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Analysis of Protein Complexes by Mass Spectrometry

Analysis of Protein Complexes by Mass Spectrometry. John Yates The Scripps Research Institute LaJolla, CA. Protein Complex Discovery. Who : identity of proteins in complex? What : biological process involved? Where : is the complex localized?

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Analysis of Protein Complexes by Mass Spectrometry

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  1. Analysis of Protein Complexes by Mass Spectrometry John Yates The Scripps Research Institute LaJolla, CA

  2. Protein Complex Discovery • Who: identity of proteins in complex? • What: biological process involved? • Where: is the complex localized? • When: are proteins involved in the complex? • How much: stoichiometry of proteins in complex, quantity- relative vs absolute • Regulation: modifications (kinase,etc) proteolysis (protease)

  3. Protein or Peptides General Strategy for Protein Characterization Purification/ Enrichment 1-DE 2-DE Solution Measurement Mass Spectrometry • Identification • Sequencing Analysis

  4. 836.4362 1406.7220 1209.5710 766.4868 904.4685 997.5691 40000 1570.6782 2061.1366 1697.8175 30000 Counts 1890.9643 1800.9144 1221.7473 20000 10000 0 800 1000 1200 1400 1600 1800 2000 Mass (m/z) Molecular Weight Fragmentation 922.4 100 90 80 835.4 70 60 Relative Abundance 50 40 30 1051.6 778.5 333.1 20 619.0 1236.7 1074.5 468.1 961.4 10 0 200 300 400 500 600 700 800 900 1000 1200 1400 m/z …PPGTGKTLLAK AVANESGANFISVK FYVINGPEIM...

  5. Post Translational Regulation • What structural changes occur to create an active protein, alternate splicing, proteolytic processing? • How is a protein’s activity regulated? • Are modifications involved in regulation?

  6. Protein Separation methods for ProteomicsDynamic range is central issue for separations • Gel Electrophoresis • 1 and 2-Dimensional Separations • Native and Denaturing • Detection- stains • Chromatographic or Electrophoretic • Liquid Chromatography • Capillary Electrophoresis • Affinity Chromatography • Multi-Dimensional Separations • Detection

  7. Mass Spectrometry Analysis of Proteins • Analysis of Peptides – digested proteins & mixtures of proteins (“Bottom Up” Approach) • ESI – Tandem Mass Spectrometers (QIT, LIT, Q-TOFs, TSQs) • MALDI-Tandem Mass Spectrometers (LIT, QIT, Q-TOFs, TOF/TOFs) • ESI-FTMS • MALDI-FTMS • Analysis of Intact Proteins (“Top Down” Approach) • FTMS • ESI-TOF • MALDI-TOF • Analyis of Protein Complexes • Ion Mobility mass spectrometers • GEMMA • Mass Spectrometry technology evolves at a constant rate • Product cycles are 18-24 months

  8. Computational Proteomics • Mass Spectrometry (MS/MS) data pre-processing • Sequence Signatures • Modification Signatures • Spectral quality • Protein and Modification Identification • Well established methods exists • Faster, More Sensitive, More Accurate • High throughput and large scale de novo analysis • Global Quantification Software • Software is needed based on established rules for isotopomer analysis • Statistical Significance of Matches • Empirical v. Non-empirical methods

  9. Analytical Challenges • Cell biology techniques to isolate structures • Sensitivity • Dynamic range: low affinity binders • Throughput • Biochemical Throughput • Analytical Throughput • Direct measurement of intact complex • Quantitation of components and modifications

  10. Protein Agarose Ig-G Agarose GST Protein Interaction Chromatography TAP-Tagged Proteins Multiprotein Complex Agarose Ig-G TEV C L Comprehensive Analysis of Protein-Protein Interactions Co-immunoprecipitation Proteolysis LC/MS/MS LC/LC/MS/MS Identification of Protein Components Identification of Modifications Dynamics of components and modifications Cell Biology/ Genetics

  11. Digestion Separation MS/MS SEQUEST ymr201 ymr154 ymr130 ymr142 Second Generation Proteomics TechnologyShotgun ProteomicsIdentification of Proteins in Mixtures LC Complex Peptide Mixture Protein Identification data acquired at ~1 peptide per 1-3 secs Eng, McCormack, Yates, JASMS 1994

  12. Integrated Multi-Dimensional Liquid Chromatography 200 nL/min 100 micron ID 5 micron Tip Solvent Flow

  13. MS-MS of Peptide Mixtures LC MS MS/MS

  14. Database x8 100 1007.4 GGILAQSPFLIIK IIGHFYDDWCPLK 80 AFDSLPDDIHEK SPAFDSIMAETLK real spectrum Cross- Correlated with model spectrum 1155.5 60 662.3 1226.8 892.6 805.5 255.7 40 360.9 403.0 519.1 20 185.3 1324.8 250 500 750 1000 1250 m/z 1410.6

  15. Phosphopeptide Total Ion Chromatogram Identifying PTMs – Limitations MS Spectrum at Scan 1650 • Dynamic Range • Complex Peptide Mixtures • Low Stoichiometry • Tandem mass spectra showing clearly the modified site

  16. Molecular Analysis of Kinetochore Composition and Organization Cheeseman, Drubin, Barnes- UCB

  17. Ctf19p Mcm16p Chl4p Mcm21p Mcm22p Mcm19p Okp1p Ctf3p Ghosh et al. 2001 Ortiz et al. 1999 Measday et al. 2002 Additional Central Kinetochore Proteins ** Defined by 2-hybrid and co-immunoprecipitation **

  18. The Ctf19p Complex • 12 Subunits NEW!

  19. in vivo Phosphorylation Sites Complexes Ipl1p Targets 6 Dam1p Complex - 13 Ndc80p Complex - 1 1 Ctf19p Complex - 0 0 Ipl1p Complex - 3 4 Bim1p Complex - 2 1? Mif2p Complex - 3 1? Mapped Phosphorylation Sites

  20. C-terminally tagged at the endogenous locus ProtA CBP cdc2 Bind to IgG Beads Elute w/ TEVCleavage ProtA CBP IgGSepharose cdc2 CalmodulinResin Bind to CalmodulinResin CBP cdc2 Elute w/ EGTA Direct Id of Protein Modifications in a Tandem Affinity Purified (TAP) Complex

  21. Relative Quantitation of Proteins • Metabolic labeling 15N • Chait et al. PNAS 1999 • Covalent labeling to introduce mass labels • CD3OH, CD3CO-, CD2=CDCONH2 • James, Nicotinic-NHS D0/D4: Anal. Chem. ‘00 • 18O labeling during Proteolytic Digestion • Fenselau, Anal. Chem 2001 • Covalent labeling with affinity enrichment • Gygi et. al. Nature Biotech. 1999 • Regnier J. Chromatography 1999

  22. Large Scale Mass Spectrometry Analysis of Complexes • Serial technique- throughput increases derive from adding additional instruments • Analyses requiring high sensitivity and large dynamic range require more deliberate techniques- generally more manual • LC/MS/MS 0.5-1hr • LC/LC/MS/MS 3hr-6 hr • Protein identification robust and accurate esp in metazoans • Automation allows throughput increases, but decreases sensitivity, dynamic range

  23. Technology Advances for Mass Spectrometry of Complexes • Throughput • Biochemical • Mass Spectrometry • Sensitivity • Low copy number complexes • Low stoichiometry modifications • Identify proteins with fast on-off rates • Direct Analysis of Intact Proteins • Isoforms • Modified forms • Direct Analysis of Intact complexes • Stoichiometry • Shape • H/D exchange to determine interaction surfaces

  24. Multiprotein Complex/Organelle Comprehensive Analysis of Complex Protein Structures in the Cell Total Protein Characterization • Protein Identification: What’s there • Post Translational Modifications: Regulation • Quantification: Dynamics

  25. Funding • NIH NCRR RR11823 Yeast Resource Center, University of Washington

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