Solutions for Protein Biomarker Research
Protein Biomarker Discovery, Verification and Validation Powerful workflows built on solid science Biomarker research includes quantitative characterization of protein mixtures in order to understand complex biological systems and determine relationships between the functions of proteins across a large population of samples. The goal of biomarker research is to discover and validate markers that can be used in clinical research applications such as patient stratification, diagnosis and therapeutic monitoring, or in pharmaceutical development to fully characterize the behavior of candidate drugs.
As a leader in LC/MS/MS proteomics solutions, Applied Biosystems/MDS Analytical Technologies is committed to the development and application of powerful tools and workflows to help researchers move quickly and confidently from putative biomarker discovery to candidate verification and validation. New directions in biomarker research
Eliminating the Discover to Validation Bottleneck In general, biomarker research follows a continuum that to validation, a verification step can screen potential begins with the discovery and proceeds through validation biomarkers to ensure that only the highest quality to the eventual implementation of the biomarkers in leads from the discovery phase are taken into the a clinical setting. Biomarker discovery requires high costly validation stage. The ability to take fewer, confidence identification of biomarker candidates with higher quality leads into the validation stage will have simultaneous quantitation information to indicate which significant cost and time saving benefits. A verification proteins are changing to a statistically relevant degree in stage requires a high throughput workflow that response to a given environmental change, drug treatment, provides high specificity, sensitivity, requires minimal or disease. Biomarker candidates identified in discovery sample preparation, and can provide identification need to be validated using larger sample sets covering a confirmation. Additionally, a verification stage can broad cross section of patients or populations. To avoid a result in measurement methods that can be used potential bottleneck of taking a large number of candidates in validation.
Biomarker Workflow Overview
clinical discovery verification validation application
Advancing Biomarker Research Applied Biosystems/MDS Analytical Technologies is setting new standards for biomarker research with powerful mass spectrometry based workflows for biomarker discovery, verification, and validation. Faster, more efficient discovery Protein biomarker discovery
Low Abundance Demands In-Depth Coverage Identify and Quantify Biomarkers in One Experiment One of the biggest challenges in biomarker discovery is the difficulty of identifying medium to low abundance proteins in complex biological samples. For example, human plasma has over 1x106 different protein molecules with an estimated dynamic range of 1010 (Anderson, J. Physiol 563.1 (2005), 23-60). Of these, the 22 most abundant proteins make up 99% of the protein content of plasma, and the abundance of a given biomarker will fluctuate within a sample population. A robust biomarker discovery workflow must be capable of uncovering a panel of biomarkers in samples such as human plasma, with unambiguous identification and quantitative characterization. The Applied Biosystems biomarker discovery workflow meets this requirement, with high quality data and in-depth coverage. An Easier Way to Get Better Results Multiple proteins can be quantified and identified across multiple samples in a single run. In this study Chaperonin 10 was confirmed to be up-regulated Faster Discovery Through Multiplexing in endometrial cancer tissue, while several other proteins were down- regulated. The 114 and 115 peaks are from control samples. The 116 and 117 Applied Biosystems’ biomarker discovery workflow peaks are from cancer tissue. overcomes many of the time and reproducibility challenges of profiling-based experiments. It takes BIOiTRAQ™ systems are optimized to enable high advantage of the multiplexing capabilities of iTRAQ® confidence biomarker discovery workflows, with the ability reagents to provide high-confidence identification of to perform both label-based and label-free experiments. medium to low-level candidate biomarkers and relative Industry leading MS platforms, combined with the new quantitation of multiple samples in a single run. Tempo™ MDLC system and iTRAQ® reagents, provide high sensitivity and reliability. The innovative ProteinPilot™ • Multiple samples in a single analysis software with the novel Paragon™ search algorithm • Simultaneously identify and quantify candidate increases the number of proteins identified, while biomarkers in biological fluids, tissues, and cells minimizing false positives, and Pro Group™ algorithm • Perform multiple quantitative measurements per improves protein ID accuracy and minimizes the reporting protein for added precision and confidence of incorrect protein isoforms, and MarkerView™ software is • Run samples and/or controls in duplicate available for statistical data analysis and MALDI and LC/MS profiling workflows.
discovery verification validation
Starting out with Confidence The Applied Biosystems biomarker discovery workflow has been used to identify markers such as protein kinases, signal transduction proteins, and structural proteins in a broad range of biological samples, including plasma, urine, saliva, and tissues 11 3 192 11 3
11 4 191 m/z 11 4
11 5 190 11 5 MS MS/MS 11 6 189 + PRG 11 6 + Peptide + peptide Fragments 11 7 188 11 7
11 8 187 11 8 +PRG +peptide 11 9 186 11 9
121 184 121
8 samples labeled (identical m/z) Peptides of same m/z from all Reporter Ions for quantitation and and mixed 8 samples coelute peptide fragments for sequencing
116 118
115
121 113 114
117 119 y5 y2 y3 % Intensity b1 b3 y6 b7 y7 y4 y1 y8 b6 b2 b4 b5
Mass (m/z)
iTRAQ® Reagent Protein Discovery Workflow iTRAQ reagent labeled peptides produce strong signature ions for multiplexed quantitation, while also providing sequence information for peptide identification.
Systems for Biomarker Discovery
The BIOiTRAQ™ Discovery System QS with The BIOiTRAQ™ Discovery System TT with the the QSTAR® Elite LC/MS/MS System 4800 Plus MALDI TOF/TOF™ Analyzer
• Greatest speed and accuracy • Greatest depth of coverage • Powerful, no compromise versatility • Intelligent LC/MALDI capabilities • High sensitivity and dynamic range • High sensitivity with MALDI ease of use A faster path from discovery to validation Biomarker candidate verification and validation
Narrowing the List to the Most Promising Candidates Because of normal clinical or biological variability, be moved forward to more rigorous validation, focusing candidate biomarkers identified in the discovery stage entirely on fully identified, verified markers, saving time and need to be validated across a large number of samples. cost while also improving assay quality. The challenge is to develop a fast, targeted analysis method capable of analyzing as many identified candidates ™ as possible in hundreds or even thousands of samples. MIDAS Workflow for Hypothesis-Driven Verification ™ This can create a time-consuming and expensive The MIDAS workflow develops high sensitivity, high bottleneck, especially if antibodies or synthetic peptides specificity assays with the automated ability to confirm ™ are required. peptide identification. MIDAS workflow overcomes the need for separate methods development and provides A biomarker candidate verification phase eliminates this the ability to go directly from proteomic or genomic-based bottleneck by ensuring that only the most promising discovery workflows to the development of biomarker putative biomarkers found in discovery go on to the verification assays. validation phase. The MIDAS™ workflow enables the multiplexed verification of 10s to 100s of candidate • Assay broad panels of markers with minimal sample biomarkers per analysis prior to validation. Complex preparation samples such as plasma or tissue samples require • Design multiplexed MRM assays in silico minimal sample preparation and can be rapidly analyzed in • Eliminate the need for antibodies, synthetic peptides minutes. After narrowing the number of candidates in this or protein standards verification step, the most promising candidates can then • Move quickly from biomarker discovery to validation
MIDAS Workflow
MSAIQAAWPSGTECIAKYNFHGTAEQD 10 0 90
LPFCKGDVLTIVAVTKDPNWYKAKNKV R A V V A H A A V 80 615.4 631.4 GDVLTIVANVTK GREGIIPANYVQKREGVKAGTKLSLMP 70 WFHGKITREQAER LLYPPETGLFLVRE 763.9 814.5 LLYPPETGLRLVR 60 50 STNYPGDYTLCVSCDGKVEHYRIMYHA y1
% Intensity b9 743.4 813.4 SIDEEVYFENLK 40 b2 b3 b4 SKLSIDEEVYFENLK MQLVEHYTSDAD b6 % Intensity % 30 b7 y5 b5 b8 GLCTRLIKPKVMEGTVAAQDEFYRSGW 679.8 754.4 GEFGDVMLGDYR b1 20 y2 y4 y6 y3 y7 y8 ALNMKELKLLQTIGKGEFGDVMLGDYR 10 y9 y10 GNKVAVKCIKNDATA… 0 9.0 260.4 511.8 763.2 1014.6 1266.0 Time (min) Mass (m/z) Protein Sequence In silico MRM transitions MRM-Relative quantitation MS/MS Identification
discovery verification validation Move on to Validation with a Proven Method Methods developed in the verification phase with the MIDAS™ workflow are directly applied to the validation phase, using isotopically labeled synthetic peptides as standards. The MIDAS™ validation workflow leverages the long-term strength of the MRM quantitation strategy, providing the highest sensitivity and accuracy for targeted quantitation. The high throughput, high sensitivity MRM scan function of the MIDAS TRAQ™ Validation systems with 104 linear dynamic range is ideally suited for the quantitation demands of a successful validation phase.
The MIDAS workflow enables development of methods that can monitor Systems Optimized for MIDAS Workflows 10s to 100s of protein biomarker candidates in a single run, covering a ™ wide dynamic range of abundances with high sensitivity and specificity. MIDAS TRAQ Validation systems simultaneously measure Candidate biomarker identification can also be confirmed in the same large numbers of putative protein biomarkers in complex experiment. The 104 linear dynamic range provides excellent quantitative ™ performance. biological samples. MIDAS Workflow Designer software enables in silico multiplexed MRM assay design based on gene or protein sequences and allows rapid identification of the best peptides for any sequence-defined protein target before synthetic peptides are required.
Systems for Candidate Verification and Validation
The MIDAS TRAQ™ Validation System with the The MIDAS TRAQ™ Validation System with the 4000 QTRAP® and Tempo™ nano MDLC System 3200 QTRAP® and Tempo™ nano MDLC System
• Highest sensitivity • Single experiment quantitation and ID confirmation • Outstanding qualitative performance • Economical solution • Wide dynamic range for quantitation • High sensitivity and quantitative performance
discovery verification validation
Taking Better Candidates to Validation The Applied Biosystems candidate verification workflow saves time by filtering potential biomarkers so that only the best candidates are taken forward to validation. Leading the way in biomarker research
Powerful workflows open up new possibilities for biomarker research that may lead to improved clinical assays and faster, more robust drug discovery and development. Applied Biosystems/MDS Analytical Technologies can help your laboratory turn these possibilities into reality with a broad portfolio of chemistries, software, LC/MS/MS systems and expert applications support. When you choose Applied Biosystems/MDS Analytical Technologies as your partner, you’re choosing to work with the leading developer of solutions for every stage of biomarker research. And, we are committed to remaining at the forefront by providing you with the tools you need to succeed.
Support You Can Depend on—Whenever and Wherever You Need it Our products are backed by one of the world’s most extensive service and support organizations staffed by professionals who are trained and dedicated to keep your system running at peak performance and productivity—24 hours a day, seven days a week.
For more information about Applied Biosystems/MDS Analytical Technologies mass spectrometry solutions, please call the sales office nearest you or visit www.appliedbiosystems.com
For Research Use Only. Not for use in diagnostic procedures. Applied Biosystems, AB (Design) and iTRAQ are registered trademarks and BIOiTRAQ is a trademark of Applied Biosystems or its affiliates in the US and/or certain other countries. QSTAR and QTRAP are registered trademarks and Tempo, ProteinPilot, Pro Group, MarkerView, MALDI TOF/TOF, MIDAS and MIDAS TRAQ are trademarks of Applied Biosystems/MDS Analytical Technologies, a joint venture between Applied Biosystems and MDS Inc. Other trademarks are the sole property of their respective owners. © Copyright 2009 Applied Biosystems and MDS Inc. Joint Owners. All Rights Reserved. Printed in the USA, 02/2009 Publication 114BR14-04
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