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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)
?
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
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