Proteomics Core Lab an Introduction by Emmure

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									  Proteomics Core Lab
      an Introduction



            Proteomics Core Lab
  Institute of Plant and Microbial Biology
Agricultural Biotechnology Research Center


             Tuan-Nan Wen

                                             8. 21. 2009
           Proteomics

Global –    Studying all of the expressed proteins encoded
            by a genome of an organism

Targeted – Focus on protein expression within a particular
           cell type, growth conditions and subcellular
           localization.




                               Proteins and Proteomics, R.J. Simpson, CSHL
Why proteomics in addition to genomics and transcriptomics


    1. Verification of gene products.

    2. No simple correlation between changes in mRNA
      expression levels and protein levels.

    3. Post-translational modification, protein-protein
      interaction, and protein-ligand interaction increase
      the complexity in proteomics.


                               Proteins and Proteomics, R.J. Simpson, CSHL
   Approaches :

1. Protein mining – Large-scale identification and
   characterization of proteins in a sample.

2. Protein-expression profiling – Differential analysis, e.g.
  normal vs. diseased.

3. Protein-network mapping – Protein-protein interaction,
   protein functional networks, e.g. signal-transduction
   cascades.

4. Protein modification – Posttranslational modification
   (PTM), e.g. phosphorylation (phosphoproteome) and
   glycosylation (glycoproteome).
Technology platforms for Proteomics

1. Protein separation technology – SDS-PAGE, 2D-
  PAGE, LC, affinity capture.

2. Mass spectrometry (MS)

3. Software (Algorithms for protein ID from MS data
  and statistical validation, e.g. SEQUEST, Mascot,
  Trans-Proteomics Pipeline)

4. Database (Protein or nucleic acid sequences, such
  as NCBI, SwissProt)
General Workflow                                                                         Digest
                                                                                         protein
                      Protein extract                                                    mixture to
                                                                                         peptides

                    Electrophoresis                 LC                   Multi-dimensional
                    1DE/2DE                                              peptide separation
protein/peptide
separation
                                                                 SCX                          RP
strategies




 Digest           in-gel trypsin digest     in-solution trypsin digest
 proteins to
 peptides


 capillary
 HPLC peptide
                                          Mass Spectrometer
 separation
 and MS                     RP-LC


 Data analysis
2D-PAGE – protein separation




IEF (1st dimension)
                                           SDS-PAGE (2nd dimension)
         pH 3     IEF   pH 10
                                100 kDa
                                SDS-PAGE




                                10 kDa
Proteome Mining



    IEF

           SDS-PAGE


                      excise,   Peptide
                                            Database search &
                      in-gel    analysis
                                           Protein identification
                      digest    by MS

2-D PAGE
Pea chloroplast thylakoid proteins




       peripheral                            lumenal




                             Peltier, J-B, et al. 2000 The Plant Cell 12, 319
200 to 230 proteins
61 identified
33 known function
10 unknown funtion
18 not in the database   Peltier, J-B, et al. 2000 The Plant Cell 12, 319
         Protein expression profiling
Control    IEF




                   SDS-PAGE



                              excise                    Peptide analysis
                                                             by MS

 Test      IEF
                                           In-gel
                                       digestion with
                   SDS-PAGE




                                          trypsin
                                                         Database search &
                                                        Protein identification


        2-D PAGE
 2-D Fluorescence Difference Gel Electrophoresis system (DIGE)


  Protein Ext 1
      Cy3




  Protein Ext 2                                            Software
      Cy5
                                                           analysis

                         mix      2-D PAGE
Internal standard
(Pooled Ext 1 & 2)
      Cy2

                                              Image scan

                               spot excise, digest
                                MS analysis, ID
  Protein-Protein Interaction



                              excise
                              bands,     MS
                              digest   analysis
Cell lysate


                 SDS-PAGE
                                           Database search,
                                           Identification of
                                           protein components


                                LC-MS/MS


 coimmuno-      proteolysis
 precipitate
Protein identification


1. Functional analysis

2. N-terminal sequencing (Edman degradation)

3. Mass spectrometry
Mass Spectrometer in Proteomics

-   Protein identifications by MS and MS/MS - the most significant
    breakthrough in proteomics

-   Has essentially replaced the classical technique of Edman
    degradation

- Why ?

       - High sensitivity (~femtomoles or even atomoles)

       - High resolution

       - High accuracy --- 0.001 – 1 amu

       - Can deal with protein mixture

       - High throughput
       The Nobel Prize in Chemistry 2002

"for the development of methods for identification and
structure analyses of biological macromolecules"




   John B. Fenn     Koichi Tanaka      Kurt Wüthrich
   1/4 of the prize 1/4 of the prize   1/2 of the prize
        USA             Japan            Switzerland
  "for their development of soft       "for his development of nuclear
  desorption ionisation methods,       magnetic resonance
  ESI (Fenn) and soft laser            spectroscopy for determining the
  ionization (Tanaka), for mass        three-dimensional structure of
  spectrometric analyses of            biological macromolecules in
  biological macromolecules"           solution"
    Mass Spectrometer


Sample

          Ion source      Mass analyzer   Detector


                       Vacuum chamber



                        Mass spectrum




         intensity




                             m/z
  Ion source
     MALDI (Matrix-Assisted Laser Desorption Ionization)

     ESI (Electrospray Ionization)


Mass analyzer                        Tandem mass analyzer
  Quadrupole                           Triple quadrupole (Q-Q-Q)

  TOF (Time-Of-Flight)                 Q-TOF

  Ion Trap                             TOF-TOF

  Sector (Magnetic, Electrostatic)     Ion Trap

  Fourier Transform MS
Protein identification by MS


1. Peptide-mass mapping (peptide-mass fingerprinting)
  by MALDI-TOF MS


2. Peptide sequence analysis by tandem mass spectrometry
  (MALDI-Q/TOF, ESI-Ion trap MS/MS)
  Peptide-mass mapping (peptide-mass fingerprinting)

           2DE                                   Database
                                                 computer
                                                 calculated
                             MS                  peptide
                             measured            mass list
                             peptide
     in-gel trypsin digest   mass list           Protein X
                                                 790.21
                              899.33
                             1120.31
                                                 899.34
                             1251.71             987.11
MALDI-TOF MS                 1529.83             1010.21
                             1833.88             1120.31
                             1922.39             1251.79
 Full scan MS                 ____               1333.45
                              ____               1501.11
                              ____               1529.81
                              ____
                                                 1705.00
                              ____
                                                 1833.84
                                                 1922.34
           m/z
                                                 2100.45
Protein identification by MS


1. Peptide-mass mapping (peptide-mass fingerprinting)
  by MALDI-TOF MS


2. Peptide sequence analysis by tandem mass spectrometry
  (MALDI-Q/TOF, ESI-Ion trap MS/MS)
    Automated acquisition of MS-MS spectra

                       TIC
                                                                      1         Full scan
                                                        2                       MS
                                                                  3

                                                  4




          Time (min)                                        m/z


                                   Fragmentation of precursor ion &
                                   MS-MS spectra collection


1               2                             3                           4




    m/z                      m/z                      m/z                     m/z
Peptide sequence analysis by tandem mass spectrometry
(MALDI-Q/TOF, ESI-Ion trap MS/MS)

   Fragmentation of peptide ions


                   a    b    c

                        O          R2                O
               H                            H
               N                            N
                             N
                             H
                                        O
                   R1                           R3




                         x   y      z
     BSA trypsin digest
     100 fmol




MS
MS/MS
Database search and peptide identification
- Peptide MS2 spectrum and fragment ion matches



                              b ions

                           HLVDEPQNLIK
                               y ions
Database search result
Protein mix sample in one LC-MS/MS run

                              5 protein mix, 20 fmol/ea




        Coverage
                          100 fmol     50 fmol      20 fmol

   BSA (69.2 kDa)         72% (44)    71% (43)     68% (41)

    -Casein (23.5 kDa)    27% (5)      17% (2)      17% (2)

   Myoglobin (16.9 kDa)   44% (5)      44% (5)      44% (5)

   Ribonuclase A (13.6    61% (5)      53% (4)      36% (3)
   kDa)
   Insulin (5.1 kDa)      43% (1)      43% (1)      43% (1)
Peptide Phosphorylation Site Mapping


                                       shows neutral loss
                             MS2          (-49 Da)




                                           MS3
          MS
FQS*EEQQQTEDELQDK
        -Casein
Thanks

								
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