Quantitative Analysis of Two Cancer Signaling Pathways Using Multiplex-Immunoprecipitation and Targeted Mass Spectrometry

Transcription

1 Quantitative Analysis of Two Cancer Signaling Pathways Using Multiplex-Immunoprecipitation and Targeted Mass Spectrometry John C. Rogers, PhD Senior R&D Manager Thermo Fisher Scientific The world leader in serving science

2 Overview Background Targeted proteomics and mass spectrometry Advantages and challenges for targeted mass spectrometry (MS) assays Immunoprecipitation to mass spectrometry (IP-MS) Verification of antibody specificity and affinity Optimization of target enrichment protocols Targeted MS assay development Quantitation of AKT-mTOR and Ras pathway targets by mip-tms assays and benchmarking 2

3 How Do Biologists Currently Measure Signaling Proteins? Desire to monitor entire pathways Low-abundance targets and posttranslational modifications (PTMs) Many targets Many conditions AACR 2015 Poster Figure 3

4 What Does Protein Mass Spectrometry (MS) Offer to Biologists? Detect protein isoforms and modifications that cannot be detected by other methods Identify and quantitate multiple proteins at a time Identify posttranslational modifications and protein interactions Can be combined with antibody-dependent methods to improve specificity (immunoprecipitation, flow cytometry) 4

5 What Are the Challenges in MS-based Proteomics? Challenge: Quantitation of complex samples using MS is challenging due to: Wide dynamic range of abundance Sensitivity limits of instrumentation Ionization suppression Missing information for quantitation Solutions: Reduce sample complexity through enrichment (Immunoprecipitation) Introduce isotopically labeled internal standards 5

6 How Do You Reduce Sample Complexity? Biofluids Cells Tissue Protein abundance High Digestion Medium enrichment Depletion Depletion + + digestion Digestion Digestion + Fractionation Low Enrichment + Digestion 6

7 Target-Specific Protein Extraction and Enrichment Lyse cells Target Cell line Detected by Q Exactive HF Antigen-Ab complex Bind to beads Wash 3x Elute Reduce, alkylate, digest IR IGF1R IRS1 AKT1 AKT2 PTEN TSC2 mtor p70s6k PRAS40 Neat Enriched-IP A HCT A549 + (4) + (22) HCT A HCT116 + (4) + (10) A HCT A HCT A HCT A HCT A549 + (2) + (82) HCT116 + (9) + (110) A549 + (2) + (7) HCT A HCT116 + (2) + (8) Antibody enrichment enhances the detection of targets 7

8 Recommended Antibody Validation Strategies Nat Methods, doi: /nmeth.3995? Methods: Genetic Orthogonality Correlation Tagging IP-MS The use or any variation of the word validation refers only to antibodies that were subject to functional testing to confirm that a biological target can be appropriately recognized and can be used with the research techniques indicated. The use or any variation of the word validation does not ensure that the product was validated to fulfill defined user needs and intended uses. Only IP-MS can identify off-targets, interacting proteins, and protein modifications 8

9 Overview Background Targeted proteomics and mass spectrometry Advantages and challenges for targeted MS assays Immunoprecipitation to mass spectrometry (IP-MS) Verification of antibody specificity and affinity Optimization of target enrichment protocols Targeted MS assay development Quantitation of AKT-mTOR and Ras pathway targets by mip-tms assays and benchmarking 9

10 Workflow for Antibody Verification by IP-MS Select targets, cell lines, and antibodies Immuno-capture and LC-MS analysis Data analysis 10

11 Fold-Enrichment Calculation Ranks Identified Proteins Fold enrichment = Target protein abundance in IP Total protein abundance in IP Target protein abundance in whole lysate Total protein abundance in whole lysate - 11

12 Cross-Verification of IP-MS Results Wikipedia.org β-catenin antibody captures cadherins and many native interactors 12

13 Comparison of β-catenin Interactors Across Antibodies Hierarchical clustering Comparison across antibodies verifies co-ip results Clusters of interacting proteins suggest unique complexes or epitopes 13

14 Broad Coverage of Known Cancer Signaling Pathway Proteins 14

15 Summary of IP-MS Antibody Verification Efforts Over 1,000 antibodies screened to >250 signaling pathway targets IP-MS verifies the antibody target and provides additional information about antibody performance Fold-enrichment provides a simple way to verify antibody performance, assess interactions, and identify off-targets 15

16 Protein Immunoprecipitation (IP) Methods Protein A/G IP Streptavidin IP biotin Protein A/G IP Antibodies with any formulations (BSA) Best for screening multiple antibodies Not suited for biological fluids or vascular tissues Streptavidin IP Requires biotinylated antibody Best for protein A/G-verified antibody with no carrier (BSA) Cells, tissue, and biological fluids Agarose Higher capacity Magnetic Small-scale enrichment Low throughput Easy to handle, automatable 16

17 Evaluation of Different Resins for Protein Immunoprecipitation (IP) A431 lysate Hydrazide Aldehyde NHS-ester Epoxy Direct IP Indirect streptavidin IP EGFR Heavy Chain (P) (A) (M) (M) (P) (M) P: Polyacrylamide, A: Agarose,M: Magnetic +: Anti-EGFR IP, - : Rabbit IgG IP 17

18 Comparison of Direct and Indirect IP Methods In-solution LC-MS/MS results Success criteria: Resin # proteins identified Anti-EGFR EGFR % sequence coverage # proteins identified Rabbit IgG EGFR % sequence coverage Hydrazide (P) # proteins identified <60 EGFR % sequence coverage >60% Direct IP Indirect IP Aldehyde (A) NHS-Ester (M) Epoxy (M) Streptavidin (P) Streptavidin (M)

19 Advantages of Using Magnetic Beads Large surface = efficient capture Can be used for samples with different viscosities Possible to concentrate the sample Easy to automate Suitable for low to high throughput applications 19

20 Overview of the Thermo Scientific KingFisher Technology Magnetic beads are transferred in the process Magnet Disposable tip Paramagnetic particles Thermo Scientific Pierce magnetic beads and kits Catch Move Wash Incubate Thermo Scientific KingFisher instrument family 20

21 Targeted MS Methods Selected reaction monitoring (SRM) with a triple quadrupole (QQQ) mass spectrometer Q1 Q2 Q3 Peptide Selection Fragmentation Fragment selection Parallel reaction monitoring (PRM) with the Thermo Scientific Q Exactive hybrid quadrupole-mass spectrometer Isolation with Fragmentation with Detection with the Identification using Quantification the quadrupole the HCD cell Orbitrap analyzer MS/MS spectrum of XIC 21

22 Targeted MS Assays Enable Absolute Quantitation Concentration (fmol) Peptide 1 Peptide 1 Peptide 1 Peptide 2 Peptide 1 Peptide 2 Peptide 1 Peptide 2 Peptide 1 Peptide 1 Peptide 1 Peptide 1 Peptide 2 Peptide 1 Peptide 2 Peptide 1 Peptide 1 Peptide 2 0,25 0,2 PRM LOD (fmol) SRM LOD (fmol) 0,15 0,1 0,05 0 AKT1 AKT2 mtor IGF1R IR PRAS- p70s6k TSC2 PTEN GSK3ɑ GSK3β IRS1 40 All 12 target peptides were monitored with linear quantitation and 2-3 orders of magnitude PRM and SRM methods provided essentially the same level of sensitivity 22

23 Overview Background Targeted proteomics and mass spectrometry Advantages and challenges for targeted MS assays Immunoprecipitation to mass spectrometry (IP-MS) Verification of antibody specificity and affinity Optimization of target enrichment protocols Targeted MS assay development Quantitation of AKT-mTOR and Ras pathway targets by mip-tms assays and benchmarking 23

24 Workflow for Antibody Verification by IP-MS Total and phosphopeptide pathway profiling Multiplexed protein immuno-capture LC-MS analysis Biotin Akt/mTOR & Ras/MAPK pathway targets in cells and tissues Validated antibodies and peptides in automated magnetic bead workflows Multiplexed-directed discovery or targeted PRM/SRM quantification 24

25 Enrichment of AKT-mTOR and RAS/ERK Pathway Targets IP antibody Phospho AKT Target # of unique peptides Relevant phospho sites AKT1 20 Ser473 AKT2 14 Ser474 AKT AKT1 AKT AKT PRAS40 PRAS40 8 Thr246 Targeted MS assays Phospho PRAS40 Phospho mtor Pan Ras PIK3R2 PRAS40 6 Thr246 mtor 82 Thr2446, Ser2448 RICTOR 2 - SIN1 3 - Gbl 4 - HRAS 15 - KRAS 13 - NRAS 14 - PIK3R2 32 Ser262, Ser263 PIK3CA 29 - PIK3CB 30 - RAF1 RAF1 42 Ser259, Ser621 Dominic Esposito and Frederick National Laboratory for Cancer Research RSK1 RPS6KA1 60 Ser221, Ser363 25

26 Multiplex IP-MS for Total and Phospho-AKT/mTOR and RAS/ERK Pathway Targets Top 3 peptides area (log) Top 3 Peptides area (log) Top 3 peptides area (log) A 1,0E+10 mip-ms total AKT/mTOR pathway targets 1,0E+09 1,0E+08 1,0E+07 B 1,0E+10 1,0E+09 1,0E+08 1,0E+07 1,0E+06 mip-ms phosphorylated AKT/mTOR pathway targets C 1,0E+10 1,0E+09 mip-ms total RAS/ERK pathway targets 1,0E+08 1,0E+07 26

27 PRM Quantitation Limits of Peptides for AKT-mTOR and RAS/ERK Pathway Proteins LLOQ (fmol) LLOQ (fmol) A. 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 Peptide # 0,69 0,69 0,69 0,69 0,23 0,23 0,23 0,23 0,23 0,23 0,23 0,08 0,08 0,08 0,08 0,08 0,08 0, AKT1 AKT2 mtor IGF1R IR IR TSC2 PTEN GSK3ɑ GSK3β IRS1 B. 1,4 1,2 1 0,8 0,6 0,4 0,2 0 Peptide # 1,23 1,23 0,41 0,41 0,41 0,41 0,14 0,05 0,05 0,14 0,05 0,05 0,14 0, RAF1 HRAS KRAS NRAS MAP2K1 MAP2K2 MAPK1 MAPK3 RPS6KA1 27

28 IP-MS Enriches Targets, Interactors, and Modifications IP antibody Antibody # Target Cell line CTNNB1 Pan CDH CDKN1A 4407M PA MA PAK CTNNB1 APC - 35 CDH1 HCT IP enriched # of unique peptides Relevant phosphopeptide ID Nest Enriched-IP CDH Ser19, Ser551, Ser552, Ser675 CTNNA Ser641, Thr654, Thr658 CDH1-11 CDH CDH4 A549-7 CTNNB CTNNA Ser641 CDKN1A 1 4 CDK CDK2 HCT CDK4 2 6 CCND1 3 7 PAK1 GIT1 HEK GIT Ser174, Thr230 EGFR AHR5062 EGFR A Ser991, Ser1026, Ser1039 TP TP53 BT Ser9 28

29 Orthogonal Detection Methods Show High Correlation Protein LFQ Volumecorrected intensity Protein LFQ Western blot 2,0E+04 1,5E+04 1,0E+04 5,0E+03 0,0E+00 IP-MS 1,2E+09 8,0E+08 4,0E+08 0,0E+00 IGF1R protein LFQ after IP-MS Direct MS 8,0E+08 4,0E+08 0,0E+00 IGF1R deep proteome analysis 29

30 Comparison of Western Blot vs. Targeted IP-MS % Untreated Marker HCT116 untreated HCT116 stimulated HCT116 inhb./stim. Marker HCT116 untreated HCT116 stimulated HCT116 inhb./stim. Marker HCT116 untreated HCT116 stimulated HCT116 inhb./stim. Marker HCT116 untreated HCT116 stimulated HCT116 inhb./stim. i) ii) iii) iv) Untreated Stimulated Inhib Inhibit./Stim. / 0 ppras40 pakt1 pp70s6k pmtor pirs1 pgsk3a pgsk3b 30

31 AKT/mTOR and RAS/ERK Pathway Expression Changes HCT116 (stim.) HCT116 (inhib.) No change P P except GSK3 Targetdependent changes No change A549 (stim.) A549 (inhib.) No change P except mtor P except IRS1 T P P Cell line dependent differences in response to PI3K inhibition 31

32 Conclusions Enrichment is necessary for identification and MS quantitation of signaling pathway proteins, interacting partners, and PTMs Thermo Scientific Pierce MS-Compatible Magnetic Immunoprecipitation IP Kits (Protein A/G and Streptavidin) resulted in a higher yield of AKT/mTOR and RAS/ERK pathway proteins and fewer nonspecific binding proteins than with other beads/resins Total and phosphorylated target mip-tms assays allowed simultaneous quantitation of multiple AKT/mTOR and RAS/ERK pathway proteins and modifications in treated cell lines Elucidating specific pathway differences between A549 and HCT116 cells will lead to better understanding and treatment of lung and colon cancer. mip-tms assays were benchmarked against western blot: Overall good correlation observed for AKT/mTOR and RAS/ERK pathway proteins Variability between techniques for some targets could be due to antibody specificity 32

33 Acknowledgements Bhavin Patel Penny Jensen Greg Potts Leigh Foster Abid Hasseb Kay Opperman Matt Baker Brian Johnson Wayne Considine Carrie Clothier Kevin Harvey 33

34 Q&A The world leader in serving science

35 Thank you For Research Use Only. Not for use in diagnostic procedures Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COL The world leader in serving science