1 / 43

NEW SOLUTIONS FOR HIGHLY MULTIPLEXED PROTEIN ASSAYS VIA MRM Webinar June 04, 2012

NEW SOLUTIONS FOR HIGHLY MULTIPLEXED PROTEIN ASSAYS VIA MRM Webinar June 04, 2012. CAPRION PROTEOMICS. Leading proteomics-based service provider Biomarker and target discovery Multiplexed MRM assays Biomarker verification and validation Recently acquired ImmuneCarta

vivek
Download Presentation

NEW SOLUTIONS FOR HIGHLY MULTIPLEXED PROTEIN ASSAYS VIA MRM Webinar June 04, 2012

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. NEW SOLUTIONS FOR HIGHLY MULTIPLEXED PROTEIN ASSAYS VIA MRM Webinar June 04, 2012

  2. CAPRION PROTEOMICS • Leading proteomics-based service provider • Biomarker and target discovery • Multiplexed MRM assays • Biomarker verification and validation • Recently acquired ImmuneCarta • Multiparametric GLP flow cytometry for immune monitoring • Located in: • Montreal, Canada • Menlo Park, California • Founded in 2000 • 62 employees (55 scientists) • Extensive experience • Over 45 industry and government partners • Over 100 large scale studies completed (clinical and pre-clinical) • All major therapeutic areas and sample types • Expertise in study design, execution, statistical analysis, biological insights and comprehensive reporting

  3. TECHNICAL APPROACHES IN MASS SPEC-BASED PROTEOMICS • Biomarker Discovery (LC-MS/MS) • Non-hypothesis based discovery approach • Label-free, gel-free quantitative mass spectrometry • Shotgun sequencing • Profile 1000’s of proteins in 100’s samples • Identify differentially expressed proteins as candidate targets and biomarkers • Multiplexed Assays (MRM) • Targeted quantification of up to 350 specific proteins • Target list from MS, literature, transcriptomics • Rapid assay development • Ab-free or ab enrichment • Synthetic labeled standards for absolute quantification

  4. IDENTIFY MARKERS DIRECTLY IN FLUID OR IN THE SOURCE TISSUE SAMPLE-SPECIFIC PROCESSING Deplete abundant proteins, Antibody enrichment, or exosome isolation Plasma, serum, CSF, urine, other fluids Trypsin digestion to peptides LC-MS analysis Dissolve tissue sections FFPE slides Protein Sample Peptide Sample Enrich plasma membranes, Secreted proteins, phosphoproteins, other organelles or proteins Cells and tissues

  5. UNBIASED DISCOVERY LC-MS ANALYSIS AND MATCHING PEPTIDES ACROSS ALL SAMPLES RP-HPLC and QTOF or Orbitrap Mass Spectrometry Peptide samples Mass / charge ratio Retention time 10,000-20,000 peptides observed, matched and quantified

  6. Expression Profiling AND PROTEIN ID Identification of 500-2,500 proteins Sequencing by LC-MS/MS Statistical analysis to identify differentially expressed peptides Differentially expressed proteins in disease and drug treatment

  7. PEPTIDE EXPRESSION CORRELATION 76% of proteins with median r > 0.6 Dataset of 915 proteins (9,964 peptides) from cell lysate (cytosol) study

  8. PEPTIDE EXPRESSION CORRELATION: BEST CASE EXAMPLE Median r=0.96 (24 peptides) Comparison of 4 groups (n = 6)

  9. PEPTIDE EXPRESSION CORRELATION: WEAKER BUT ROBUST EXAMPLE Median r=0.75 (13 peptides)

  10. MRM ASSAY DEVELOPMENT STRATEGY List of Candidate Proteins Highly multiplexed: Up to ~350 proteins

  11. MRM ASSAY DEVELOPMENT Select up to 5 peptides for each protein Peptide Synthesis List of Candidate Proteins MSAIQAAWPSGTECIAKYNFHGTAEQDLPFCKGDVLTIVAVTKDPNWYKAKNKVGREGIIPANYVQKREGVKAGTKLSLMPWFHGKITREQAERLLYPPETGLFLVRESTNYPGDYTLCVSCDGKVEHYRIMYHASKLSIDEEVYFENLKMQLVEHYTSDADGLCTRLIKPKVMEGTVAAQDEFYRSGWALNMKELKLLQTIGKGEFGDVMLGDYRGNKVAVKCIKNDATA… Resolubilizationand pooling of peptides Determination of best transitions Sample Analysis CE-Optimization Intensity Intensity Intensity Selection of best 2 peptides and transitions RT RT m/z

  12. MRM SELECTS SPECIFIC PEPTIDE PEAKS FOR ANALYSIS Less Intense MoreIntense MRM selects only specific peaks at each retention time Transitions, in ordered by scheduled RT Discovery detects all peaks Retention Time

  13. TOOLS FOR MRM AUTOMATION:ELUCIDATOR PEAK DETECTION AND QUANTIFICATION Align one peptide peak across hundreds of samples Caprion has developed proprietary tools to automate MRM assays and improve results

  14. AVOIDING FALSE POSITIVES WITH SYNTHETIC PEPTIDE STANDARDS Correct peak Interfering peak

  15. 1498 PEPTIDES (~700 PROTEINS) IN THE SAME MRM ASSAY • 1498 peptides spiked at 200 pmol/mL • MRM assay generated • Transitions analyzed for reproducibility

  16. LINEARITY AND SENSITIVITY Quantitative detection of up to ~350 proteins in each assay

  17. Options for MRM assays

  18. 13C, 15N-LABELED STANDARDSRATIOMETRIC QUANTIFICATION Co-inject labeled synthetic peptides for better quantification Synthetic labeled standards can be used to quantify biomarkers over 4 orders of magnitude

  19. QUANTIFICATION OF ENDOGENOUS PEPTIDES IN PLASMA: “PEPTIDOMICS” Remove all proteins, Analyze only native peptides (< 10,000 kDa) 100 pg/ml • Protein precipitation followed by SPE cleanup of endogenous peptides • 500 ul rat plasma • LOQ 1-10 pg/ml (CV ~15%): Very sensitive approach

  20. IMMUNOAFFINIY CAPTURE: QUANTIFYING PROTEINS IN THE LOW PG/ML RANGE Addition of beads Binding Washing Trypsin digestion LC-MRM-MSanalysis of s/n • Quick assay development • 2-3 weeks • Parallel sample processing in 96 well-plates • 200 samples per person per 24 h • MRM analysis time ~ 5-15min • LLOD in the low pg/mL range • Precision < 25 % • Accuracy < 15 % • Assay can be multiplexed Cytokine spiked at 10 pg/mL Peak Area 0 10 20 40 60 80 100 120 140 160 180 200 Concentration of Cytokine in pg/mL

  21. ANALYSIS AND QUANTIFICATION OF FFPE SAMPLES Tumor Normal • Direct detection by MRM • Quantitative assessment • Detect and compare multiple candidate targets and control proteins • 5 micron FFPE thin sections

  22. Case Study 1:Non-Targeted Discovery in PlasmaFollowed by MRMMarkers of active TB in plasma and serum

  23. 360 SAMPLE COMPARISON OF TB STATUS IN 3 INTERNATIONAL COHORTS US cohort is early stage disease HIV background in South African cohort Extrapulmonary TB in Brazil cohort Samples from each site were analyzed independently

  24. SUMMARY OF DISCOVERY RESULTS • High abundance proteins depleted with IgY14/Supermix resin • Single LC-MS analysis per sample • Identified and quantified ~450 proteins • Compared active TB to other cohorts • Number of differentially expressed proteins: • US: 105 • Brazil: 146 • S. Africa: 49 • Non-redundant total: 200 • Most differentially expressed proteins assigned to defined processes: • Immune response ~50% • Lipid metabolism ~20% • Lung damage & wound healing ~20%

  25. ANALYSIS OF SAMPLES BY MRM Selected 89 differentially expressed proteins 247 peptides were monitored MRM assay run on 360 discovery samples Markers from each site tested on all 3 sites

  26. RESULTS OF MRM QUALIFICATION/VERIFICATION STUDY 54 proteins differentially expressed in at least one site Active TB vs. Latent TB and Asymptomatic 34 US 38 Brazil 24 South Africa 13 proteins differentially expressed in all three sites Immune response (3), lipid metabolism (4), lung damage (6)

  27. Case Study 2:Targeted Discovery of Biomarkers by MRM in PlasmaMarkers of H5N1 vaccine response

  28. DISCOVERY STUDY DESIGN: SAMPLES NIAID-funded clinical trial involving 451 healthy adults 291 samples from 97 patients receiving the highest dose (90 µg) of inactivated subvirion influenza A (H5N1) vaccine: Baseline (pre-vaccination) Samples taken 28 days post-dose 1 Samples taken 28 days post-dose 2 Immunogenicity determined by hemagglutination inhibition (HAI) and microneutralization (MN) Response: increase in HAI or MN titer by 4-fold or more between the baseline and post-dose 2 values

  29. ESTABLISHED IMMUNE RESPONSE MRM PANEL 336 proteins categorized according to the biological functions and pathways Protein classifications taken from the Ingenuity Pathway Analysis database

  30. PROJECT STATUS 291 samples from the discovery study analyzed using the multiplexed MRM assay 101 immune response proteins tracked by MRM 20 differentially expressed proteins correlated with response to vaccine at 28 days following second inoculation AUC = 0.79 • Currently improving detection of immune response proteins • Verification assay planned for different trials with earlier time points collected

  31. Case Study 3:Discovery in the Source TissueFollowed by MRM in Plasmaa) Identify initial candidates in secretome of NSCLC tumorsb) Detect and quantify circulating markers in plasma

  32. DISTINGUISHING BENIGN FROM MALIGNANT LUNG NODULES Problem: 150,000 lung nodules detected in the US each year 60% are benign and 40% are malignant Standard of care is CT scans performed every 6 months If the nodule enlarges, a biopsy may be ordered Goal: Develop blood-based test for early detection of small, malignant nodules to be used in conjunction with imaging Approach: Identify 347 candidate markers for testing in patient plasma Lung cancer tissue-based discovery studies plasma membrane secretory vesicles Transcriptomics Literature review Establish MRM assay and screen patient plasma

  33. SECRETED PROTEINS: >1,000x MORE CONCENTRATED IN GOLGI THAN IN BLOOD Identify secreted proteins before release from the tissue www.mie.utoronto.ca

  34. LUNG TUMOR SECRETED PROTEINS Study Design Collaboration with local hospitals IRB approved, patient consented 24 patients Comparison of tumors and adjacent normal Results 1196 proteins identified in lung secretome 200 up-regulated in tumor

  35. “BODY ATLAS” APPROACH TO TISSUE-SPECIFIC SECRETED PROTEIN IDENTIFICATION • Rationale • Identify proteins secreted by each tissue to identify the uniquely and commonly secreted • Aid in candidate prioritization • Method • Isolate secretory vesicles from appropriately preserved normal human tissue • Extract secreted proteins for MS analysis • Minimum 6 subjects per tissue, low abundance tissue was pooled Heart 749 proteins Lung 1196 proteins Breast 713 proteins Liver 1010 proteins Kidney 1509 proteins Kidney 1244 proteins Prostate 1534 proteins Muscle 653 proteins Fat 883 proteins Bladder 739 proteins Pancreas 856 proteins Colon 1549 proteins

  36. 90-PROTEIN LUNG CANCER MRM PANEL • Development of MRM assay • Predict best 10 peptides/protein, synthesize • Determine empirically the best 2, monitor 2 transitions each • Pre-verification study • Depleted plasma from 20 cancer and 20 control subjects • Results • 64 targeted proteins (71%) successfully detected in cancer and control samples • Candidate classifiers identified

  37. CANDIDATE CLASSIFIERS IDENTIFIED Current study: Full set of 347 candidate markers and more samples (>600) from multiple sources

  38. Current Studies:Integrated Diagnostics Alzheimer’s PanelAD Biomarker Discovery in Plasma

  39. ALZHEIMER’S DISEASE DIAGNOSTIC DISCOVERY Initial study incorporated 50 candidate markers, tested on 60 samples AD patients healthy controls Currently testing an expanded panel of 350 proteins on 300 samples AD patients MCI healthy controls other dementias)

  40. MRM PANEL FROM INITIAL STUDY ACHIEVES 80% SENSITIVITY AND SPECIFICITY

  41. Off the Shelf Assays:Plasma ProteinsCSF ProteinsSecreted Proteins, Various TissuesRat Toxicology

  42. Kidney AHSG ALB AMBP AQP1 ASS1 B2M CALB1 CLU CST3 CTSL1 EGF ENO1 FABP3 GCLM GSTA1 GSTM1 GSTP1 HAVCR1 HGD HIST2H2AA3 126 DETECTED proteins IN MRM RAT Tox panel Liver A1I3 A1M ACTB ALAD ALB ALPL ANXA3 ANXA4 ANXA5 APOA1 APOE APOH BCKDHA C3 C4 CA2 CAT COMT CTSD FAH Brain APOA1 APOB BDNF CRP D3ZNQ7 FGA FGB FGG IL6 MMP9 Pappalysin-3 PARK7 SERPINA3L TGFB1 TNF VCAM1 VEGFA Vascular A2M CAV1 CRP HP ICAM1 ORM1 PLAT SAA SELE THBD THBS1 VCAM1 VEGFA FETUB GC GDA GGT1 GGT5 GOT1 GPD1 GSN GSS GSTA1 HP HPD HPX HSPA8 HSPD1 LAP3 MAT2A MDH1 MDH2 MUP HSPA5 LCN2 MGEA5 MUP PBLD PHB RBP4 SELENBP1 SPP1 UMOD VEGFA PON1 PSMA4 PYGL RBP4 RGN SERPINA1 SERPINA3L SERPINC1 SORD SUCLG1 SULT1A1 TF THBS1 TTR Urocanase Heart CKB ENO1 FABP3 LDHA MB S100A9 Skeletal muscle FABP3 GOT1 MB TNNI2

More Related