Control Over All Laboratory Data
Full Security – Get both data and functional security. Control who
can access data and what they can do with the data they access.
Flexible Workflows – Define methodology and the various tasks
that need to be accomplished. Life Science LIMS software
methods are flexible and dynamic - no programming is necessary.
The software also provides method version control with approval.
Tracking – Complete location and analyst tracking. Know where
samples are located and which analyst controls which samples.
Historical tracking of location, workflow and analyst movement
allows you to recreate the events that produced your results.
Scientific Context User Interfaces – Multiple user interfaces have
been developed to tailor the user experience to the scientific
application. Use a 2D Gel or LC/MS context for proteomics,
a subject or marker context for genotyping or have our
professional service group build a user context that specifically
fits your lab’s workflow.
Third-Party Instrument and Software Integration – A wide variety
of instrument hardware and software applications typically need
to be integrated to provide efficient data flow and seamless data
analysis. The Applied Biosystems Professional Service Group has
experience in integrating many different instruments, robots and
third-party analysis tools.
FDA 21 CFR Part 11 Compliance – If business needs require
compliance with the FDA’s electronic record rule, Life Science
LIMS software supports FDA 21 CFR Part 11 with integrated
audit trail and electronic signature.
A Track Record of Innovation
With over 15 years of LIMS experience, Applied Biosystems has
over 1,000 LIMS customers and over 20,000 users worldwide
and is the preferred LIMS vendor for 17 of the top 20
Pharmaceutical Companies. This experience and track record of
innovation ensures that RIS systems from Applied Biosystems
are designed rapidly and cost-effectively.
Going from experimental results to new scientific discoveries to
patented, profitable new drugs is typically a long and expensive
process. The RIS program and Life Science LIMS software from
Applied Biosystems can help you get there quicker.
S T A N D A R D
For more information on:
AmpliTaq Gold DNA Polymerase, LD enter:
16
No. 515
For more information on:
Life Science LIMS enter:
No. 516
17
new product review
new product review
The API QSTAR XL
Hybrid LC/MS/MS System
®
A new dimension in flexibility and performance
T
he API QSTAR® XL Hybrid LC/MS/MS System is the
premier quadrupole time-of-flight LC/MS/MS system,
setting a new dimension of flexibility and performance.
The enhanced ion optics and new detector provide answers to
the most challenging analytical questions at the highest
sensitivity. This high sensitivity, coupled with excellent mass
accuracy, yield unequivocal molecular weights and high-quality
structural information for both protein and small molecule
analysis. Novel scan functions offer a high degree of
selectivity for low-level protein analysis, together with
the most sensitive precursor ion scanning capability
for accurate analysis of post-translational modifications
(PTMs) and target compound analysis for drug metabolites.
The QSTAR XL system is the most flexible MS/MS platform,
offering fast, easy switching between the broadest range
of ionisation, including NanoSpray™ source, oMALDI™ source,
Key Features of the QSTAR XL System:
APCI, PhotoSpray™ source, and TurboIonSpray® source.
➜ Unique trapping pulsing capability for maximum duty
➜ Most flexible MS/MS platform for both electrospray and
MALDI analysis
➜ New oMALDI 2 ion source with enhanced collisional
cooling for better sensitivity
➜ New NanoSpray source for increased productivity with
capillary and nanoflow HPLC
➜ New ion optics and detector to improve ruggedness in
24/7 working environment
➜ Increased quadrupole mass selection of up to 6,000 amu
and time-of-flight mass range of up to 40,000 amu
➜ Increased efficiency of high mass transmission
➜ Improved low mass fragment ion transmission
New Increased Mass Selection Capability
The high-mass ion transmission properties have been
significantly improved in the new generation QSTAR XL system.
Ions of up to 40,000 amu can now be analysed by the time-offlight detector, and their efficiency of transmission has been
increased. The CID capabilities have been enhanced so that ions
of up to 6,000 amu can be isolated and fragmented for sequence
analysis, with improved transmission of low mass fragments
(see figure 2).
Novel Multiple Charge Separation
A unique, proprietary charge separation method is applied to the
QSTAR XL system to improve detection limits of peptides and
proteins when analysed from complex mixtures. Multiple charge
separation (MCS) eliminates singly charged ions in the spectra,
thereby enhancing the signal-to-noise ratio of multiply charged
ions at very low levels. This high degree of separation offers
significant gains in signal-to-noise for species that have a charge
state higher than 1. The benefits of charge separation are
particularly apparent at low femtomole concentration levels,
where in regular TOF MS spectra peptide ions are often lost in
a sea of chemical noise. The suppression of chemical noise
reduces the need for chromatography and makes peptide mass
fingerprinting using an electrospray source equivalent to peptide
mass fingerprinting by MALDI.
The new QSTAR XL system can be operated with the new
oMALDI 2 ion source, a schematic of which is shown in Figure 1.
The oMALDI 2 source possesses a high pressure (1-2 Torr)
interface that collisionally cools the MALDI-generated ions as
they are generated. This effectively quenches the fragmentation
that is commonly seen when employing ‘hot’ matrices such as
alpha-cyano-hydroxycinnamic acid. The new design offers
control over the front-end fragmentation and cleaner spectra
due to better declustering of matrix/solvent ion adducts.
Additionally, control over the energy of the oMALDI 2 source
generated ions allows psuedo-MS3 to be performed in the source
region, as indicated in Figure 4.
Figure 4.
Illustrates the ability of the QSTAR XL system equipped with
the oMALDI 2 source to perform pseudo-MS3 experiments.
By increasing the plate and skimmer voltage, in-source
CID occurs, providing enhanced structural information.
cycle and ultimate sensitivity
Figure 1.
Schematic of the new QSTAR XL system featuring the
oMALDI 2 source. Other enhancements include a
DC quad and new detector for improved ruggedness
Q0. Patented collisional focusing
maximises ion transmission for
superior sensitivity
DC Quad The quadrupole lens provides
a marked improvement in the ability to
optimise the ion beam profile
Ultra Stable
Quadrupole
Mass Filter
Carrier
Plate
New Detector
High Efficiency
LINAC
Collision Cell
Laser
➜ Unique scan functions for enhanced selectivity and
sensitivity for low level compound analysis
Figure 2.
Fragmentation of the synthetic peptide Bovine Corticotropin
Releasing Factor (CRF) at 4695.5 Da, showing
the high mass fragment ion sequence information
obtainable from large parent peptides
Figure 3a.
Shows a standard TOF MS spectrum of BSA digest at 4 fm/mL;
very few, if any, multiply charged ions can be detected above
the background
Figure 3b.
Was recorded with MCS “on” and shows a significant
suppression of background, in addition to a significant
increase in signal-to-noise of the multiply charged peptides
Figure 3c.
Depicts practically complete suppression (by 2 to 3 orders
of magnitude) of all singly charged ions
The QSTAR XL system is the latest generation QSTAR system
from Applied Biosystems/MDS SCIEX. Featuring improvements
including extended mass range, oMALDI 2 source, and Multiple
Charge Separation Scans, the QSTAR XL system is the premier
instrument for proteomics research. The QSTAR XL system
which has high mass accuracy, resolution and sensitivity,
combined with Metabolite ID software, makes the QSTAR XL
system ideal for drug metabolism analysis.
Q2 Patented LINACTM High Pressure collision
cell provides increases sensitivity and unique
trapping pulsing capability for maximum
duty-cycle.
For more information on:
The QSTAR XL system enter:
18
No. 517
19
new product review
new product review
Advanced Informatics Solutions
for Genomics and Proteomics
Data Management and Analysis
P
rogress in our functional understanding of human
biology is possible as never before. But this
understanding, and new opportunities in drug discovery
and diagnostics, can only be realised if we can build
on the information from the genome and implement it
at a practical level in the laboratory. The greatest
challenge is to enable scientists to use global
genomics and protein information with a hypothesis
and conducting experiments
Automation Capabilities to realise Your Goals Faster
Informatics Rapid Integration Solutions (RIS), can also help
you realise greater efficiency and productivity in your laboratory
by simplifying, and even automating, repetitive tasks like sample
management and tracking, analyses, and database searching.
Eliminate scattered files, manual data collection and other
bottlenecks in your laboratory. Make your data more secure
and simpler to manage with a unified database that allows
you to mine your data for relationships and correlations you
might otherwise miss.
RIS Integration for the Gene Expression Laboratory
Optimising throughput and improving quality of data are critical
success factors for medium- to high-throughput gene expression
laboratories. RIS include Gene Expression Data Management,
Instrument Integration including ABI PRISM® 7000, 7700 and
7900 HT Sequence Detection Systems. Support for third-party
robotic platforms and Gene Expression and data analysis.
RIS Integration for the Genotyping Laboratory
A combination of new analytical techniques and lab automation
has made the genotype analysis of tens of thousands of samples
a day, routine. RIS include Gene Expression Data Management,
Instrument Integration including ABI PRISM® 3100 and 3700
family of Genetic Analyzers. This has increased the need for
better data management and better analytical tools to aid in the
visualisation of the meaning behind the raw data. The solutions
address data and phenotype management aspects, and are able
to graphically present pedigrees, samples and results including
LOD scores and haplotypes. Bottlenecks and problem areas in
the lab flow may be identified.
RIS Integration for the Proteomics Laboratory
Proteomics has rapidly become an important strategic
component to any successful drug discovery and development
programme or disease research study. Whether you are
engaged in quantifying relative changes in protein expression in
healthy versus diseased tissues, identifying subsets of protein
candidates that may play a role in the etiology of a specific
disease, or identifying and isolating massive numbers of
cellular proteins in parallel, our Informatics solutions will
accelerate your research process.
For more information on:
Rapid Integration Solutions for the Proteomics Lab
Rapid Integration Solutions for the Gene Expression Lab
Rapid Integration Solutions for the Genotyping Lab
20
No. 518
No. 519
No. 520
BLASTING your way out
of a compute bottleneck
Using your existing IT infrastructure, Paracel™ BLAST software
achieves much higher BLAST speed and throughput,
making compute power available for other applications
More effective use of existing hardware
When extending your IT infrastructure, costs do not stop
at the computer purchase stage; there is floor space,
maintenance, power supply, cooling and network issues.
These real organisational costs are often overlooked in
cost/performance analysis.
Why not make use of your existing hardware by implementing
Paracel BLAST, which has been carefully engineered to provide
a cost-effective solution?
Get results faster on your data
For some benchmarks, Paracel BLAST provides in excess of
6x performance improvement over NCBI, even on a single CPU.
With the use of a multi-node cluster system, the performance
difference can exceed 10–100x. Paracel BLAST allows you
to dramatically improve your search performance without
significant investments in new hardware. Contact us for
additional benchmarking information, or to arrange for an
evaluation copy of Paracel BLAST.
NCBI compatibility makes integration simple
Paracel BLAST is built around re-engineered NCBI code,
so the outputs and ‘user feel’ is identical with NCBI BLAST,
including the GUI. However, two key areas make Paracel
BLAST significantly different:
➜ Paracel BLAST supports parallel execution on cluster
systems. This allows you to achieve high-throughput on
cost-effective commodity hardware systems.
➜ Paracel has made algorithmic enhancements to BLAST,
removing significant bottlenecks and making it possible
to complete analyses that were previously impossible.
These enhancements are not available in any other BLAST
solution based on unmodified NCBI BLAST.
Release additional IT resources for tasks other than BLAST
➜ Most research groups have a wide range of software tasks,
but frequently BLAST compute tasks tie up significant
application processes. By directing BLAST processes to a
dedicated BLAST server and/or using Paracel BLAST
software, significant power resource will become
available for other applications.
Graphical User Interface Enhancements
Paracel BLAST comes with BioView® WorkBench, a graphical
user interface (GUI) for launching similarity searches
and for viewing search results. The launcher allows users
to paste in query sequences and select databases,
automatically determining the list of applicable search
algorithms. The Java-based interactive viewer facilitates
the analysis of complex search results, freeing the user
from the task of interpreting the plain-text BLAST report.
For more information:
or to request an evaluation copy of Paracel BLAST,
visit http://www.paracel.com/products/paracel_blast.html
or enter
No. 521
21
new product review
new product review
volumes onto the MALDI sample plate. The SymBiot XVI Sample
Workstation is taking care of your sample preparation routine and
can be part of an integrated workflow with the 4700 Proteomics
Analyzer, the API QSTAR Pulsar, and Voyager Systems.
➜ Provides consistent reliable spotting regardless of
plate squareness or flatness
GPS Explorer Workstation
The GPS Explorer Workstation is the high-performance
global proteome server that provides automated database
searching capabilities at maximum throughput with an
integrated Mascot Database Search Engine.
➜ Up to ten MALDI plates can be spotted at one time
➜ Supports automated database searching for MS data only,
MS/MS data only, or both MS and MS/MS data
➜ 96-well plates spotted in 2-4 minutes
➜ Supports LC/MALDI and 2D Gel Workflows
➜ 200nl – 2µl multi-probe spotter
➜ Provides ICAT™ Reagents quantitation and labelled display
of ICAT reagent pairs
➜ Ease-of-use: Pre-configured methods for dilution,
matrix addition, and sample deposition
➜ Stand alone software with sample tracking and easy
integration to LIMS
➜ Includes sample tracking, Mascot and Oracle Database
for Report Generation and Results Analysis
➜ Includes computer and barcode reader
SymBiot XVI Sample Workstation Software.
Introducing automation capabilities
for your proteomics research
The 4700 Autoloader, SymBiot® XVI Sample Workstation
and GPS Explorer™ Workstation
A
pplied Biosystems recently introduced the 4700
Proteomics Analyzer with TOF/TOF™ optics,
providing researchers the tool for rapid protein
identification and extensive protein characterisation
with unparalleled speed. Biological information of
up to 1,000 samples per hour can be obtained.
With the introduction of the 4700 Autoloader, the SymBiot XVI
Sample Workstation and GPS Explorer Workstation,
researchers can now take full advantage of the 4700
Proteomics Analyzer by automating sample preparation,
plate loading and data handling even further.
22
4700 Autoloader
➜ Automatically loads MALDI sample plates,
that are held in a 24-plate cassette
➜ Autoloader is kept under vacuum for rapid
plate-to-plate transfer
➜ Integrated barcode reader to scan plates
and match plate to a submitted job list
SymBiot XVI Sample Workstation
The SymBiot XVI Sample Workstation is a 16 probe fixed tip
multi-channel liquid handling robot developed by
Applied Biosystems to rapidly dispense sub-micro-litre
For more information on:
4700 Autoloader enter:
SymBiot XVI Sample Workstation enter:
GPS Explorer Sample Workstation enter:
No. 522
No. 523
No. 524
23
new product review
new product review
Diving Deeper into the Proteome
Next generation ICAT™ reagents coupled with MDLC
and high throughput mass spectrometry
T
he ultimate goal of proteomics is to identify and quantify
proteins that are relevant to a given biological state and
unearth their networks of interactions in an effort to
understand that biological state at the molecular level.
With this in mind, a great deal of interest has been generated
on the use of Isotope Coded Affinity Tags (ICAT) for the
simultaneous quantification and characterisation of proteins in
a complex sample. In this approach, protein samples from
two states are labelled separately with the heavy and the light
ICAT reagent, combined, digested and then analysed by MS.
This approach is used to study protein expression levels in
yeast 1 and differentiation-induced microsomal proteins 2.
We present a new ICAT reagent that incorporates two major
advances: (a) use of 13C instead of deuterium in the heavy
reagent and (b) incorporation of an acid cleavable linker
that allows for the removal of the biotin affinity tag after
affinity purification and before MS analysis.
Figure 2.
ESI elution profile of QNCDQFEK (a) labelled with H/L ICAT
reagent and (b) labelled with H/L new ICAT reagent
2a
serum albumin and serotransferrin were analysed in more
detail (Table 2) and shows Standard Deviation to be in the
order of 10% compared with 20% for the current deuterium
ICAT reagent, illustrating importance of co-eluting isotopes
for internal standard quantification.
Figure 4.
MS/MS performance of (a), (c) two peptides labelled with
ICAT reagent and (b), (d) the corresponding peptides
labelled with the new ICAT reagent
2b
4a
4b
4c
4d
Figure 3.
H/L peptide elution profile across MALDI wells
To illustrate the chromatographic separation of ICAT reagent
peptide pairs, BSA was labelled with heavy and light ICAT
reagent and processed through the ICAT reagent workflow as
shown in Figure 1. An example of the elution profile from one
of those ICAT pairs (Figure 2a) clearly shows the separation of
the light and the heavy isotopes. The same peptide labelled with
the new ICAT cleavable reagent demonstrates the co-elution of
the isotopes (Figure 2b). This was observed throughout the
LC/MS run for every peptide observed.
Û
This co-elution of the isotopes is even more important when
considering LC/MS MALDI analysis, as separation of the
resolved pairs across different wells can lead to quantification
errors. The elution profile of an ICAT pair labelled with the new
reagent and analysed via LC/MS MALDI is shown in Figure 3.
It can be seen that the heavy/light ratio remains constant
across all wells.
24
This fragmentation was enough to produce several diagnostic
ions, which can be used to confirm an ICAT reagent labelled
peptide. The improved MS/MS performance of the new reagent
led to 34 out of the possible 35 cysteines being identified
by MS/MS database searching using Pro ICAT software for BSA
(Table 1). Based on this encouraging result, the new reagent
was tested on increasingly complex samples. A 6 protein mix
was processed through the workflow using both ESI and
MALDI MS instruments. The quantitation data for
BSA Peptides
H:L
CCAADDKEACFAVEGPK
0.93
CCTESLVNR
1.02
CCTKPESER
1.03
DAIPENLPPLTADFAEDKDVCK 0.90
DDPHACYSTVFDK
0.93
EACFAVEGPK
0.86
ECCDKPLLEK
1.02
ECCHGDLLECADDR
0.93
ECCHGDLLECADDRADLAK
1.05
ETYGDMADCCEK
1.07
GACLLPK
0.93
LCVLHEK
0.99
LFTFHADICTLPDTEK
0.88
LKECCDKPLLEK
0.97
LKPDPNTLCDEFK
0.92
LKPDPNTLCDEFKADEK
0.91
NECFLSHKDDSPDLPK
0.91
QNCDQFEK
0.89
RPCFSALTPDETYVPK
0.83
SHCIAEVEK
0.92
SLHTLFGDELCK
0.93
TCVADESHAGCEK
1.08
VHKECCHGDLLECADDR
1.34
YICDNQDTISSK
0.94
YNGVFQECCQAEDK
0.97
AVG
0.97+/-0.1
Transferrin Peptides
H:L
ADRDQYELLCLDNTR
1.00
CDEWSVNSVGK
0.79
CLKDGAGDVAFVK
0.84
CSTSSLLEACTFR
0.94
DCHLAQVPSHTVVAR
0.92
DLLFRDDTVCLAK
0.91
DYELLCLDGTR
0.83
EGTCPEAPTDECKPVK
0.89
FDEFFSEGCAPGSK
0.88
IECVSAETTEDCIAK
0.93
KPVEEYANCHLAR
0.94
KASYLDCIR
0.92
LKCDEWSVNSVGK
0.95
NLNEKDYELLCLDGTR
0.94
SVIPSDGPSVACVK
0.86
WCALSHHER
0.91
WCAVSEHEATK
0.91
AVG
0.90+/-0.05
CONCLUSIONS
The incorporation of an acid cleavable linker into the ICAT
molecule allows for removal of the biotin affinity tag
before MS and MS/MS analysis. This results in improved
MS/MS performance leading to identification of more proteins
with higher confidence scores. The incorporation of 13C
rather than deuterium into the ICAT heavy reagent molecule
produces co-elution of the heavy and light isotopes enabling
quantitation by MALDI. This new ICAT cleavable reagent
generates a greater number of significant protein
identifications with much smaller ICAT ratio standard
deviations compared to the normal ICAT reagent.
Figure 1.
Overview of the ICAT process
To evaluate the removal of the biotin tag after affinity
purification on MS/MS performance, the same BSA sample
underwent LC MS/MS analysis. Peptides labelled with the ICAT
cleavable reagent can be seen to generate MS/MS spectra that
are significantly richer in peptide backbone sequence ions
(Figure 4). Fragmentation due to the new ICAT tag was
dramatically reduced to <5% of the ion current.
Table 2.
ICAT ratios for all BSA and transferrin peptides identified via MS/MS
References
1. S. Gygi, B. Rist, S. Gerber, F. Turecek, M. Gelb and R. Aebersold,
Nat. Biotechnol., 17, 1999, 994
2. D. K. Han, J. Eng, H. Zhou and R. Aebersold, Nat. Biotechnol., 19, 2001, 946
Table 1.
BSA Pro ICAT results
Acc #
Name
gi|1351907 SERUM ALBUMIN
Unique Peps
Avg. H:L
#Cys ID/Total
35
0.9225
34/35
➜ 85 different MS/MS spectra identified BSA
For Special Introductory Pricing for the
new Cleavable ICAT Reagents, see next page
➜ 35 unique peptides were identified
– 21 peptides were fully cleaved
– 14 peptides had 1 missed cleavage
➜ 34 out of 35 Cysteines were identified
For more information on:
Cleavable ICAT Reagents enter:
No. 525
25
promotions
promotions
™
New Cleavable ICAT Reagents
Special Introductory Pricing* – valid until 31 December 2002
C
leavable ICAT reagents incorporate a number of key
technological advances that significantly improve
protein expression profiling. Incorporation of an acid
cleavable linker into the ICAT reagent molecule
(See Figure 1) allows for removal of the biotin affinity
tag before MS and MS/MS analysis. This improves
MS/MS performance and significantly increases the
number of proteins identified and quantified with
higher confidence scores in a single experiment.
Part number 4339035
Cleavable ICAT Reagent Methods Development Kit
Contains cleavable ICAT Reagents, affinity and
cation-exchange buffers and cartridges,
and cartridge/hardware accessories.
Contains enough reagents for methods/protocol development
plus two complete expression analysis assays.
Part number 4339036
Figure 1.
Cleavable ICAT reagent structure
Cleavable ICAT Reagent, 10-Assay Kit
Contains cleavable ICAT Reagents, affinity and
cation-exchange buffers and cartridges.
Contains enough reagents for 10 assays at 100µg labelling
227 (236) amu
Affinity Tag
(Biotin)
Acid Cleavage Site
(C10H17N3O3)
Protein
Isotope Coded Tag
Heavy: 9 x 33C (236 amu) Reactive Group
Light: 9 x 12C (227 amu) (lodoacetamide)
Part number 4339038
Cleavable ICAT Reagent, 10-unit bulk reagent
Contains Cleavable ICAT Reagent Light and Heavy,
Cleaving Reagents A and B.
Contains enough reagents for 10 assays at 100µg labelling
These significant improvements in the reagent design, along
with the availability of Pro ICAT software for data analysis,
enable researchers to fully exploit the potential of this
technology in protein expression profiling studies.
from the 50th ASMS Conference, Orlando
E
Cleavable ICAT Reagents Special Introductory Pricing*
E
Part number 4339035
Cleavable ICAT Reagent Methods Development Kit
Switzerland
CHF
Denmark
DKK
940.00
1'455
7,030
Part number 4339036
Cleavable ICAT Reagent 10-Assay Kit
1,880.00
2'901
14,000
Part number 4339038
ICAT Reagent, 10-unit bulk reagent
1,250.00
1'938
9,360
UK
GBP
Norway
NOK
586.00
7,760
1,170.00 15,500
780.00
10,300
Sweden
SEK
8,630
$**
870
17,200 1,750
11,500 1,150
**For pricing in other non EU countries.
*Terms and conditions for this special offer:
Orders can be placed by email, fax or phone quoting a purchase order number.
Offer valid until 31 December 2002 for first order. No other discounts apply.
All prices exclude delivery and local tax. E&OE.
26
Posters and presentations
available...
For more information on this offer:
please contact your local Applied Biosystems office
ach year Mass Spectrometrists from around the world
migrate to the annual meeting of the American Society
for Mass Spectrometry (ASMS). This year the meeting was held
in the sunny climes of Orlando, Florida.
A new revolutionary instrument: the linear Ion Trap, Q TRAP™
LC/MS/MS system was launched by Applied Biosystems/
MDS SCIEX during this conference. This instrument has the
advantages of both a 3D trap and triple quadrupole instrument
and was featured in Biosystems Solutions Issue 4 (see pages
5-6 and 20-21). This new mass spectrometry instrument was
the most innovative technique introduced during the ASMS
meeting and as such generated significant interest and
discussion among delegates.
Applied Biosystems/MDS SCIEX hosted a User Meeting
on Sunday, 2 June, which was attended by nearly
1,000 scientists. We also presented 53 Scientific Posters
and gave 8 presentations during the different sessions,
many of which are now available on CD. We are
proud to have contributed so much good science to
this meeting.
Applied Biosystems/MDS SCIEX will continue to provide
scientists with new and cost-effective technology in Mass
Spectrometry. We strive to meet the developing needs of those
scientists who are now using mass spectrometry technology
for a wider range of application areas.
For more information on:
The Q TRAP system enter:
No. 526
or to request the Poster & Presentation CD enter:
No. 527
27
promotions
promotions
Invitation to attend the meeting
on ‘Genetics of Complex Diseases
and Isolated Populations’
Sunday, 25 May 2003 to Thursday, 29 May 2003, Tortolì, Sardinia, Italy
T
he identification of genes concerned with
susceptibility to common, genetically complex,
diseases is one of the most exciting but challenging
areas of human genetics and medicine. One of the
strategies being adopted to meet the difficulties inherent in
mapping genes of minor effect is to study isolated populations.
Isolated populations often show favourable characteristics,
such as reduced genetic diversity and greater environmental
uniformity, compared with large continental populations.
There are, however, diverse types of isolated populations and
their environments differ in important ways, providing further
opportunities for exploring interactions between genes and
environment. This meeting is designed to explore the major
issues arising in such studies.
Scientific Committee
John Blangero, Ph.D. Scientist, Department of Genetics,
Population Genetics Laboratory, Southwest Foundation for
Biomedical Research, San Antonio, Texas, USA
Paolo Gasparini, M.D. Associate Professor of Medical
Genetics, 2nd University of Naples and Telethon Institute
of Genetics and Medicine, Napoli, Italy
Topics to be covered
Methods for the study of isolated populations, analysis of large
genealogies, quantitative traits, genotyping methods and
technologies, gene/environment interactions in complex
diseases, twin research, studies in animal models, bioethics,
exploring the relationship between academia and private sector.
Satellite Events
Workshop on Genotyping technology organised by
Applied Biosystems.
A one-day ‘International Partnering Event’ to support technology
and know-how exchange, organised by the Innovation Relay
Center Network, will be held on 29 May 2003.
Conference Location
The Conference will be held at Villaggio Telis in Tortolì (Nuoro
province), in the Ogliastra region of Eastern Sardinia, Italy.
The village is a seaside resort with conferencing facilities.
The village offers affordable accommodation rates based on a
one week stay (Friday, 23 May to Friday, 30 May 2003) which
can be associated with inexpensive charter flights from Milan.
Other travelling and accommodation arrangements can be
organised upon request.
ake advantage of our End of Year Offer* until
31 December 2002 save 50% from List Price off your
first order for the cAMP-Screen Direct Immunoassay System.
T
plates and reagents for 200 assays.
Save Time and Money with the
cAMP-Screen Direct Immunoassay System
➜ Wide dynamic range – measure femtomole to nanomole
levels without dilution
➜ Precise – produce consistent results with typical
CVs below 5%
For more details on location and for travel information visit
the conference web page www.genosconference.it
Fellowships
A limited number of accommodation and registration
fellowships will be available for young researchers presenting
an abstract selected by the Scientific Committee.
Sensitivity of the cAMP-Screen Direct Chemiluminescent Immunoassay system
106
Each kit (part number T1505), includes two pre-coated 96-well
➜ Sensitive – detect femtomole levels of the cAMP
standard sample
Mario Pirastu, M.D. Director of the Institute of Population
Genetics, Italian National Research Council, Alghero (Italy),
President of Parco GENOS
28
New cAMP-Screen Direct™
Immunoassay System
➜ Saves time – get results up to 30 minutes faster than
other assays
Leena Peltonen, M.D., Ph.D, Professor and Chair,
Department of Medical Genetics and Molecular Medicine,
University of Helsinki and National Public Health Institute,
Finland
Alan Wright, MBChB, Ph.D., FRCP, Professor, Cell and
Molecular Genetics Section, MRC Human Genetics Unit,
Western General Hospital, Edinburgh, UK
Save 50% from List Price off your first order*
➜ Simplified protocol – use one assay plate for both cell
growth and lysis steps
Carole Ober, Ph.D. Professor, Department of Human Genetics,
University of Chicago, Chicago, Illinois, USA
Joseph Terwilliger, Ph.D. Assistant Professor, Columbia
Genome Center and Department of Psychiatry,
Columbia University, New York, NY, USA
end of year offer
To place your order either email us with your contact details, including
your purchase order number quoting ‘cAMP’ as your email subject to:
abdirect@eur.appliedbiosystems.com or contact your local Sales Engineer.
105
Relative Light Units
Organised by Parco GENOS, the Ogliastra Genetic Park Consortium
104
103
0 cells
5,000 cells
10,000 cells
20,000 cells
10-2
10-1
1
10
102
103
104
cAMP Standard (pmol/well)
The sensitivity of the cAMP-Screen Direct system was studied with four
levels of HEK293 cells grown in a cAMP-Screen Direct system plate.
The assay was performed with cAMP standards and signals were
measured with the TR717™ microplate luminometer.
Detection sensitivity of the exogenously added cAMP standards is
unchanged following growth of the cells.
Signal intensity differences result from basal cellular levels of cAMP.
To order online, visit our store at: https://store.appliedbiosystems.com
Please contact your local Applied Biosystems office if you don’t find your country in
the drop down menu on the registration page.
*Terms and conditions for this special offer: Orders can be placed by email, fax or
phone quoting a purchase order number. Offer valid until 31 December 2002 for
first order of up to 4 x cAMP-Screen Direct Immunoassay Kits. No other discounts
apply. All prices exclude delivery and local tax. E&OE.
For more information on:
cAMP-Screen Direct Immunoassay System enter:
No. 528
29
The whole genome, ready-to-use
The new Applied Biosystems Genomic Assays-on-Demand products are our unique effort to leverage information from both public and
Celera Genomics genome sequencing efforts. For the first time, this wealth of information and knowledge has been made accessible
through the most comprehensive on-line Assays-on-Demand database.
With the revolutionary offering of 200,000 human SNP assays, Assays-on-Demand SNP genotyping products will comprise of the largest
collection of biologically informative, validated, pre-designed assays available anywhere and anytime. We have already done all of the
background work so that you can focus on your research.
Following the approximately 14,000 well-characterised RefSeq transcripts, assays for the remaining human genes will be available,
finally resulting in a total of 30,000 human gene expression assays, including major splice variants of these genes.
Candidate gene association, candidate region association and linkage disequilibrium mapping studies that were never before possible,
are now within your reach! Explore the wealth of Genomic Assays and take advantage of our half price promotional offer.
*See next page.
30
31
The whole genome – it’s just a mouse click away:** https://store.appliedbiosystems.com
Receive a discount of 50% on one on-line order for up to 10 Assays-on-Demand™ products (either Gene Expression or SNP Genotyping).
*Registration, Terms & Conditions: For on-line ordering please register at the above web page. After registration, please contact your local
Applied Biosystems office or mail directly from the promotion button on http://europe.appliedbiosystems.com to receive your voucher with
your personal promotion ID number. Orders can only be placed through https://store.appliedbiosystems.com
For registration and ordering in France, please send an email with your full contact details to: oligofr@eur.appliedbiosystems.com
32
**Please contact your local Applied Biosystems office if you don´t find your country in the
drop down menu on the registration page.
The offer is valid until 31 December 2002. One offer only per customer number applies.
No other discounts apply. All prices exclude delivery and local tax. Prices are valid in local
currencies only. E&OE.
33
promotions
promotions
World Drug Discovery and
Development Summit 2003
T
he World Drug Discovery and Development
summit brings together the world’s leading drug
discovery and development practitioners from major
pharmaceutical and biotechnology companies with
providers of technology, equipment and services.
Applied Biosystems will be participating at the
next meeting, which will take place in Copenhagen,
17-19 February 2003.
John S. Loyer
Director, Worldwide Pharmaceutical Business Development
John’s business development interests include platforms and
services for high-throughput genotyping and gene expression.
Business interests in Mass Spectrometry include novel
applications in ADME, toxicology and biomarker discovery in
the drug development process.
Dennis A. Gilbert, Ph.D.
Vice President, Genomics
Dennis’ major responsibilities are to lead Applied Biosystems’
efforts to commercialise validated assays based on discoveries
from human genome for use by the research community in
studying gene expression and genetic variation.
Team Photo: L to R top row: Peter Strapps (UK), Daniel Rogers (UK), Kai-Uwe Brodersen (Germany), Kirsten Illum (Denmark), Viktoria Lindstrom (Finland)
L to R bottom row: Claire Gibbs (UK), Celine Assalit (Benelux), Rupa Ark (Team Leader In-House Sales, Northern & Southern Europe), Karine Bentot (France),
Jeanette Hallenbring (Sweden), Heike Kison (Germany). Other team members not in photo: Muriel Vincent and Christel Lelait (France), Elisabeth Cousin (Switzerland),
Rachel Pickles (UK) and Brigitte Fortnagel (Team Leader In-House Sales, Central Europe)
The programme also incorporates a selection of presentations
including top level Keynote Speakers.
KEYNOTE SPEAKERS
The event differs from the conventional conference or exhibition
as the summit is designed around the individual requirements
of the delegate and supplier representatives. The time-efficient
format incorporates conference sessions, one-to-one business
meetings, round table discussions and interactive workshops.
Time spent at the summit will represent an outstanding learning
and networking opportunity for all participants. Each attendee
will work with the organisers to generate a tailored programme
of activities that will ensure personal and business needs are
met. Attendees will be able to meet with other delegates and
suppliers on an individual basis to discuss topics and issues of
importance to them and their business.
Key Senior Management members from Applied Biosystems will
be attending this summit to meet with delegates. By attending
you will have the opportunity to meet with these key players in
genomics and proteomics.
Stephen A. Martin, Ph.D.
Director, Discovery Proteomics
Stephen is responsible for Proteomics at Applied Biosystems
and is working to better understand the key technologies that
will revolutionise this field.
34
Introducing you to the
European In-House Sales Team
A New Sales Channel from Applied Biosystems
Dr J Craig Venter
President of the Center
for the Advancement
of Genomics
Jutta Heim Ph.D.
VP and Sr. Scientific
Expert Molecular
Biology
Novartis Pharma AG
Klaus Lindpaintner
Executive Vice
President, Director
Roche Genetics
Roche
So book the dates in your diary and attend this prestigious event.
Supported by
14 Shepherdess Walk, London, N1 7LB, UK
Tel: +44 (0)20 7566 4800 Fax: +44 (0)20 7336 0707
www.wdd.worldtradeco.com
For more information on:
World Drug Discovery and Development Summit enter:
No. 529
A
s our product portfolio is growing, at Applied Biosystems we
are constantly looking to improve our quality and efficiency
of sales service to our customers. We know that scientists’ time
in the laboratory is very valuable and occasionally you need to
make urgent purchasing decisions for products like thermal
cyclers, PCR reagents and Oligos. So keeping this in mind,
we have set up a new, fast and effective sales channel for all
of these products, called ‘The European In-House Sales Team’.
All team members are qualified and experienced molecular
biologists and are able to help you choose the right
product for all your PCR needs. The In-House Sales Team
works in partnership with the Field Sales Team to provide a
fast and efficient customer service.
To contact your local In-House Sales person see details below:
By contacting your local In-House Sales Team, you can
get the latest information on PCR products, discuss your
requirements, obtain a competitive quotation and place an
order. From time-to-time your local In-House Sales Team will
keep you informed about special offers and promotions.
This new sales channel is currently available in the following
countries: Austria, Belgium, Denmark, Finland, France, Germany,
The Netherlands, Norway, Sweden, Switzerland and UK.
UK
Tel: +44 (0)1925 282601
Denmark
Tel: +45 45 58 60 00
Norway
Tel: +47 23 16 25 75
Finland
Tel: +358 (0)9 693 79427
Sweden
Tel: +46 (0)8 619 4451
France
Tel: +33 (0)1 69 59 95 00
Benelux
Tel: +31 (0)180 392 357
Germany
Tel: +49 (0)6151 96 700
Austria
Tel: +43 (0)1 867 35 75 0
Switzerland Tel: +41 (0)41 799 77 77
35
customer relations
promotions
Michael D. O'Neill, Editor
BioBeat Online Magazine
®
BioBeat Online Magazine Wins
Publishing Excellence Awards
A
pplied Biosystems' BioBeat Online Magazine and its
writers have just received six APEX 2002 Awards for
Publication Excellence. This marks the fourth consecutive year
that BioBeat Online Magazine has been recognised by these
prestigious industry awards. The number of awards represents
the highest number ever received by BioBeat in a single year,
doubling the number of awards received last year.
Other APEX Award winners this year included the Walt Disney
Company, WGBH-TV in Boston, FedEx, Daimler-Chrylser, IBM,
ESPN, NASA, Exxon Mobil, Time, the American Red Cross,
Abbott Diagnostics, the World Wildlife Fund, Hemispheres
Airline Magazine, Princeton University, Compaq Computer,
Merck, Roche Diagnostics, and Toyota.
Offer Clues to Primate Evolution, Gene Birth, and Human
Disease” by Michael D. O'Neill); and News Writing
(“Sweet-Tooth Gene Identified” by Michael D. O'Neill).
The “Sweet-Tooth Gene” story was published in Biosystems
Solutions magazine issue 3, page 13. The other articles were
published in BioBeat Online Magazine (http://www.biobeat.com)
and may be viewed online.
BioBeat Online Magazine covers the world of life
science research that is enabled by technology from
Applied Biosystems. BioBeat has approximately 6,000
subscribers in 109 countries around the world, and currently
provides over 120 articles on research advances enabled
by Applied Biosystems’ technology.
APEX 2002 is the 14th annual awards program recognising
excellence in publication work by professional communicators.
APEX Awards are presented on the basis of excellence in
graphic design, editorial content, and the ability to achieve
overall communications excellence.
BioBeat Online Magazine won its APEX Awards this year for
excellence in the categories of Online & Electronic
Magazines & Journals and Web & Intranet Site Content &
Writing. BioBeat writers won for excellence in the categories
of Online Writing (“Chemokines Implicated in Spread of
Cancer Cells” by Mark Springer; “Genome Scan of
Centenarians Reveals Locus for Exceptional Human Longevity”
by Michael D. O'Neill); Technical Writing (“Morpheus Genes
36
Free Subscription to BioBeat Online Magazine
To obtain a free subscription to BioBeat Online Magazine,
simply go to the BioBeat site (http://www.biobeat.com),
click on the SUBSCRIBE button, and fill out the online
subscription form. BioBeat subscribers receive email notices of
story postings, periodic emailings of a calendar of upcoming
conferences (with URL links to conference web sites), and lists
of recent journal articles featuring use of products from
Applied Biosystems (with URL links to article abstracts).
Release of powerful new ABI PRISM®
3100 Data Collection Software v1.1
Available free-of-charge to ABI PRISM 3100 Genetic Analyzer Users
T
he upgrade includes a new module that allows ultra-rapid
sequencing in just 40 minutes. It also requires only one
polymer type and array length to perform multiple applications.
Sequencing and genotyping samples can now be analysed
automatically, quickly, and with high accuracy, without
changing either the polymer type or capillary array length.
Designed for the ABI PRISM 3100 Genetic Analyzer, the new
3100 Data Collection Software v1.1 provides much greater
flexibility for laboratories engaged in a wide variety of
applications, including SNP genotyping and validation,
microsatellite analysis, and sequencing.
If you are an ABI PRISM 3100 Genetic Analyzer User and
haven’t registered for your free upgrade then please email us
with your contact details and your instrument serial
number, quoting ‘3100 collection software’ as your subject:
abdirect@eur.appliedbiosystems.com. Or alternatively, contact
your local Sales Engineer.
37
customer relations
customer relations
Positioned to integrate
Product line(s) & technology
Drive creation of new/expanded systems
The results enable us to qualify the benefits of new approaches
to proteomics from sample preparation through to data
analysis. Joint papers and presentations have been released
with additional work in process.
Future Directions
Over 100 groups involved in proteomics have now visited the
PRC and met with the scientists to discuss alternative
approaches and future directions of proteomics and advanced
protein analysis. These tours and meetings have allowed
constructive two-way exchanges of ideas and opinions
concerning a wide variety of techniques and scientific
objectives. In this way Applied Biosystems looks to ensure
research and development are in line with customer needs and
future expectations.
The Technology
Application workflows of greatest interest today include
LC/ESI MS/MS and LC-MALDI MS/MS workflows as well as new
approaches such as ICAT reagent based quantitation and
identification. New platforms such as the 4700 Proteomics
Analyzer, the Q TRAP™ LC/MS/MS System and the API QSTAR®
Pulsar Hybrid LC/MS/MS System are the workhorses,
with multiple units running almost continuously. Evaluation of
new innovations or early versions of new features is blended
into the daily and weekly project schedules. Various MDLC or
gel based workflows are part of the capability within the center.
Applied Biosystems Proteomics
Research Center advances into
its 3rd year of operation
T
he Proteomics Research Center (PRC) located
in Framingham, Massachusetts continues to
engage in projects that push the limits of technology
with real world proteomics applications to ensure
Applied Biosystems remains on the ‘cutting edge’ of
Proteomics as it moves into its third year of operation.
The team has had a significant and diverse list of
accomplishments since the founding 2 years ago, and its
charter remains the same; a focus on maximising
Applied Biosystems’ existing proteomics expertise and
internal research capabilities, while facilitating collaborations
with complementary biology and technology partners.
The goal is to achieve advances in the production of high
quality proteomic information by means of significant
improvements in throughput, automation, protein coverage
and cost efficiency.
38
The Group
The PRC Staff is currently 14 dedicated scientists with expertise
in mass spectrometry, sample preparation, separations,
protein chemistry, LIMS and bioinformatics analysis.
In addition, associate members from Applied Biosystems'
multiple technology groups collaborate with the PRC on
projects such as ICAT™ reagent technology, automated protein
ID and characterisation, and software development.
Most important are the external collaborators and scientists
from institutions and companies world wide, working with the
PRC staff in Framingham. Armed with qualified and interesting
biological samples, including liver cancer, bacteria, yeast,
plasma, rice, human fibroblasts and other complex materials,
these collaborations bring real-world research problems into
new, advanced proteomic workflows.
The PRC is also the scientific and technological hub for
Applied Biosystems’ proteomics activities. A partial list of
PRC collaborations include:
➜ Celera Genomics
➜ University of Geneva
➜ Northeastern University
➜ Millipore Corporation
➜ Myriad Proteomics
➜ University of California, San Francisco
➜ Institute of Medical Science, University of Tokyo
For a virtual tour and more information about the PRC visit:
www.appliedbiosystems.com/apps/proteomics/prc/nav.swf
Learn more about some of the latest advances in instruments,
systems and proteomics workflows currently being used to help
identify and break the bottlenecks that inhibit the pace of
proteomics research. The PRC tour is continually updated to
include the most advanced tools available, and how they are
being integrated into workflows to address key problems in
proteomics today.
The research collaborations span the areas of:
➜ Sample Preparation
➜ New Separation Techniques
➜ Mass Spectrometry Software
➜ Protein Identification
➜ Phosphorylation Analysis
➜ Complex Differential Expression Workflows
➜ Labelling Chemistries including ICAT Reagents
Advances from these projects are expected to become key
elements of the next generation proteomics projects from
Applied Biosystems.
For more information on:
The Proteomics Research Center enter:
No. 530
39
customer relations
customer relations
Many products and services introduced this year offer increases
in automation, throughput and enhanced ability to identify and
distinguish proteins. These and a lot more besides will be
available for customer services or for demonstrations.
In addition to its proximity to nearby Frankfurt, Darmstadt,
also referred to as the Science City (Wissenschaftsstadt),
has a number of important pharmaceutical organisations,
such as Merck KgaA, and smaller biotechnology companies,
as well as the Technical University of Darmstadt.
Model of new building In Darmstadt, Germany, due to open in October 2002,
the Darmstadt site in July 2002, and an artist’s impression of the spacious new customer welcoming area.
New extension to the main European Distribution Centre
The Netherlands.
The Applied Biosystems’ site at Nieuwerkerk a/d IJssel
(Applera Nederland) is situated a few kilometres north of
Rotterdam and houses the main European central distribution
facility as well as the European Repair Centre (ERC), opened in
2000. Once again there was a need to expand to make way for
additional staff and space for looking after the receipt
and shipping of all instruments for Europe and beyond,
including the European Managed Territories of the Middle East,
West Asia, Africa and Central/SE Europe and CIS.
The combined Sales, Support and Service organisation remain
on-site as well as the European Headquarters for Information
Technology. Warehouse space totals 1,500 sq. meters along
with a little known site at Schiphol airport of 500 sq. meters.
Applied Biosystems expanding...
pplied Biosystems Europe has been in existence
almost as long as the company itself, which was
established in 1981 in California, USA. The European
operations have grown from one office in Germany in
1983 to operations in over 30 countries. Over the last
five years the company has grown significantly and has
been able to expand its services and to add employees
exponentially to meet customer demands. Sustained growth
and expansion of the company and its product portfolio
in the Life Science sector has meant that larger and more
integrated facilities were required.
The need to stretch out from the original and start-up working
environments that were created at the early stage of
developments in Europe had been essential at the three major
facilities within Europe, some of which were already the largest
and are amongst the most important sites within the company.
The process started late last year with the UK organisation
acquiring a new facility in Warrington, an existing site at our
European Distribution Centre (EDC) near Rotterdam being
scheduled for redevelopment and lastly, a large and
completely new development that was opened in October of
this year in Germany.
40
Two into one in Germany
In October 2001 Applied Biosystems, (Applera Deutschland
GmbH), announced that the building of a new facility
for its corporate offices, laboratory and training facilities
in Darmstadt, Germany, would start. The company,
which employs approximately 230 people in Germany,
currently has its offices in Langen and Weiterstadt,
(both sites being in close proximity to Frankfurt).
The 6,100 square metre facility will integrate all
organisational capabilities and functions under one roof,
effectively enhancing communications between the
composite parts of its sales, support and service network
along with the Oligo manufacturing facility.
The facility in Darmstadt (30 km south of Frankfurt) will have
a new state-of-the-art laboratory infrastructure that will allow
Applied Biosystems the opportunity to further develop its
services in the Life Science sector. Applied Biosystems will
continue to use these facilities as training premises for its
growing customer base in Germany, as well as opening a
new European Proteomics Research Centre. The new training
facilities, with state-of-the art presentation equipment will
contribute to the expected success of this initiative.
New Sales, Support and Service site in Warrington, United Kingdom
The UK office has been based in Warrington, Cheshire,
since 1985 and although our nearby manufacturing site
at Woolston will remain in its current location, the Sales,
Support and Service Organisation has continued to expand
and relocated to larger premises in November 2001.
Close to the UK’s newly acquired office at the recently
opened North West Technology Park in Warrington, our
manufacturing facility is now offering a new Assays-by-DesignSM
service and Assays-on-Demand™ products – one of the first in
Europe. This enables scientists to obtain ready-made assays
for their research needs.
to support our European Customers
A
organisation. With significant new developments in DNA
research and DNA Sequencing techniques being introduced
from Applied Biosystems since 1993, the UK site has achieved
a sales and service growth rate averaging 23% year-on-year;
maintaining its position as a core life science instrument,
software and services business in Europe.
Facilities contained within the overall 40,000 square metre
site, is a conference room for 140 people, video conferencing,
11 meeting rooms, 5 training rooms and 8 large demonstration
laboratories most of which are conveniently positioned on
the ground floor of the new building.
Applied Biosystems in Nieuwerkerk, The Netherlands
The new office wing will be added to the existing building
and total an additional 1,250 sq. meters of space,
adding space for 65 new or existing employees. As in all new
facilities in Applied Biosystems, conference rooms with
separate video conferencing is available, and a new training
centre added, the largest conference room holding up to 120
people. Employees and visitors alike should enjoy a larger
restaurant. The new wing opened in mid-July of this year.
Undaunted by the market trends of 2002, which has seen
many companies treading carefully or reducing capacity,
Applied Biosystems in Europe has been able to move with the
times and by the end of this year the company will be in a very
strong position to maintain and improve services to customers
and employees alike. Thus, bringing together the essential
components of a highly successful and potent life science
company, fully equipped and prepared to support existing and
new customers.
A newly established site in the UK
The UK branch of Applied Biosystems (Applera UK) was
established in 1985 and since that time has grown
considerably to become a major resource within the European
41
customer relations
customer relations
GEN U I N E
SERVICE CO N T R A C TS
Applied Biosystems at the
34th European Conference of
Human Genetics, Strasbourg, 2002
A
pplied Biosystems was one of the main sponsors
of the 34th European Conference of Human
Genetics held in Strasbourg, France, 25-28 May 2002.
Our new tools for enabling discoveries using the genome
were presented at our Applied Biosystems’ 28 square metre
booth and during one of the satellite meetings. The ABI PRISM®
3100-Avant Genetic Analyzer, the ABI PRISM® 7000 Sequence
Detection System and the exciting Genomic Assays initiative
were highlighted and exhibited to the 1,500 international
attendees of the conference.
Lajos Kalma'r is the lucky winner of the XBOX. He is a 25 year
old Ph.D. student from the National Medical Center of
Budapest, Hungary. His research work is currently at the
Institute of Haematology and Immunology, supervised by
Prof. Attila Tordal. Lajos’ main projects are Primary Congenital
Glaucoma (PCG) and Hereditary Angioneurotic Eudema (HAE).
He works with an ABI PRISM® 310 Genetic Analyzer from
Applied Biosystems, for DNA sequencing and mutation
detection. The HAE project is in collaboration with a
European concern led by Prof. Mario Tosi from the Faculté de
Médecine of Rouen, France.
After completing his Ph.D., Lajos intends to look for a
research position. Applied Biosystems wishes him well with
his future career and hope that they can contribute to his
research success.
Who better to service your instruments
than the company that built them?
I
n an ever more regulated environment, the need to
maintain and track the performance of your
instrument is vital. This and many other lessons learned from
our customers have led us to a complete review of our service
offerings. You can now choose between 5 different agreement
plans that are tailored to your needs. Along with the benefits of
each agreement, you will be assured of priority breakdown
response and free access to our help desks for service and
technical support issues.
New Service Agreement Plans
(commenced September 2002)
BioAssurance™ Premium Plan
Parts + Labour + Travel + 2 Planned Maintenance (PM) Visits
Provides the complete package for all your service and
maintenance needs. Travel and site time, as well as all parts,
are included in the agreement price. Your instrument will be
regularly maintained with 2 PM visits.
BioAssurance™ Plus Plan
Parts + Labour + Travel + 1 Planned Maintenance Visit
Provides a similar package as described for BioAssurance
Premium, with a single PM visit.
BioParts™ Plan
Parts + Planned Maintenance Visit(s)
Provides cover for all replacement service parts plus an annual
PM visit (2 PM visits for some instrument types)
BioRepair™ Plan
Labour + Travel + Planned Maintenance Visit(s)
Covers all engineer travel and site time and provides an
annual planned maintenance visit (2 PM visits for some
instrument types)
BioMaintenance™ Plan
Regular Planned Maintenance Visit(s)
Provides an annual fixed-price planned maintenance visit
(2 PM visits for some instrument types)
For more information on:
New Service Agreement Plans enter:
No. 531
Customer Training Courses
Call Eve Lightfoot 01925 282551 or Josephine Kinsey 01925 282475
The lucky winner of the XBOX at our booth, Lajos Kalma'r with Fabienne LeFloch,
Applied Biosystems, France. Katheryne Dong (in front), daughter of Penny Dong
who is working in R&D Foster City, drew the winner’s entry in the prize draw.
Visitors to our booth were invited to participate in a prize draw.
They were asked to answer a number of questions on products
and applications from Applied Biosystems. This gave them the
chance to win a Microsoft® XBOX™ game station.
42
ABI PRISM® 3100 Genetic Analyzer
12-13 December
No. 532
ABI PRISM 7700 Sequence Detection System User Training
03-04 December
No. 533
10-11 December
No. 534
ABI PRISM® 7900 Sequence Detection System User Training
05-06 December
No. 535
HID Training
09-11 December
No. 536
®
ABI PRISM® 7000 Sequence Detection System User Training
21-22 November
43