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Proteomics of Tumor Extracellular Matrix The “Matrisome Project” Karl Clauser

Proteomics of Tumor Extracellular Matrix The “Matrisome Project” Karl Clauser Proteomics Platform of the Broad Institute Alexandra Naba Koch Institute for Cancer Research @ MIT – Hynes Lab. The ECM is a prominent part of the tumor microenvironment. Tumor cells. Normal epithelial cells.

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Proteomics of Tumor Extracellular Matrix The “Matrisome Project” Karl Clauser

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  1. Proteomics of Tumor Extracellular Matrix The “Matrisome Project” Karl Clauser Proteomics Platform of the Broad Institute Alexandra Naba Koch Institute for Cancer Research @ MIT – Hynes Lab

  2. The ECM is a prominent part of the tumor microenvironment Tumorcells Normal epithelialcells Basement membrane Immune cells: macrophages, lymphocytes… Fibroblasts ECM Lymphaticvessels Blood vessels endothelialcells, pericytes Adaptedfrom Joyce JA. and Pollard JW., Nature ReviewCancer (2009)

  3. The ECM gene expression is dysregulated in tumors Fibronectin/Smooth Muscle Actin Normal pancreaticislet arteriole Normal Pancreas Tumor  Marked up-regulation of fibronectin-rich matrix around vascularizedRIPTAgpancreatic tumors Astrof S. et al., MCB (2004)

  4. ECM organization is often altered in tumors Merge (blue: DAPI – nuclei) GPR56 TGM2 Non-metastatic melanoma Metastatic melanoma Lei Xu

  5. The Extracellular Matrix: Not Just Pretty Fibrils! ECM GF GF Integrins GF receptors Focal Adhesion Talin, Paxillin, Vinculin, FAK, Src… Survival Migration Proliferation Morphology

  6. Why study the tumor matrisome? • The ECM provides biophysical and biochemical cues promoting cell growth, invasion and metastasis. • Goals: • What is the source of the tumor ECM (tumor or stroma?) • What are the changes in the matrisome during tumor progression? •  Invasion? •  Angiogenic switch? •  Metastatic dissemination? • Can ECM proteins serve as prognostic/diagnostic tool?

  7. The extracellular matrix

  8. Why using a proteomics-based approach? • Correlation of the protein expression with the mRNA data? • The insolubility of the ECM proteins: an advantage!

  9. Enrichment of ECM protein from normal tissue Purification Steps C N M CS Whole lung extract ECM-rich Fraction Tissue: Mechanical Lysis ç Collagen VI (ECM) 180kDa ç Laminin(ECM) Chemical Lysis (High salt Buffer) 180kDa  ECM proteins: 8-fold enrichment ç Integrinb1 (PM) 120kDa ç Tf Receptor(PM) 83kDa Membrane protein solubilization (DOC, NP-40) 55kDa ç Tubulin(Cytoskeleton) 49kDa ç Actin(Cytoskeleton) Cytoskeletal protein solubilization (SDS) 38kDa ç GAPDH (Cytosol) 16kDa Insoluble fraction = ECM-enriched fraction ç Histones (Nucleus)

  10. The proteomics workflow Agilent 3100 Offgel Electrophoresis Separation by peptide pI Lys-C, trypsin Digestion to peptides 2M urea Spectrum Mill identification of peptides and proteins Solubilize in 8M urea PNGase-F Deglycosylation 2M urea Reversed phase Desalting Thermo LTQ-Orbitrap LC-MS/MS

  11. LC/MS/MS LC MS 8MS/MS Intensity Relative Abundance m/z Retention time (min) Quantitation Identification 1 cycle: 3sec. 1MS scan 8 most abundant peptides 2nd MS 8 MS/MS: peptide sequence

  12. Database search parameters

  13. Content of the lung matrisome - unfractionated Mass Spec Intensity Number of Peptides Number of Proteins Protein sub-cellular location classified from literature knowledge and GO annotation

  14. The lung matrisome

  15. Off Gel Electrophoresis: principle IPG gel strip pH gradient Pi (B) Pi (A) • Capacity 50-100ug total peptide • Separation into 12 fractions, pI 3-10 • Each fraction is analyzed by LC/MS/MS • On average ~ 1800 proteins identified (6 times more than without OGE)

  16. OGE fractionation: normal lung 5557 1 frxn 9178 83% Unseparated sample Overlap of Distinct Peptides in Fractions pI resolution

  17. The lung matrisome before/after OGE Mass Spec Intensity Number of Peptides Number of Proteins Before OGE 59,41% 1291 peptides ~ 2x more ~ 2x more After OGE 9,74% 105 prot. 26,94% 2328 peptides 72,19% 27,81% 73,06% 90,26% ECM Non-ECM

  18. Coverage and Sensitivity Improvements from OGE Increased coverage 1 peptide before OGE Undetected before OGE

  19. Lung matrisome after OGE Additional ECM proteins detected after OGE

  20. A very reproducible approach • The comparison of 2 samples processed in parallel lead to a > 90% identity • The difference between the matrisome of 2 different organs represents less than 10% of the proteins identified. • Comparison of the lung and colon matrisome: • Identification of proteins exclusively present in the lung and participating in the TGFb regulation axis • LTBP2: binds TGFb family member • Thrombospondin-1: the TSP1 knock-out mice get pneumonia that can be ameliorated by TGFb activation

  21. The complex domain structures of ECM proteins Fibronectin S S Fibrillin-1 LTBP-1 Thrombospondin-1 Hynes RO., Science (2009)

  22. Focusing on ECM proteins - Help from Bioinformatics? Combination of the two approaches: Complete Matrisome We will miss unknown proteins 85 domains found in proteins involved in : cell adhesion, GF, enz. (InterPro IDs) OGE proteomics data Extraction of the proteins that contains at least one domains We will miss unknown proteins that have no domains! Knowledge-based Annotation

  23. Annotations of the protein sub-cellular location • The GO annotations for cellular compartment unsatisfactory • Can be inconsistent for mouse vs. human • Tgm2 Protein-glutamine gamma-glutamyltransferase 2 (human) mitochondrion|mitochondrion|plasma membrane|plasma membrane| • Tgm2 Protein-glutamine gamma-glutamyltransferase 2 (mouse) proteinaceous extracellular matrix|cytosol|membrane| • Lamb2 laminin, beta 2 (human) extracellular region|basal lamina|extracellular space|nucleus|cytoplasm|endoplasmic reticulum|laminin-11 complex| • Lamb2 laminin, beta 2 (mouse) basement membrane|basement membrane| • Fbn1 fibrillin 1 (human) microfibril|microfibril|extracellular region|basement membrane|extracellular space| • Fbn1 fibrillin 1 (mouse) microfibril|extracellular region|proteinaceous extracellular matrix| • EMILIN1 (human) extracellular region|proteinaceous extracellular matrix|extracellular space|nucleus|nucleolus|centrosome| • Emilin1 (mouse) extracellular region|proteinaceous extracellular matrix|extracellular space|

  24. Characterization of the tumor matrisome • Understanding the origin of tumor ECM • Can we observe changes in the matrisome during the course of tumor progression? •  Invasion? •  Angiogenicswitch? •  Metastatic dissemination •  How different is the ECM from when compared to the ECM of the primary tumor? • Can we correlate changes in the matrisome to the invasiveness of a tumor? •  Can ECM serve as a diagnostic / prognostic tool in clinics?

  25. Of mouse or man? SC Injection of A375 HumanMelanoma Cells “NSG” mouse NOD/SCID/IL2R Tumor Collection --- Tumor ECM preparation Proteomics pipeline Proteins secreted by the tumor cells: human sequence Proteins secreted by the stromal cells: mouse sequence

  26. VTN - Vitronectin is secreted by the stroma Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse 10 peptides detected 0 human only 8 mouse only 2 shared A375-1A

  27. Emilin-1 is predominantly secreted by the tumor Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse 37 peptides detected 20 humanonly 9 mouseonly 8 shared A375-1A MS intensity (H/M) = 6.3

  28. BGN - Biglycan is Predominantly Secreted by the Stroma Mouse Human Mouse Human Mouse Human Mouse Human 11 peptides detected 2 mouse only 1 human only 8 shared A375-1A MS intensity (M/H) = 460

  29. Challenges in MS Quantitation of Tumor vs. Stroma Secretion 16 peptides detected 10 mouse only 5 human only 1 shared A375-1A Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Human Use all distinguishing peptides # peptides MS intensity (M/H) 10M, 5H 1.6 3M, 3H 0.7 Skip - cleavage site alteration - unpaired peptides Use only similar pairs with same charge

  30. Protein Grouping for Species in Xenograft Tumors protein group subgroup 2 subgroup 1 shared shared mouse human mouse human human • Subgroup specific - ON • total MS1 intensity for only human • total MS1 intensity for only mouse • Subgroup specific - OFF • total MS1 intensity for human + shared • total MS1 intensity for mouse + shared

  31. Of mouse or man? More: >5x Similar: -5 to 5x

  32. Of mouse or man? More: >5x Similar: -5 to 5x

  33. Can we identify trends? • Basement membrane produced by combination of tumor/stroma • Predominantly produced by the tumor • ECM modifying enzymes • Laminins • Growth Factors • Predominantly produced by the stromal cells • Proteoglycans • Most Collagens

  34. Characterization of the tumor matrisome • Understanding the origin of tumor ECM • Can we observe changes in the matrisome during the course of tumor progression? •  Invasion? •  Angiogenicswitch? •  Metastatic dissemination •  How different is the ECM from when compared to the ECM of the primary tumor? • Can we correlate changes in the matrisome to the invasiveness of a tumor? •  Can ECM serve as a diagnostic / prognostic tool in clinics?

  35. Ongoing work… • Comparing metastatic and non-metastatic tumors: • Limitation of the A375 xenograft model: • Subcutaneous injection • Metastatic only by tail vain injection • What would be the control normal matrisome? Xu L. et al., PNAS (2006)

  36. Mouse model of mammary carcinoma: MMTV-PyMT 4 wks 8 wks 15 wks • A transgenic mouse strain that expresses the polyoma middle T oncogen (PyMT) under the mouse mammary tumor virus promoter (MMTV) in the mammary gland. • Carcinomas develop in the mammary gland and mimics human disease stages. • In parallel, mammary gland of age-matched WT FVB mice are collected. Metastatic tumor Premalignant mammary gland No tumor palpable Palpable tumor (1wk pp) Late Stage tumor Guy CT. et al., MCB (1992) Lin EY. et al., (2003)

  37. The MMTV-PyMT mRNA signature Molecular expression profiling of tumors initiated by transgenic overexpression of polyoma middle T antigen (PyMT) targeted to the mouse mammary gland. Procollagen type I, a2 Procollagen type III, a1 Biglycan Fibrinogen-like Nidogen-1 LOXL MMP-2 Laminin B1 subunit 1 Collagen Procollagen type XI, a1 Syndecan-2 And: Procollagen type IV, a2, 3 and 6, procollagen type XV, lumican, nephronectin, MMP-14  How well do the array datareflect in protein-level changes? Desai KV. et al., PNAS (2002) Qiu TH. et al., Cancer Res (2004)

  38. Acknowledgment • Richard Hynes Lab Alexandra Naba John Lamar Hui Liu • Bioinformatics Core Facility (KI) Charlie Whittaker • Proteomics Platform Steve Carr Jake Jaffe TMEN (TUMOR MICROENVIRONMENT NETWORK NCI)         U54-CA126515 

  39. Xenotransplant model of mammary carcinoma • MDA-MB-231: Human mammary carcinoma cell line • LM2: Highly metastatic derivative[Massagué Lab] • Orthotopic injection in the mammary fat pad [John Lamar] • Comparison of the primary tumor matrisome to the “normal” mammary gland matrisome • Identification of the ECM proteins synthesized by the tumor-associated stroma and not by the normal stroma. • Manipulation of gene expression: validation

  40. Characterization of the ECM changes at the angiogenic switch • Model system: RIP-Tag mouse 9 wks 12 wks • A transgenic mouse strain that expresses the simian virus 40 large T antigen (TAg) under the rat insulin II promoter (RIP) in the b-pancreatic islet cells. • Carcinomas develop in the pancreatic islets and progress through characteristic stages. • Human disease: Insulinoma (only in very rare case malignant) Folkman, J. et al. (1989) Nature 339

  41. Characterization of the tumor matrisome • Understanding the origin of tumor ECM • Can we observe changes in the matrisome during the course of tumor progression? •  Invasion? •  Angiogenicswitch? •  Metastatic dissemination •  How different is the ECM from when compared to the ECM of the primary tumor? • Can we correlate changes in the matrisome to the invasiveness of a tumor? •  Can ECM serve as a diagnostic / prognostic tool in clinics?

  42. Can we correlate changes in the matrisome to the invasiveness/aggressiveness of a tumor? • Collaboration with MGH: • Colon cancer sample +/- Liver metastasis • Patient history • Can ECM proteins serve as prognostic/diagnostic tool?

  43. iPRG: Informatic Evaluation of Phosphopeptide Identification and Phosphosite Localization ABRF 2010, Sacramento, CA March 22, 2010 A B R F Proteome Informatics Research Group

  44. P(m/z) -H3PO4 879 A B A Challenging Problem R F Proteome Informatics Research Group 4/7 DSAIPVEsDtDDEGAPR 3/7 DSAIPVESDtDDEGAPR 14/21 said can identify peptide but can not localize site

  45. Solution A B R F Proteome Informatics Research Group • Not fun to do by hand! • Software available that evaluates ‘site-determining ions’ • Generate per residue localization scores • Examples: Ascore, PTM Score (MaxQuant), pFind, PhosphoScore, etc.

  46. Study Goals A B R F Proteome Informatics Research Group Evaluate the consistency of reporting phosphopeptide identifications and phosphosite localization across laboratories Characterize the underlying reasons why result sets differ Produce a benchmark phosphopeptide dataset, spectral library and analysis resource

  47. Study Design A B R F Proteome Informatics Research Group Use a common dataset Use a common sequence database Allow participants to use the bioinformatic tools and methods of their choosing Use a common reporting template Fix the identification confidence (1% FDR) Require an indication of phosphosite ambiguity per spectrum Ignore protein inference – for now

  48. Soliciting Participants and Logistics A B R F Proteome Informatics Research Group Study advertised on the ABRF website and listserv, Molecular and Cellular Proteomics blogsite, GenomeWeb and by direct invitation from iPRG members 1. Email participation request to ‘iPRGxxxx@gmail.com’ Participant 2. Send official study letter with instructions iPRG members Questions / Answers 3. All further communication (e.g., questions, submission) through ‘iPRGxxx.anonymous@gmail.com’ “Anonymizer”

  49. Study Materials and Instructions to Participants A B R F Proteome Informatics Research Group • 1 Orbitrap XL dataset (3 files) • RAW, mzML, mzXML, MGF, pkl or dta – conversions by ProteoWizard • 1 FASTA file (SwissProt human seq’s. v57.1) • 1 template (Excel) • 1 on-line survey (Survey Monkey) Analyze the dataset Report the phosphopeptidespectrum matches in the provided template Complete an on-line survey Attach a 1-2 page description of your methodology

  50. Reporting Template A B R F Proteome Informatics Research Group

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