Selected Reaction Monitoring Christine A. Jelinek, Ph.D.
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1 Selected Reaction Monitoring Christine A. Jelinek, Ph.D. Johns Hopkins University School of Medicine Department of Pharmacology and Molecular Sciences Middle Atlantic Mass Spectrometry Laboratory
2 Selected Reaction Monitoring Lecture Agenda Selected Reaction Monitoring: Technique Selected Reaction Monitoring: Instrumentation Quantitation: Absolute vs. Relative Selected Reaction Monitoring: Method Development Selected Reaction Monitoring: Application Selected Reaction Monitoring: Case Studies
3 Selected Reaction Monitoring: Technique
4 Selected reaction monitoring (SRM) SRM is a mass spectrometry technique for the detection and quantitation of specific, pre-determined analytes SRM selectively monitors specific analyte molecular ion and several fragment ions generated from the analyte by collisional dissociation Most effectively used in an LC-coupled MS system Exploits the unique capabilities of triple quadrupole instruments to act as mass filters Originally applied to the measurement of small molecules SRM allows for the measurement of proteins in low µg or high ng/ml range Picotti P and Aebersold R Nat Methods :
5 Selected Reaction Monitoring Mass Spectrometric Assay Elliott MH et al. J Mass Spectrom : Q0 Q1 Q2 Q3 RF only Scanning RF/DC RF only Collision Cell Scanning RF/DC November 27,
6 Selected Reaction Monitoring Mass Spectrometric Assay Elliott MH et al. J Mass Spectrom : Each peptide has a unique mass and unique sequence fragments G A G Q N I I P A S T G A A K X X X Q1 passes only the molecular mass Q3 passes three selected fragments y11 y9 y7 6
7 Selected Reaction Monitoring Mass Spectrometric Assay Output Peak Area November 27,
8 Selected Reaction Monitoring Mass Spectrometric Assay Output Peak Area Peptide Sequence Transition Ion AUC HQQQFFQR y HQQQFFQR y HQQQFFQR y HQQQFFQR y HQQQFFQR y November 27,
9 Selected Reaction Monitoring Mass Spectrometric Assay Platform Selectivity Specificity Sensitivity Mass filters pass only targeted peptides even when ion signal is below noise Pre-select unique transitions ions during detection High duty cycle. LODs 2-3 orders of magnitude better than scanned spectra 9
10 Selected Reaction Monitoring: Instrumentation
11 Selected Reaction Monitoring Using Triple Quadrapole Mass Spectrometers
12 Triple Quadrapole Mass Spectrometers Jim Morrison of LaTrobe University, Australia first developed the QQQ arrangement for the purpose of studying the photodissociation of gasphase ions. The first triple-quadrupole mass spectrometer was developed at Michigan State University by Dr. Christie Enke and graduate student Richard Yost in the late 1970s.
13 Quantitation: Absolute vs. Relative
14 Quantitation Strategies Picotti P and Aebersold R Nat Methods : Quantitation Relative (differential) quantitation Labeling Label-free Metabolic stable-isotope labeling Quantitation strategy [ 15 N]ammonium sulfate SILAC Absolute quantitation Chemical stable-isotope labeling Enzymatic stable-isotope labeling Metabolic stable-isotope labeling Chemical stable-isotope labeling ICAT, itraq mtraq [ 18 O]water QconCAT, PSAQ AQUA synthetic peptides
15 Method Development Strategy Peptide Standards Labeled Standards: PEPTIDEK* LAGKPEPTIDEKLAG*.KPEPTIDEK * PEPTIDE* Internal Standards: Non-biomarker peptide present within sample Used to minimize run-to-run variability from processing and instrumentation Panel or single peptide can be selected External Standards: Heavy isotope labeled standards can be added to each sample Panel of biomarker peptides, a single biomarker peptide, a panel of biomarker proteins, or a single protein can be used Isotope labeling directs absolute quantitation schema Non-labeled non-biomarker peptide or protein can also be used
16 Relative Quantitation: Replicate Comparison 16
17 Relative Quantitation: Replicate Comparison 17
18 Relative Selected Target Quantitation: Peptide Calibration Curve using External Standard 18
19 Area Relative Quantitation: Calibration Curve using External Standard LOD In Solvent LOD of Apomyoglobin in Solvent y = x R² = Concentration of apomyoglobin (fmol/ul) Target Peptide NDIAAK Conc (fmol/ul) RT/min FWHM Start Time End Time Area Background Height
20 Area Relative Quantitation: Calibration Curve using External Standard LOD In Matrix LOD of Apomyoglobin in digested CSF y = x R² = Concentration (fmol/ L) Target Peptide NDIAAK Concentration (fmol/ L) Average Area Standard Deviation
21 Absolute Quantitation: Calibration Curve using heavy labeled IS peptides Replicate IEAPQDIK IEAPQDIK* (heavy) Standard_1 40 fmol/ul 40 fmol/ul Standard_ fmol/ul 12.5 fmol/ul Standard_3 5 fmol/ul 5 fmol/ul Standard_4 2.5 fmol/ul 2.5 fmol/ul Standard_5 1 fmol/ul 1 fmol/ul Standard_6 0.5 fmol/ul 0.5 fmol/ul Standard_7.25 fmol/ul.25 fmol/ul Standard_8.1 fmol/ul.1 fmol/ul Stergachis, A. et al. Nature Methods. 2011; 8:
22 Absolute Quantitation: Calibration Curve using heavy labeled IS peptides Peak Area Retention Time Representative Ion Chromatogram Stergachis, A. et al. Nature Methods. 2011; 8:
23 Absolute Quantitation: Calibration Curve using heavy labeled IS peptides Peptide Sequence Protein Name Replicate Name Ratio to Standard IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag FOXN1-GST Stergachis, A. et al. Nature Methods. 2011; 8:
24 Absolute Quantitation: Calibration Curve using heavy labeled IS peptides Peptide Sequence Protein Name Replicate Name Ratio to Standard [fmol /ul] IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag Standard_ IEAIPQIDK GST-tag FOXN1-GST Stergachis, A. et al. Nature Methods. 2011; 8:
25 Topic: Assay Development Selected Reaction Monitoring
26 Selected Reaction Monitoring Mass Spectrometric Assay Workflow 18 Hour Tryptic Digestion Solid Phase Extraction Speedvac and Resuspension C18 Reverse Phase HPLC SRM-MS Detection SRM- Assay Dissertation Defense November 27, 2012 April 5,
27 NDIAAK (11.3) ASEDLK (12.9) LFTGHPETLEK (21.4) VEADIAGHGQEVLIR (23.9) ELGFQG (24.6) ALELFR (28.0) HGTVVLTALGGILK (31.2) YLEFISDAIIHVLHSK (37.7) Method Development Strategy Signature Peptide selection Digested horse apomyoglobin Tryptic Myoglobin Peptides 27
28 Method Development Strategy Signature Peptide selection Signature Peptide Selection Criteria: Minimum peptide length Maximum peptide length Exclude N-terminal processing peptides Exclude potential ragged ends Exclude peptides containing: o Met o Cys o His o NXT/NXS o RP/KP 28
29 Method Development Strategy Transition Ion selection Selected Transitions y2 TCVADESAENCDK y2 y3 y4 y5 y6 y7 y8 y9 y10 y11 y7 Transition Selection y10 Total Ion Chromatogram 29
30 Method Development Strategy Transition Ion selection Iterative Method Development Initial 1 Peptide SRM Transition Selection Poor performing Transitions eliminated from subsequent experiments 30
31 Intensity (10 3 ) Typical Limit of Detection β-galactosidase signature peptide in sera matrix GDFQFNISR Experimental Detection Range y = x R² = b4 y3 y Retention Time (mins) Sample Concentration (fmol) November 27,
32 Method Development Strategy Scheduling SRM Assay
33 Selected Reaction Monitoring Mass spectrometric validation assay platform SRM Platform Limitations: SRM-Assay protocol NOT standardized across instrument manufacturers Precision of Assay dependent upon technical skill and expertise of technician. Assay precision varies WIDELY across users, laboratories, test sites. Successful target detection in biological fluids is dependent upon front-end sample preparation Laborious sample preparation limits total throughput capability 33
34 Selected Reaction Monitoring Bioinformatics Solutions Skyline is an open-source tool available at Skyline is a windows-based application that allows researchers to build methods and analyze data results from Selected Reaction Monitoring experiments. Originally developed in conjunction with NCI's Clinical Proteomic Technology Assessment for Cancer network (CPTAC) program. Skyline users can directly access raw mass spectrometry data from data files generated by almost all instrument manufactures.
35 Selected Reaction Monitoring Bioinformatics Solutions
36 Selected Reaction Monitoring: Application
37 Biomarker Assay Workflow Role of Selected Reaction Monitoring in Biomarker Research Picotti P and Aebersold R Nat Methods. 2012; 9:
38 Clinical Biomarkers Definition Biomarker - a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease (National Cancer Institute) Predictive biomarker gives an indication of the probable effect of a treatment on a patient; helps to assess the most likely response to a particular treatment type Diagnostic biomarker determines whether disease already exists Prognostic biomarker shows disease progression with or without treatment
39 Clinical Biomarkers Working Hypothesis Disease Process Localized Tissue Damage Molecular Cues Mol Oncol. 2009; 3(1): November 27,
40 Biomarker Assays Clinical Sample Types Tissue Tissue-derived proteins become highly diluted in the systemic circulation Proximal fluids o Urine prostate, bladder or kidney diseases o CSF intracranial processes o Nipple aspirate breast cancer o Local drainage sites for disease-derived proteins Serum Primary component of biobank archives Clotting process may lead to neo-generation of peptides Plasma >90% of plasma proteome is comprised of ~10 proteins
41 Sandwich ELISA Validation Assay Platform of Choice ELISA Platform Advantages: Quantitative measurements High sensitivity for desired target High selectivity for target antigen Rapid diagnostic High-throughput platform Easily performed diagnostic Easily interpretable results Platform requirements are compatible with clinical testing facilities 41
42 Sandwich ELISA Validation Assay Platform of Choice ELISA Platform Limitations: Target constrained by antibody availability, epitope recognition and binding specificity Quality of available antibodies often varies across manufacturers. Constrained to single-plex analysis Assay development costs are prohibitively high for most biomarker target development projects When initiated, assay development proceeds for multiple years 42
43 Biomarker Assay Workflow Role of Selected Reaction Monitoring in Biomarker Research Picotti P and Aebersold R Nat Methods. 2012; 9:
44 Case Study #1: A. Hoofnagle, HDL Assay Hoofnagle, A. et al. Multiple-Reaction Monitoring Mass Spectrometric Assays Can Accurately Measure the Relative Protein Abundance in Complex Mixtures. Clinical Chemistry. April 2012; 58(4):
45 HDL Assay SRM-Assay measurements Hoofnagle, A. et al. Clinical Chemistry. April 2012; 58(4): November 27,
46 HDL Assay: Study Methodology SRM-Assay measurements Hoofnagle, A. et al. Clinical Chemistry. April 2012; 58(4): Comparing Assay Platforms for Apolipoproteins HDL purified from plasma from 30 donors All isotope-labeled peptides incorporated Single isotope-labeled peptides incorporated 6-plex MRM- Assays 1-plex Immuno- Assays Label-free shotgun Mass Spectrometric Assay November 27,
47 HDL Assay: Study Results SRM-Assay measurements Hoofnagle, A. et al. Clinical Chemistry. April 2012; 58(4): November 27,
48 HDL Assay: Study Results SRM-Assay measurements Hoofnagle, A. et al. Clinical Chemistry. April 2012; 58(4): Comparing ApoA1 Assay Results Isotope dilution MRM-MS Assay vs Immunoassay November 27,
49 HDL Assay: Study Conclusions SRM-Assay measurements Hoofnagle, A. et al. Clinical Chemistry. April 2012; 58(4): Relative quantification by shotgun proteomics approaches correlated poorly with the 6 protein immunoassays. Peak area ratios measured in multiplexed MRM-MS assays correlate well with immunochemical measurements. o o The isotope dilution MRM-MS approaches showed correlations with immunoassays of r The MRM-MS approaches had high reproducibility and linearity-- 13% CV and linearity (r 0.99) A single protein internal standard applied to all proteins performed as well as multiple protein-specific peptide internal standards. The ISprot approach was capable of correcting for digestion variability but the ISpep approach was not. 49
50 Case Study #2: CPTAC Program Clinical Proteomic Tumor Analysis Consortium Addona, T. et al. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring based measurements of proteins in plasma. Nature Biotechnology. June 2009; 27(7):
51 CPTAC Program Program Mission and Objectives Mission: The Clinical Proteomic Tumor Analysis Consortium (CPTAC) is a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust, quantitative, proteomic technologies and workflows. Objectives: Objective 1: Identify and characterize the protein inventory from tumor and normal tissue biospecimens Objective 2: Integrate genomic and proteomic data from analysis of common cancer biospecimens Objective 3: Develop assays against proteins prioritized in the discovery stage as potential biomarker candidates Objective 4: Perform testing of verification assays in relevant cohorts of biospecimens November 27,
52 CPTAC Study: Study Objectives Assessing SRM Assay Reproducibility and Precision Addona, T. et al. Nature Biotechnology. June 2009; 27(7): Study 1 synthetic peptides Study 2 digested synthetic proteins Study 3 undigested synthetic proteins Comparing SRM-MS Assay Protocols within and between laboratories: Location Nano-LC MS QQQ 8 Sites Eksignet nanolc-1d Plus Eksigent nano LC-2D Agilent 1100 Nanosystem ABI-Sciex 4000 QTRAP Thermo TSQ Quantum Ultra November 27,
53 CPTAC Study: SRM-MS Assay Targets Assessing SRM Assay Reproducibility and Precision Protein CPTAC Target Proteins and Signature Peptides Abbrev Species Signature peptide MH+ (mono) Q1 MRM transitions (m/z) Q3 Aprotinin APR-AGL Bovine AGLCQTFVYGGCR Leptin LEP-IND Mouse INDISHTQSVSAK Myoglobin MYO-LFT Horse LFTGHPETLEK Myelin basic protein Prostate-specific antigen MBP-HGF Bovine HGFLPR MBP-YLA Bovine YLASASTMDHAR PSA-IVG Human IVGGWECEK PSA-LSE Human LSEPAELTDAVK Peroxidase HRP-SSD Horseradish SSDLVALSGGHTFGK C-reactive protein CRP-ESD Human ESDTSYVSLK CRP-GYS Human GYSIFSYATK CRP-YEV Human YEVQGEVFTKPQLWP November 27,
54 CPTAC Study: Study Workflow Assessing SRM Assay Reproducibility and Precision Addona, T. et al. Nature Biotechnology. June 2009; 27(7): Comparing SRM-MS Assay Protocols within and between laboratories: November 27,
55 CPTAC Study: Study Workflow Assessing SRM Assay Reproducibility and Precision Addona, T. et al. Nature Biotechnology. June 2009; 27(7): Comparing SRM-MS Assay Protocols within and between laboratories: November 27,
56 CPTAC Study: Study Results Assessing SRM Assay Reproducibility and Precision Addona, T. et al. Nature Biotechnology. June 2009; 27(7): Comparing SRM Assay Results Intralaboratory Assay CVs November 27,
57 CPTAC Study: Study Results Assessing SRM Assay Reproducibility and Precision Addona, T. et al. Nature Biotechnology. June 2009; 27(7): Comparing SRM Assay Results Intralaboratory Assay CVs November 27,
58 CPTAC Study: Study Results Assessing SRM Assay Reproducibility and Precision Addona, T. et al. Nature Biotechnology. June 2009; 27(7): Comparing SRM Assay Results Intralaboratory Assay LOQs November 27,
59 CPTAC Study: Study Results Assessing SRM Assay Reproducibility and Precision Addona, T. et al. Nature Biotechnology. June 2009; 27(7): Reproducibility and Precision of the quantitative measurements for 9 of 10 peptides ranged from 4 14%, 4 13%, and 10 23%. Intralaboratory CVs were predominantly <15% and <25% at the identical concentration for studies I/II and III, respectively. Interlaboratory and intralaboratory CVs improved with increasing analyte concentration. Under real plasma biomarker conditions (study III): o Performance was below that expected for clinical assays (<10 15%). o Sensitivity was sufficiently low for verifying candidate biomarkers present in plasma with a concentration higher than 2 6 g/ml in plasma o Assay demonstrated a linear dynamic range spanning three orders of magnitude 59
60 CPTAC Study: Study Conclusions Assessing SRM Assay Reproducibility and Precision Addona, T. et al. Nature Biotechnology. June 2009; 27(7): The most frequent cause of poor peptide performance was the presence of interference from the background plasma digest matrix. Monitoring a minimum of three transitions per analyte is critical in maintaining assay selectivity and recognizing signal interference. SRM Assay method development and optimization must be completed independently for all instrument platforms included within a multi-site validation effort. SRM Assay design impacts assay measurements, assay LOQ, and reproducibility.. SOP compliance is critical for assay reproducibility. 60
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