Analysis of Protein Complexes by Mass

SpectrometryJohn Yates

The Scripps Research InstituteLaJolla, CA

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)

General Strategy for Protein CharacterizationGeneral Strategy for Protein Characterization

Purification/Purification/EnrichmentEnrichment

1-DE1-DE 2-DE2-DE SolutionSolution

• IdentificationIdentification• SequencingSequencing

PeptidesPeptidesProteinProtein oror

MeasurementMeasurement

AnalysisAnalysis

Mass SpectrometryMass Spectrometry

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1406.72201406.7220

……PPGTGKTLLAK PPGTGKTLLAK AVANESGANFISVKAVANESGANFISVK FYVINGPEIM... FYVINGPEIM...

Molecular WeightMolecular Weight

200200 300300 400400 500500 600600 700700 800800 900900 10001000 12001200 14001400m/zm/z

00101020203030404050506060707080809090

100100

Rel

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922.4922.4

835.4835.4

1051.61051.6778.5778.5333.1333.1 619.0619.0 1074.51074.5 1236.71236.7468.1468.1 961.4961.4

FragmentationFragmentation

Post Translational RegulationPost Translational Regulation

• What structural changes occur to create What structural changes occur to create an active protein, alternate splicing, an active protein, alternate splicing, proteolytic processing?proteolytic processing?

• How is a protein’s activity regulated?How is a protein’s activity regulated?• Are modifications involved in regulation? Are modifications involved in regulation?

Protein Separation methods for ProteomicsProtein Separation methods for Proteomics

Dynamic range is central issue for Dynamic range is central issue for separationsseparations

• Gel ElectrophoresisGel Electrophoresis– 1 and 2-Dimensional Separations1 and 2-Dimensional Separations– Native and DenaturingNative and Denaturing– Detection- stainsDetection- stains

• Chromatographic or Electrophoretic Chromatographic or Electrophoretic – Liquid ChromatographyLiquid Chromatography– Capillary ElectrophoresisCapillary Electrophoresis– Affinity ChromatographyAffinity Chromatography– Multi-Dimensional SeparationsMulti-Dimensional Separations– DetectionDetection

Mass Spectrometry Analysis of ProteinsMass Spectrometry Analysis of Proteins

• Analysis of Peptides – digested proteins & mixtures of proteins Analysis of Peptides – digested proteins & mixtures of proteins (“Bottom Up” Approach)(“Bottom Up” Approach)– ESI – Tandem Mass Spectrometers (QIT, LIT, Q-TOFs, TSQs)ESI – Tandem Mass Spectrometers (QIT, LIT, Q-TOFs, TSQs)– MALDI-Tandem Mass Spectrometers (LIT, QIT, Q-TOFs, TOF/TOFs)MALDI-Tandem Mass Spectrometers (LIT, QIT, Q-TOFs, TOF/TOFs)– ESI-FTMS ESI-FTMS – MALDI-FTMSMALDI-FTMS

• Analysis of Intact Proteins (“Top Down” Approach)Analysis of Intact Proteins (“Top Down” Approach)– FTMSFTMS– ESI-TOFESI-TOF– MALDI-TOFMALDI-TOF

• Analyis of Protein ComplexesAnalyis of Protein Complexes– Ion Mobility mass spectrometersIon Mobility mass spectrometers– GEMMAGEMMA

• Mass Spectrometry technology evolves at a constant rateMass Spectrometry technology evolves at a constant rate– Product cycles are 18-24 monthsProduct cycles are 18-24 months

Computational ProteomicsComputational Proteomics

o Mass Spectrometry (MS/MS) data pre-processingMass Spectrometry (MS/MS) data pre-processingSequence SignaturesSequence SignaturesModification SignaturesModification SignaturesSpectral qualitySpectral quality

o Protein and Modification IdentificationProtein and Modification IdentificationWell established methods existsWell established methods exists

• Faster, More Sensitive, More AccurateFaster, More Sensitive, More AccurateHigh throughput and large scale High throughput and large scale de novode novo analysis analysis

o Global Quantification SoftwareGlobal Quantification SoftwareSoftware is needed based on established rules for isotopomer Software is needed based on established rules for isotopomer analysisanalysis

o Statistical Significance of MatchesStatistical Significance of Matcheso Empirical v. Non-empirical methodsEmpirical v. Non-empirical methods

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

Comprehensive Analysis of Protein-Protein InteractionsComprehensive Analysis of Protein-Protein Interactions

Co-immunoprecipitation Co-immunoprecipitation

ProteolysisProteolysisLC/MS/MSLC/MS/MS

LC/LC/MS/MSLC/LC/MS/MS

Identification of Protein ComponentsIdentification of Protein ComponentsIdentification of ModificationsIdentification of Modifications

Dynamics of components and modificationsDynamics of components and modifications

Multiprotein ComplexMultiprotein ComplexProtein Interaction Protein Interaction ChromatographyChromatography

AgaroseAgarose ProteinGST

AgaroseAgarose Ig-G

AgaroseAgarose Ig-G

C

LTEV

TAP-Tagged Proteins

Cell Biology/Genetics

Second Generation Proteomics TechnologySecond Generation Proteomics TechnologyShotgun ProteomicsShotgun Proteomics

Identification of Proteins in MixturesIdentification of Proteins in Mixtures

MS/MSMS/MS

SEQUESTSEQUEST

ymr130ymr130 ymr142ymr142ymr154ymr154 ymr201ymr201

DigestionDigestion SeparationSeparation

Complex PeptideComplex PeptideMixtureMixture

LCLC

Protein Identification data acquired at ~1 peptide per 1-3 secsProtein Identification data acquired at ~1 peptide per 1-3 secs

Eng, McCormack, Yates, JASMS 1994Eng, McCormack, Yates, JASMS 1994

Integrated Multi-Integrated Multi-Dimensional Liquid Dimensional Liquid ChromatographyChromatography

100 micron ID100 micron ID 5 micron Tip

200 nL/min

SolventFlow

MS-MS of Peptide MixturesMS-MS of Peptide Mixtures

LCLC

MSMS

MS/MSMS/MS

1410.61410.6

DatabaseDatabase

IIGHFYDDWCPLKIIGHFYDDWCPLK

SPAFDSIMAETLKSPAFDSIMAETLKAFDSLPDDIHEKAFDSLPDDIHEK

GGILAQSPFLIIKGGILAQSPFLIIK

real spectrum Cross-real spectrum Cross-Correlated with modelCorrelated with modelspectrumspectrum

250250 500500 750750 10001000 12501250

2020

4040

6060

8080

100100x8x8

185.3185.3

255.7255.7

360.9360.9403.0403.0 519.1519.1

662.3662.3

805.5805.5

1007.41007.4

1155.51155.5

1226.81226.8

1324.81324.8

892.6892.6

m/zm/z

• Dynamic RangeDynamic Range

-Complex Peptide Complex Peptide MixturesMixtures

-Low StoichiometryLow Stoichiometry

• Tandem mass spectra Tandem mass spectra showing clearly the showing clearly the modified sitemodified site

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Identifying PTMs – LimitationsIdentifying PTMs – Limitations

Molecular Analysis of KinetochoreComposition and Organization

Cheeseman, Drubin, Barnes- UCBCheeseman, Drubin, Barnes- UCB

Additional Central Kinetochore Proteins

Ctf19p

Mcm21p

Okp1p

Ortiz et al. 1999

Mcm19p

Chl4p

Ghosh et al. 2001

** Defined by 2-hybrid and co-immunoprecipitation **

Ctf3p

Mcm22p

Mcm16p

Measday et al. 2002

The Ctf19p Complex

• 12 Subunits

NEW!

Mapped Phosphorylation Sitesin vivo

Phosphorylation Sites

13

1

4

0

2

3

Ipl1p Targets

6

1

3

0

1?

1?

Dam1p Complex -

Ndc80p Complex -

Ctf19p Complex -

Ipl1p Complex -

Bim1p Complex -

Mif2p Complex -

ComplexesComplexes

Direct Id of Protein Modifications in a Tandem Affinity Purified (TAP) Complex

C-terminally tagged atthe endogenous locus

ProtAcdc2

CBP

IgGSepharose

ProtA

cdc2CBP

Bind toIgG Beads

CalmodulinResin

cdc2CBP

Bind toCalmodulinResin

Elute w/ TEVCleavage

Elute w/ EGTA

Relative Quantitation of ProteinsRelative Quantitation of Proteins

• Metabolic labeling Metabolic labeling 1515NN– Chait et al. Chait et al. PNASPNAS 1999 1999

• Covalent labeling to introduce mass labelsCovalent labeling to introduce mass labels– CDCD33OH, CDOH, CD33CO-, CDCO-, CD22=CDCONH=CDCONH2 2

– James, Nicotinic-NHS DJames, Nicotinic-NHS D00/D/D44: : Anal. Chem.Anal. Chem. ‘00 ‘00

• 1818O labeling during Proteolytic Digestion O labeling during Proteolytic Digestion – Fenselau, Fenselau, Anal. ChemAnal. Chem 2001 2001

• Covalent labeling with affinity enrichmentCovalent labeling with affinity enrichment– Gygi et. al. Gygi et. al. Nature BiotechNature Biotech. 1999. 1999– Regnier Regnier J. ChromatographyJ. Chromatography 1999 1999

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

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

Comprehensive Analysis of Complex Protein Structures in the Cell

Total ProteinCharacterization

• Protein Identification: What’s there• Post Translational Modifications: Regulation• Quantification: Dynamics

Multiprotein Complex/OrganelleMultiprotein Complex/Organelle

Funding

• NIH NCRR RR11823 Yeast Resource Center, University of Washington