1. PROTEIN CHARACTERIZATION
PURIFIED A PROTEIN
Extraction
Capture
Intermediate purification
Polishing
Assay
CHARACTERIZATION
SDS PAGE
Amino Acid Analysis
Edman Sequencing
Analytical centrifuge
NMR
X-ray crystal
A single protein
NOW:
PROTEOMICS……..
Defined as Protein Characterization using
Mass Spectrometry

2. Differential Expression & protein-ligand interactions
Proteomics
Mining
Differential Expression ? R
PROTEOMICS
PROBLEMS X Y Z
Protein Complexes
& protein-ligand interactions
Modifications

3. HIGH THROUGHPUT PROTEOMICS?
Analysis of all protein components; a ‘snapshot’
Useful for differential comparisons of tissues or cells
Complementary to RNA micoarray
Information about disease states
Identify useful drug target or diagnostic marker
May use robotics or semi-automated procedures (medium-high throughput)

4. How does a Mass Spectrometer Work?
Sample
Ion Source:
makes ions
Mass Analyzer:
separates ions
Mass Spectrum:
Presents
Information

5. Mass Spectrometry Schematic
Vacuum 1x10-5 to 10-11
Sample in
Inlet
System
Ion
Source
Mass
Analyzer
Detector
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
Data
System
Mass Spectrum

6. Mass spectroscopy: Ionization modes
MALDI
Sample in solid state
Mix with matrix material
Laser desorption
Salt ‘tolerant’
No pre-analysis separation
Time-of-Flight detector
Electrospray (ESI)
Liquid sample sprayed & desolvated
No matrix materials
Salt intolerant
Direct coupling to LC or HPLC
Many detectors

7. PRINCIPLES OF MASS SPECTROMETRY IONIZATION
MALDI advantages
Sample in solid state
Not time-limited for MS/MS
Analysis can be faster or slower than separation
More sophisticated workflows
Fast – lots of MS and MS/MS
MALDI disadvantages
Offline coupling of LC
Miss low MW peptides
ESI advantages
Direct coupling of LC to MS
Fast – lots of MS and MS/MS
Accepted MS/MS ionization mode
ESI disadvantages
Limited time for MS/MS (limited depth of analysis)
Lack of ability to perform result dependent analysis
Miss high MW peptides

8. MALDI-TOF Mass Spectrometer

9. MALDI-TOF Block Diagram

10. Mass spectrometry reports the mass
by Mass-to-charge ratio 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

11. MALDI-TOF Spectrum

12. MASS ACCURACY IS CRITICAL TO PROPER
IDENTIFICATION OF PROTEINS (PMF) AND
CHARACTERIZING MODIFICATIONS AND
ALTERATIONS IN PRIMARY STRUCTURE.

13. Information from a Spectrum
Angiotensin II
C50H71N13O12
Average Mass
[M + H]+ =
C =
Monoisotopic Mass
[M + H]+ =
C Defined as
100
Intensity
1,040
1,044
1,048
1,052
Mass-to-charge (m/z)

14. Mass accuracy; Important for Protein ID
Accuracy = Theoretical m/z - Measured m/z X 1000
Theoretical m/z
Theoretical (M+H)+ =
M = HGTVVLTALGGILK
73 ppm
File calibration
(M+H)+
100 50
13 ppm
External calibration
(M+H)+
100 50
% Intensity
Angiotensin I
Internal calibration
Glu1-Fibrinopeptide B
100
2 ppm
Des-arg1-Bradykinin
Neurotensin 50
(M+H)+
872.0
1040.4
1208.8
1377.2
1545.6
1714.0
Mass-to-charge (m/z)

15. Protein Separation & Detection
Separate proteins with 1D or 2D Gel Electrophoresis
Two-dimensional separation reduces likelihood of contamination; (DOES NOT ELIMINATE!)
Isoelectric focusing
Immobilized pH gradients (IPG DryStrip)
Tube gel (carrier Ampholytes)
SDS PAGE
Reduced and alkylated
Stain proteins
IEF
SDS

16. Protein Digestion & Identification
Select spot or band
EXCISE SPOT
Reduce & Alkylate
Digest
Extract
MASS SPEC ID
DATABASE QUERY

17. Concepts behind PMF Every amino acid has a different molecular mass
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
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 molecular mass
Single-stage MS measures molecular masses
Masses are ‘unique’ to protein sequence
Peptide Mass Fingerprint
G-L-S-E-T-W-D-D-H-K = Da
K-H-D-D-W-T-E-S-L-G = Da

18. Proteases 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 predicted
from database sequences (in silico digestion).
This is their Mass Fingerprint.

19. Digestion with Trypsin
Robust, cheap, stable
Specificity
Cleaves on the C-terminus of Arginine ® and Lysine (K) ONLY
Sequence information about C terminus only
~1 our 10 amino acids, average peptide mass 1100 Da
Amino acids favorable for MS (charged!)
Intact protein sequence
MASDFGHKILGFDSACV
MNQWSDFFIILRTHYWE
DTYRRLIPMASDFKTYH
MNGFDSAILIGRIISCFGK
PEQSADRTYIPLMKSDFV
CQELISEL
Digest fragments -R
-LIPMASDFK
-MASDFGHK
-THYWEDTYR
-THYWEDTYRR
-RLIPMASDFK
-SDFVCQELISEL
-PEQSADRTYIPLM
-ILGFDSACVMNQWSDFFIILR
-TYHMNGFDSAILIGRIISCFGK

20. Protein Digestion & Identification
GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKlTVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

21. Protein Digestion & Identification
GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKlTVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE
DIGEST
Experimental m/z H+

22. Protein Digestion & Identification
GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKlTVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE
DIGEST
Experimental m/z H+
Predicted m/z H+
DATABASE QUERY

23. Mass Fingerprinting: Mascot search
Using MALDI-TOF mass spec data
Database
Taxonomy
Enzyme (essential)
Modification (optional)
Protein Mass (ignore)
Peptide tolerance (100 ppm)
Data (quality over quantity)

24. Mass Fingerprinting: Mascot search

25. Mass Fingerprinting: Mascot search

26. Mass Fingerprinting: Mascot search

27. 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
Keratin
Autolysis peaks
Albumin
Actin
IgG

28. Fragmentation 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 bonds
you are breaking are the peptide bonds between residues (y ions
and b ions)

29. Fragmentation of Peptides: MS/MS
Sequence 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
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

30. How Do We Identify Proteins Using MS/MS?
Get database sequences
that match precusor peptide mass
AVAGCAGAR
CVAAGAAGR
VGGACAAAR
etc….
Actual MS/MS scan
Precursor petide
[M+H]* = 828.2
AVAGCAGAR
CVAAGAAGR
VGGACAAAR b6 y3 y2 b2 y2 b3 y3 b4 y4 b5 y5 y6 b7 b2 y2 b3 b4 y4 b5 y5 b6 y6 b7 b2 b3 y3 b4 y4 b5 y5 b6 y6
Compare virtual spectra
To real spectrum
Peptide Score
AVAGCAGAR
CVAAGAAGR
VGGACAAAR
Scoring
Detect matches between
Theoretical b- and y- ions
Compute correlation
Rank hits

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

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

33. 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)

34. DIGE; Differential Gel Electrophoresis
Labeling
2D Gel Separation
Multichannel Imaging
DeCyder Differential
Analysis Software

35. Increase in protein expression: Osteoporosis

36. DIGE; Differential Gel Electrophoresis

37. Identify Proteins of Interest

38. Pick, Digest & Spot Proteins of Interest

39. Protein Identification Using 2D-MS

40. Protein ID: Mascot search
Using LC/MS/MS nanospray mass spec data

41. Protein ID: Mascot search

42. FUNCTIONAL PROTEOMICS
Quantification (Electrospray)
ICAT
Protein-protein Interactions (SELDI)
Assemblies
Complexes
Post-translational analysis (Electrospray)
Phosphorylation
Ubiquitinylation

43. FUNCTIONAL PROTEOMICS; ICAT Isotope-Coded Affinity Tags
Isolate two populations of proteins
Tag each population with different ICAT reagents
Tags have mass differences of 8
light version with hydrogen
HEAVY version with deuterium

44. FUNCTIONAL PROTEOMICS; ICAT Isotope-Coded Affinity Tags
Pool ICAT-tagged protein populations
Cut proteins into small peptides
Purify ICAT-tagged peptides (affinity)
Use MS/MS to quantify and identify the peptides

45. Sample Quality is Key
Garbage in……………………………..Garbage out.

46. EXPRESSION PROTEOMICS Simplify mixture; Dig Deeper into Proteome
More complex sample = crowded gel pattern; lower resolution
Less complex sample = less crowded pattern; higher resolution
Pre-fractionate samples
Tissue fractionation
Cell type
Subcellular fractionation
Membrane vs. cytosol
Mitochondria
Nuclei
Ribosomes
Fluid
Protein depletion

47. Margy Glasner: Dec 1, 2009
Wow, the things you can do!