Analysis of Protein Complexes by Mass Spectrometry

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Analysis of Protein Complexes by Mass Spectrometry

• John Yates
• The Scripps Research Institute
• LaJolla, CA

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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)

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General Strategy for Protein Characterization
Purification/ Enrichment
1-DE
2-DE
Solution
Measurement
Mass Spectrometry

• Identification
• Sequencing

Analysis
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Post Translational Regulation

• What structural changes occur to create an active
  protein, alternate splicing, proteolytic
  processing?
• How is a proteins activity regulated?
• Are modifications involved in regulation?

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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

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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

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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

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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

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Comprehensive Analysis of Protein-Protein
Interactions
Co-immunoprecipitation
Proteolysis LC/MS/MS LC/LC/MS/MS
Identification of Protein Components Identificatio
n of Modifications Dynamics of components and
modifications
Cell Biology/ Genetics
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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
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Integrated Multi-Dimensional Liquid Chromatography
200 nL/min
100 micron ID
5 micron Tip
Solvent Flow
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MS-MS of Peptide Mixtures
LC
MS
MS/MS
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Identifying PTMs Limitations
MS Spectrum at Scan 1650

• Dynamic Range
• Complex Peptide Mixtures
• Low Stoichiometry
• Tandem mass spectra showing clearly the modified
  site

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Molecular Analysis of Kinetochore Composition and
Organization
Cheeseman, Drubin, Barnes- UCB
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The Dam1p Complex
9 Subunits
Cheeseman et al. 2001 Janke et al. 2002 Li et al.
2002
Dad3p
Dad4p
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Additional Central Kinetochore Proteins
Defined by 2-hybrid and co-immunoprecipitation

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The Ctf19p Complex
12 Subunits
NEW!
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Mapped Phosphorylation Sites
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Direct Id of Protein Modifications in a Tandem
Affinity Purified (TAP) Complex
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Relative Quantitation of Proteins

• Metabolic labeling 15N
• Chait et al. PNAS 1999
• Covalent labeling to introduce mass labels
• CD3OH, CD3CO-, CD2CDCONH2
• 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

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Labeled Internal Standards
Normal Media
15N Enriched Media
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Measuring Changes in Protein Expression
Experiment Cells
Labeled Standard
Control Cells
Labeled Standard
Normal Media
15N Enriched Media
Normal Media
15N Enriched Media
Mix
Mix
Cell Lysis
Cell Lysis
Proteolytic Digestion
Proteolytic Digestion
MudPIT
MudPIT
Identify Peptide
Measure
Identify Peptide
Measure
Fractional Change
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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

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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

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Comprehensive Analysis of Complex Protein
Structures in the Cell
Total Protein Characterization

• Protein Identification Whats there
• Post Translational Modifications Regulation
• Quantification Dynamics

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Funding

• NIH NCRR RR11823 Yeast Resource Center,
  University of Washington