PROTEIN CHARACTERIZATION PURIFIED A PROTEIN Extraction Capture Intermediate purification Polishing AssayCHARACTERIZATION SDS PAGE Amino Acid Analysis Edman.

PROTEIN CHARACTERIZATION

PURIFIED A PROTEINPURIFIED A PROTEINExtractionCaptureIntermediate purificationPolishingAssay

CHARACTERIZATIONCHARACTERIZATIONSDS PAGEAmino Acid AnalysisEdman SequencingAnalytical centrifugeNMRX-ray crystal

A single protein

NOW:

PROTEOMICS……..

Defined as Protein Characterization usingMass Spectrometry

PROTEOMICS

PROBLEMS

Mining Differential Expression

Protein Complexes

& protein-ligand interactions Modifications

?

X Y

Z

R

R

ProteomicsProteomics

HIGH THROUGHPUT HIGH THROUGHPUT PROTEOMICSPROTEOMICS

Analysis of all protein components; a ‘snapshot’Analysis of all protein components; a ‘snapshot’ Useful for differential comparisons of tissues or Useful for differential comparisons of tissues or

cellscells Complementary to RNA micoarrayComplementary to RNA micoarray Information about disease statesInformation about disease states Identify useful drug target or diagnostic markerIdentify useful drug target or diagnostic marker May use robotics or semi-automated procedures May use robotics or semi-automated procedures

(medium-high throughput)(medium-high throughput)

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How does a Mass Spectrometer Work?How does a Mass Spectrometer Work?

Sample

Ion Source:makes ions

Mass Analyzer:separates ions

Mass Spectrum:Presents

Information

Mass Spectrometry SchematicMass Spectrometry Schematic

Inlet Systems: Simple vacuum lock HPLC GC

Ion Sources: Matrix Assisted Laser Desorption Ionization (MALDI) Electrospray (ESI) Atmospheric Pressure Chemical Ionization (APCI) Electron Ionization (EI) FAB / LSIMS

Mass Analyzers: Multipole (Quad, Hexa, Octa) Time-of-flight (TOF) Traps (FT-ICR and QIT) Magnetic Sectors

MassAnalyzer

Ion Source

DataSystem

Inlet System

Detector

Mass Spectrum

Vacuum 1x10-5 to 10-11

Sample in

Mass spectroscopy: Ionization modesMass spectroscopy: Ionization modes

MALDIMALDI

Sample in solid stateSample in solid stateMix with matrix materialMix with matrix materialLaser desorptionLaser desorptionSalt ‘tolerant’Salt ‘tolerant’No pre-analysis separationNo pre-analysis separationTime-of-Flight detectorTime-of-Flight detector

Electrospray (ESI)Electrospray (ESI)

Liquid sample sprayed & desolvatedLiquid sample sprayed & desolvatedNo matrix materialsNo matrix materialsSalt intolerantSalt intolerantDirect coupling to LC or HPLCDirect coupling to LC or HPLCMany detectorsMany detectors

ESI advantagesESI advantages Direct coupling of LC to Direct coupling of LC to

MSMS Fast – lots of MS and Fast – lots of MS and

MS/MSMS/MS Accepted MS/MS Accepted MS/MS

ionization modeionization mode

ESI disadvantagesESI disadvantages Limited time for MS/MS Limited time for MS/MS

(limited depth of (limited depth of analysis)analysis)

Lack of ability to Lack of ability to perform result perform result dependent analysisdependent analysis

Miss high MW peptidesMiss high MW peptides

PRINCIPLES OF MASS SPECTROMETRYPRINCIPLES OF MASS SPECTROMETRYIONIZATIONIONIZATION

MALDI advantagesMALDI advantagesSample in solid stateSample in solid stateNot time-limited for Not time-limited for MS/MSMS/MSAnalysis can be faster or Analysis can be faster or slower than separationslower than separationMore sophisticated More sophisticated workflowsworkflowsFast – lots of MS and Fast – lots of MS and MS/MSMS/MS

MALDI disadvantagesMALDI disadvantagesOffline coupling of LCOffline coupling of LCMiss low MW peptidesMiss low MW peptides

MALDI-TOF Mass SpectrometerMALDI-TOF Mass Spectrometer

MALDI-TOF Block DiagramMALDI-TOF Block Diagram

Mass spectrometry reports the massMass spectrometry reports the massby by Mass-to-charge ratioMass-to-charge ratio of a molecule of a molecule

For example:

MW = 2,000

Charge = + 2

M/z = 1,000

For example:

MW = 2,000

Charge = +1

M/z = 2,000

MALDI-TOF SpectrumMALDI-TOF Spectrum

MASS ACCURACY IS CRITICAL TO PROPERIDENTIFICATION OF PROTEINS (PMF) ANDCHARACTERIZING MODIFICATIONS ANDALTERATIONS IN PRIMARY STRUCTURE.

Information from a SpectrumInformation from a Spectrum

1,0521,0481,0441,040

100

0

Mass-to-charge (m/z)

Inte

nsi

ty

Average Mass [M + H]+ = 1047.2045

C = 12.011 Monoisotopic Mass [M + H]+ = 1046.5422

C Defined as 12.000000

Angiotensin IIC50H71N13O12

Mass accuracy; Important for Protein IDMass accuracy; Important for Protein ID

872.0 1040.4 1208.8 1377.2 1545.6 1714.0

Mass-to-charge (m/z)

0

1001296.6853

1570.6776

1378.8390 (M+H)+904.4682 1672.9175

% I

nte

ns

ity 1378.8594 (M+H)+

1400.8627

1378.7409 (M+H)+

1277.6970

73 ppm

13 ppm

2 ppm

Theoretical (M+H)+ = 1378.8417M = HGTVVLTALGGILK

Des-arg1-Bradykinin

Angiotensin I

Glu1-Fibrinopeptide B

Neurotensin50

0

100

50

0

100

501277.6970

File calibration

External calibration

Internal calibration

Accuracy = Theoretical m/z - Measured m/z X 1000 Theoretical m/z

Protein Separation & DetectionProtein Separation & Detection

Separate proteins with 1D or 2D Gel Separate proteins with 1D or 2D Gel ElectrophoresisElectrophoresis

Two-dimensional separation reduces likelihood of Two-dimensional separation reduces likelihood of contamination; (DOES NOT ELIMINATE!)contamination; (DOES NOT ELIMINATE!)

Isoelectric focusingIsoelectric focusing Immobilized pH gradients (IPG DryStrip)Immobilized pH gradients (IPG DryStrip) Tube gel (carrier Ampholytes)Tube gel (carrier Ampholytes)

SDS PAGESDS PAGE Reduced and alkylatedReduced and alkylated

Stain proteinsStain proteins

IEF

SD

S

Protein Digestion & IdentificationProtein Digestion & Identification

EXCISE SPOTEXCISE SPOT Reduce & Reduce & AlkylateAlkylateDigestDigestExtractExtract

DATABASE QUERYDATABASE QUERY

MASS SPECMASS SPEC

IDID

Select spot or bandSelect spot or band

Concepts behind Concepts behind PMFPMF

G-L-S-E-T-W-D-D-H-K = 1186.5 Da

Glycine 57.0

Alanine 71.1

Serine 87.1

Proline 97.1

Valine 99.1

Threonine 101.1

Cysteine 103.1

Leucine 113.2

Isoleucine 113.2

Asparagine 114.2

Aspartic acid 115.1

Glutamine 128.1

Lysine 128.2

Glutamic acid 129.1

Methionine 131.2

Histidine 137.1

Phenylalanine 147.2

Arginine 156.2

Tyrosine 163.2

Tryptophan 186.2

Every amino acid has a different Every amino acid has a different molecular massmolecular mass

Single-stage MS measures Single-stage MS measures molecular massesmolecular masses

Masses are ‘unique’ to protein Masses are ‘unique’ to protein sequencesequence

PPeptide eptide MMass ass FFingerprintingerprint

K-H-D-D-W-T-E-S-L-G = 1186.5 Da

Proteases Cleave SpecificallyProteases Cleave Specifically

Trypsin cleaves after Lysine (K) and Arginine (R)

Chymotrypsin cleaves after aromatic amino acids

Staph aureus V8 cleaves after Aspartate (D) or Glutamate (E)

EndoLysC cleaves after Lysine (K) only

Therefore, peptide maps (m/z) can be predictedfrom database sequences (in silico digestion).

This is their Mass Fingerprint.

Digestion with TrypsinDigestion with Trypsin

Robust, cheap, stableSpecificity

Cleaves on the C-terminus of Arginine ® and Lysine (K) ONLYSequence information about C terminus only~1 our 10 amino acids, average peptide mass 1100 DaAmino acids favorable for MS (charged!)

Intact protein sequence

MASDFGHKILGFDSACVMNQWSDFFIILRTHYWEDTYRRLIPMASDFKTYHMNGFDSAILIGRIISCFGKPEQSADRTYIPLMKSDFVCQELISEL

Digest fragments

-R-LIPMASDFK-MASDFGHK-THYWEDTYR-THYWEDTYRR-RLIPMASDFK-SDFVCQELISEL-PEQSADRTYIPLM-ILGFDSACVMNQWSDFFIILR-TYHMNGFDSAILIGRIISCFGK

Protein Digestion & IdentificationProtein Digestion & Identification

GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKlTVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

Protein Digestion & IdentificationProtein Digestion & Identification

GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKlTVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

4036.89473308.56423340.55402595.35992611.35482277.03792293.03282213.10932245.09911763.80231719.87681613.90091473.77301411.7903

1201.60671233.5966909.4900869.5091829.4414805.4315795.4181811.4131760.3835739.3621694.3518688.3777685.4243

Experimental m/z H+

DIGEST

Protein Digestion & IdentificationProtein Digestion & Identification

GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKlTVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

4036.89473308.56423340.55402595.35992611.35482277.03792293.03282213.10932245.09911763.80231719.87681613.90091473.77301411.7903

1201.60671233.5966909.4900869.5091829.4414805.4315795.4181811.4131760.3835739.3621694.3518688.3777685.4243

Experimental m/z H+

DATABASE QUERY

DIGEST

4036.89473308.56423340.55402595.35992611.35482277.03792293.03282213.10932245.09911763.80231719.87681613.90091473.77301411.7903

1201.60671233.5966909.4900869.5091829.4414805.4315795.4181811.4131760.3835739.3621694.3518688.3777685.4243

Predicted m/z H+

1. Database

2. Taxonomy

3. Enzyme (essential)

4. Modification (optional)

5. Protein Mass (ignore)

6. Peptide tolerance (100 ppm)

7. Data (quality over quantity)

Mass Fingerprinting: Mascot searchMass Fingerprinting: Mascot searchUsing MALDI-TOF mass spec data

Mass Fingerprinting: Mascot searchMass Fingerprinting: Mascot search

Mass Fingerprinting: Mascot searchMass Fingerprinting: Mascot search

Mass Fingerprinting: Mascot searchMass Fingerprinting: Mascot search

What if you don’t find anything?What if you don’t find anything?

•Widen mass tolerance•Remove taxonomic limits•Go to a larger database•Increase the number of missed cleavages allowed•Increase the number of variable modifications•Get sequence information (MS/MS)

Common contaminants

KeratinAutolysis peaksAlbuminActinIgG

Fragmentation of Peptides: MS/MSFragmentation of Peptides: MS/MS

•Can give more information/confirmation

•Identification of post-translational modifications at residue level

•Generate a series of fragments of the original peptide – ideally the bondsyou are breaking are the peptide bonds between residues (y ionsand b ions)

Fragmentation of Peptides: MS/MSFragmentation of Peptides: MS/MSSequence InformationSequence Information

Glycine 57.0

Alanine 71.1

Serine 87.1

Proline 97.1

Valine 99.1

Threonine 101.1

Cysteine 103.1

Leucine 113.2

Isoleucine 113.2

Asparagine 114.2

Aspartic acid

115.1

Glutamine 128.1

Lysine 128.2

Glutamic acid

129.1

Methionine 131.2

Histidine 137.1

Phenylalanine

147.2

Arginine 156.2

Tyrosine 163.2

Tryptophan 186.2

How Do We Identify Proteins Using MS/MS?How Do We Identify Proteins Using MS/MS?

Actual MS/MS scan

Precursor petide[M+H]* = 828.2

Get database sequencesthat match precusor peptide mass AVAGCAGAR

CVAAGAAGRVGGACAAARetc….

AVAGCAGAR CVAAGAAGR VGGACAAAR

Compare virtual spectraTo real spectrum

Scoring

Detect matches betweenTheoretical b- and y- ions

Compute correlation

Rank hits

Peptide Score

AVAGCAGAR 2.56

CVAAGAAGR 0.57

VGGACAAAR 0.32

b2 b2 b2 b3b3b3y2 y2y2

y3y3

y3 b4 b4 b4 y4y4y4 b5 b5 b5 y5y5y5b6

b6 b6 y6y6y6 b7 b7

How Do We Identify Proteins Using MS/MS?How Do We Identify Proteins Using MS/MS?Sequest analysis of MS/MS Ion Trap data

How Do We Identify Proteins Using MS/MS?How Do We Identify Proteins Using MS/MS?Ion Trap MS/MS of doubly charged ions

HOW DO YOU SELECT THE PROTEINS TO STUDY?HOW DO YOU SELECT THE PROTEINS TO STUDY?

Global Expression?……………….(Expression Proteomics)Differential Expression?………….(Expression Proteomics)“Your” protein?……………..(Post-translational modification; Amount)“Your” proteins associates?…… (Functional Proteomics)

DIGE; Differential Gel DIGE; Differential Gel ElectrophoresisElectrophoresis

Labeling

2D Gel Separation

Multichannel Imaging

DeCyder DifferentialAnalysis Software

Increase in protein expression: Osteoporosis

DIGE; Differential Gel DIGE; Differential Gel ElectrophoresisElectrophoresis

Identify Proteins of Identify Proteins of InterestInterest

Pick, Digest & Spot Pick, Digest & Spot Proteins of InterestProteins of Interest

Protein Identification Using 2D-MSProtein Identification Using 2D-MS

Protein ID: Mascot searchProtein ID: Mascot searchUsing LC/MS/MS nanospray mass spec data

Protein ID: Mascot searchProtein ID: Mascot search

FUNCTIONAL PROTEOMICSFUNCTIONAL PROTEOMICS

Quantification (Electrospray)Quantification (Electrospray) ICATICAT

Protein-protein Interactions (SELDI)Protein-protein Interactions (SELDI) AssembliesAssemblies ComplexesComplexes

Post-translational analysis Post-translational analysis (Electrospray)(Electrospray) PhosphorylationPhosphorylation UbiquitinylationUbiquitinylation

FUNCTIONAL PROTEOMICS; ICATFUNCTIONAL PROTEOMICS; ICAT Isotope-Coded Affinity TagsIsotope-Coded Affinity Tags

Isolate two populations of proteinsIsolate two populations of proteinsTag each population with different ICAT reagentsTag each population with different ICAT reagentsTags have mass differences of 8Tags have mass differences of 8

light version with hydrogen

HEAVY version with deuterium

FUNCTIONAL PROTEOMICS; ICATFUNCTIONAL PROTEOMICS; ICAT Isotope-Coded Affinity TagsIsotope-Coded Affinity Tags

Pool ICAT-tagged Pool ICAT-tagged protein populationsprotein populationsCut proteins into Cut proteins into small peptidessmall peptidesPurify ICAT-tagged Purify ICAT-tagged peptides (affinity)peptides (affinity)Use MS/MS to Use MS/MS to quantify and identify quantify and identify the peptidesthe peptides

Sample Quality is KeySample Quality is Key

Garbage in……………………………..Garbage out.

EXPRESSION PROTEOMICSEXPRESSION PROTEOMICSSimplify mixture; Dig Deeper into Simplify mixture; Dig Deeper into

ProteomeProteome

More complex sample = crowded gel pattern; lower resolutionLess complex sample = less crowded pattern; higher resolution

Pre-fractionate samples

Tissue fractionationCell type

Subcellular fractionationMembrane vs. cytosolMitochondriaNucleiRibosomes

FluidProtein depletion

Wow, the things you can do!

Margy Glasner: Dec 1, 2009