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					             PROTEIN CHARACTERIZATION


PURIFIED A PROTEIN
      Extraction
      Capture
      Intermediate purification
      Polishing
      Assay                                    A single protein

CHARACTERIZATION
     SDS PAGE
     Amino Acid Analysis          NOW:
     Edman Sequencing
     Analytical centrifuge               PROTEOMICS……..
     NMR
     X-ray crystal           Defined as Protein Characterization using
                             Mass Spectrometry
                      Proteomics
Mining                              Differential Expression

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                       PROTEOMICS
                        PROBLEMS
              R
                                    X                     Y
              R

                                               Z
    Protein Complexes
& protein-ligand interactions            Modifications
     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)
How does a Mass Spectrometer Work?



 Sample




Ion Source:   Mass Analyzer:   Mass Spectrum:
 makes ions   separates ions      Presents
                                 Information
          Mass Spectrometry Schematic
                                           Vacuum 1x10-5 to 10-11

                         Inlet              Ion              Mass
Sample in                                                                 Detector
                        System             Source           Analyzer

Inlet Systems:
     Simple vacuum lock                                                   Data
     HPLC                                                                System
     GC

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

Mass Analyzers:
     Multipole (Quad, Hexa, Octa)
     Time-of-flight (TOF)
     Traps (FT-ICR and QIT)
     Magnetic Sectors
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
PRINCIPLES OF MASS SPECTROMETRY
IONIZATION

 ESI advantages                   MALDI advantages
    Direct coupling of LC to MS     Sample in solid state
    Fast – lots of MS and           Not time-limited for MS/MS
     MS/MS
    Accepted MS/MS ionization       Analysis can be faster or
     mode                            slower than separation
                                     More sophisticated workflows
 ESI disadvantages                  Fast – lots of MS and MS/MS
    Limited time for MS/MS
     (limited depth of analysis)
    Lack of ability to perform    MALDI disadvantages
     result dependent analysis
    Miss high MW peptides           Offline coupling of LC
                                     Miss low MW peptides
MALDI-TOF Mass Spectrometer
MALDI-TOF Block Diagram
 Mass spectrometry reports the mass
by Mass-to-charge ratio of a molecule


 For example:          For example:

 MW = 2,000            MW = 2,000

 Charge = + 2          Charge = +1

 M/z = 1,000           M/z = 2,000
MALDI-TOF Spectrum
MASS ACCURACY IS CRITICAL TO PROPER
IDENTIFICATION OF PROTEINS (PMF) AND
 CHARACTERIZING MODIFICATIONS AND
 ALTERATIONS IN PRIMARY STRUCTURE.
            Information from a Spectrum

                                                     Angiotensin II
                                                     C50H71N13O12
                Average Mass
             [M + H]+ = 1047.2045
                  C = 12.011                 Monoisotopic Mass
                                            [M + H]+ = 1046.5422
                                           C Defined as 12.000000

100
Intensity




 0
        1,040    1,044    1,048    1,052
                Mass-to-charge (m/z)
               Mass accuracy; Important for Protein ID
                                                               Accuracy = Theoretical m/z - Measured m/z X 1000
                                                                              Theoretical m/z
                    Theoretical (M+H)+ = 1378.8417
                    M = HGTVVLTALGGILK
                                                                               73 ppm
                                                                           1378.7409 (M+H)+
              100
                    File calibration
                                                                                     1277.6970
               50
                                                                                          1277.6970
                0

                                                                               13 ppm
                                                                           1378.8594 (M+H)+
% Intensity




              100   External calibration
               50                                                                    1400.8627

                0



                                                               Angiotensin I

              100
                    Internal calibration                       1296.6853                              Glu1-Fibrinopeptide B
                    Des-arg1-Bradykinin                                        2 ppm                       1570.6776
                                                                                                                         Neurotensin
               50   904.4682                                               1378.8390 (M+H)+
                                                                                                                         1672.9175
                0
                872.0                     1040.4     1208.8                     1377.2                  1545.6                    1714.0

                                                              Mass-to-charge (m/z)
       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)         IEF
       Tube gel (carrier Ampholytes)
    SDS PAGE
       Reduced and alkylated
        Stain proteins
 Protein Digestion & Identification
Select spot or band


                      EXCISE SPOT        Reduce & Alkylate
                                         Digest
                                         Extract




             MASS SPEC

                              DATABASE QUERY              ID
   Concepts behind
                                           Glycine         57.0
        PMF                                Alanine         71.1
                                           Serine          87.1
Every amino acid has a different          Proline         97.1
molecular mass                             Valine          99.1
                                           Threonine       101.1
Single-stage MS measures molecular
                                           Cysteine        103.1
masses                                     Leucine         113.2
Masses are ‘unique’ to protein sequence   Isoleucine      113.2
                                           Asparagine      114.2
Peptide Mass Fingerprint                  Aspartic acid   115.1
                                           Glutamine       128.1
                                           Lysine          128.2
                                           Glutamic acid   129.1
                                           Methionine      131.2
                                           Histidine       137.1
                                           Phenylalanine   147.2
G-L-S-E-T-W-D-D-H-K = 1186.5 Da            Arginine        156.2
                                           Tyrosine        163.2
                                           Tryptophan      186.2
K-H-D-D-W-T-E-S-L-G = 1186.5 Da
   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.
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                  Digest fragments

  MASDFGHKILGFDSACV                        -R
  MNQWSDFFIILRTHYWE                        -LIPMASDFK
  DTYRRLIPMASDFKTYH                        -MASDFGHK
  MNGFDSAILIGRIISCFGK                      -THYWEDTYR
  PEQSADRTYIPLMKSDFV                       -THYWEDTYRR
  CQELISEL                                 -RLIPMASDFK
                                           -SDFVCQELISEL
                                           -PEQSADRTYIPLM
                                           -ILGFDSACVMNQWSDFFIILR
                                           -TYHMNGFDSAILIGRIISCFGK
Protein Digestion & Identification
   GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK
   AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAP
   SADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKl
   TVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTC
   RLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFV
   KLISWYDNEFGYSNRVVDLMAHMASKE
Protein Digestion & Identification
      GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK
      AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAP
      SADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKl
      TVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTC
      RLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFV
      KLISWYDNEFGYSNRVVDLMAHMASKE

                                         DIGEST


 Experimental m/z H+

 4036.8947    1201.6067
 3308.5642    1233.5966
 3340.5540    909.4900
 2595.3599    869.5091
 2611.3548    829.4414
 2277.0379    805.4315
 2293.0328    795.4181
 2213.1093    811.4131
 2245.0991    760.3835
 1763.8023    739.3621
 1719.8768    694.3518
 1613.9009    688.3777
 1473.7730    685.4243
 1411.7903
Protein Digestion & Identification
      GKVKVGVNGFGRLIGRVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVK
      AENGKLVINGNPITIFQERDPKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAP
      SADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKl
      TVDGPSGKWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTC
      RLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFV
      KLISWYDNEFGYSNRVVDLMAHMASKE

                                         DIGEST


 Experimental m/z H+                                  Predicted m/z H+

 4036.8947                                            4036.8947   1201.6067
              1201.6067
 3308.5642                                            3308.5642   1233.5966
              1233.5966
 3340.5540                                            3340.5540   909.4900
              909.4900
 2595.3599                                            2595.3599   869.5091
              869.5091
 2611.3548    829.4414        DATABASE QUERY          2611.3548   829.4414
 2277.0379                                            2277.0379   805.4315
              805.4315
 2293.0328                                            2293.0328   795.4181
              795.4181
 2213.1093                                            2213.1093   811.4131
              811.4131
 2245.0991                                            2245.0991   760.3835
              760.3835
 1763.8023                                            1763.8023   739.3621
              739.3621
 1719.8768                                            1719.8768   694.3518
              694.3518
 1613.9009                                            1613.9009   688.3777
              688.3777
 1473.7730                                            1473.7730   685.4243
              685.4243
 1411.7903                                            1411.7903
     Mass Fingerprinting: Mascot search
 Using MALDI-TOF mass spec data

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 search
Mass Fingerprinting: Mascot search
Mass Fingerprinting: Mascot search
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
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)
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      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?
                                                                       Get database sequences
                                          Actual MS/MS scan            that match precusor peptide mass            AVAGCAGAR
                                                                                                                   CVAAGAAGR
                                          Precursor petide                                                         VGGACAAAR
                                          [M+H]* = 828.2
                                                                                                                   etc….




        AVAGCAGAR                                         CVAAGAAGR                                         VGGACAAAR

                             b6                             y3                                      y2
b2   y2b3 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
                                                                                  Scoring

                                                                     Detect matches between
                                                                                                                   AVAGCAGAR 2.56
                                                                     Theoretical b- and y- ions
                                                                                                                   CVAAGAAGR 0.57
                                                                     Compute correlation
                                                                                                                   VGGACAAAR 0.32
                                                                     Rank hits
How Do We Identify Proteins Using MS/MS?
 Sequest analysis of MS/MS Ion Trap data
How Do We Identify Proteins Using MS/MS?
 Ion Trap MS/MS of doubly charged ions
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 Electrophoresis

Labeling




2D Gel Separation




Multichannel Imaging




DeCyder Differential
Analysis Software
Increase in protein expression:
        Osteoporosis
DIGE; Differential Gel Electrophoresis
Identify Proteins of Interest
Pick, Digest & Spot Proteins of
            Interest
Protein Identification Using 2D-MS
Protein ID: Mascot search
Using LC/MS/MS nanospray mass spec data
Protein ID: Mascot search
  FUNCTIONAL PROTEOMICS


 Quantification (Electrospray)
    ICAT
 Protein-protein Interactions (SELDI)
    Assemblies
    Complexes
 Post-translational analysis (Electrospray)
    Phosphorylation
    Ubiquitinylation
      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
       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
 Sample Quality is Key




Garbage in……………………………..Garbage out.
       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
 Margy Glasner: Dec 1, 2009


Wow, the things you can do!

				
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