PROTEOMICS LECTURE by lL4X730

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									PROTEOMICS
  LECTURE
     The “omics” nomenclature…
             Genomics          DNA (Gene)
                                  Transcription

             Transcriptomics   RNA

Functional                        Translation
Genomics
             Proteomics        PROTEIN
                                     Enzymatic
                                      reaction
             Metabolomics      METABOLITE
              A few definitions…

Gen                                       Genes
Transcript     ~ome = Sequence of a       Transcripts
Prote                 complete set of     Proteins
Metabol                                   Metabolites


Gen          ~omics =   Analysis of the   Genome
Prote                                     Proteome
    Why study protein expression?
          (The steps of gene expression control)
                  (Gygi et al., Mol. Celll. Biol., 1990, p.1720-1730)
                   Nucleus                  Cytosol

                                                                        Inactive mRNA
                                             RNA
                                          Degradation
                                            control

           Primary
DNA          RNA             mRNA               mRNA
          transcript
                                                         Translation control
                                          RNA
                                                           protein               Modified
                       RNA             Transport
                                                                                  protein
Transcriptional     Processing          control
    control           control                                     Post-translational
                                                                       control
  Applications of Proteomics
• Mining: identification of proteins (catalog
  the proteins)
• Protein-expression profile: identification of
  proteins in a particular state of the
  organism
• Protein-network mapping: protein
  interactions in living systems
• Mapping of protein modifications: how and
  where proteins are modified.
Proteins classes for Analysis
    •   Membrane
    •   Soluble proteins
    •   Nuclear
    •   Chromosome-associated
    •   Phosphorylated
    •   Glycosylated
    •   Complexes
             SEPARATION
 General
  flow for
proteomics
             IDENTIFICATION
 analysis
  Current Proteomics Technologies
• Proteome profiling/separation
   – 2D SDS PAGE (two-dimensional sodium
     dodecylsulphate polyacrylamide gel electrophoresis)
   – 2-D LC/LC
   (LC = Liquid Chromatography)
   – 2-D LC/MS
   (MS= Mass spectrometry)
• Protein identification
   – Peptide mass fingerprint
   – Tandem Mass Spectrometry (MS/MS)
• Quantative proteomics
  - ICAT (isotope-coded affinity tag)
 2D-SDS
PAGE gel
                1) Sample loading      2) Remove the cover
                                      sheet from the IEF gel




  3)Place the strip gel             4) Place the strip on the
   in the focusing tray             top of the SDS-PAGE gel
2D-SDS PAGE gel
The first dimension
(separation by isoelectric focusing)
- gel with an immobilised pH gradient
- electric current causes charged
proteins to move until it reaches the
isoelectric point
(pH gradient makes the net charge 0)
                          Isoelectric point (pI)
                • Separation by charge:
                             Low pH:
                      4      Protein is
                             positively            At the isolectric
Stable pH gradient




                      5                            point the protein
                             charged
                      6                            has no net
                                                   charge and
                      7                            therefore no
                      8     High pH:               longer migrates
                            protein is             in the electric
                      9                            field.
                            negatively
                     10     charged
2D-SDS PAGE gel
The first dimension
(separation by isoelectric focusing)
- gel with an immobilised pH gradient
- electric current causes charged
proteins to move until it reaches the
isoelectric point
(pH gradient makes the net charge 0)



The second dimension
(separation by mass)
-pH gel strip is loaded onto a SDS gel
-SDS denatures and linearises the
protein (to make movement solely
dependent on mass, not shape)
2D-SDS PAGE gel
2D-gel technique example
 Advantages vs. Disadvantages
• Good resolution     • Not for
  of proteins           hydrophobic
• Detection of          proteins
  posttranslational   • Limited by pH
  modifications         range
                      • Not easy for low
                        abundant proteins
                      • Analysis and
                        quantification are
                        difficult
                      2D - LC/LC
                                      Peptides all bind
                          (trypsin)   to cation
Study protein                         exchange column
complexes
                                      Successive elution
without gel                           with increasing salt
electrophoresis                       gradients separates
                                      peptides by charge

                                      Peptides are
                                      separated by
Complex mixture is                    hydrophobicity on
                                      reverse phase
simplified prior to                   column
MS/MS by 2D LC
       Reverse Phase column




  Polypeptides enter the column in the mobile phase…
…the hydrophobic “foot” of the polypeptides adsorb to the
  hydrophobic (non polar) surface of the reverse-phase
  material (stationary phase) where they remain until…
   …the organic modifier concentration rises to critical
      concentration and desorbs the polypeptides
2D - LC/MS
 Methods for
   protein
identification
Mass Spectrometry (MS) Stages
• Introduce sample to the instrument
• Generate ions in the gas phase
• Separate ions on the basis of differences in m/z
  with a mass analyzer
• Detect ions
  How the protein sequencing works?
• Use Tandem MS: two mass
  analyzer in series with a collision   Ser-Glu-Leu-Ile-Arg-Trp
  cell in between
• Collision cell: a region where
  the ions collide with a gas (He,
  Ne, Ar) resulting in                         Collision Cell
  fragmentation of the ion
• Fragmentation of the peptides in      Ser-Glu-Leu-Ile-Arg
  the collision cell occur in a
  predictable fashion, mainly at the     Ser-Glu-Leu-Ile
  peptide bonds (also
  phosphoester bonds)                    Ser-Glu-Leu
• The resulting daughter ions have
  masses that are consistent with                      Etc…
  known molecular weights of
  dipeptides, tripeptides,
  tetrapeptides…
      Tandem Mass Spectrometry
            Isolates individual peptide fragments for
(trypsin)   2nd mass spec – can obtain peptide
            sequence




                             Compare peptide sequence
                               with protein databases
 Advantages vs. Disadvantages
• Determination       • High capital costs
  of MW and aa.       • Requires sequence
  Sequence              databases for
• Detection of          analysis
  posttranslational
  modifications
• High-throughput
  capability
Protein identification by Peptide
        Mass fingerprint

• Use MS to measure the masses of
  proteolytic peptide fragments.
• Identification is done by matching the
  measured peptide masses to
  corresponding peptide masses from
  protein or nucleotide sequence databases.
             Mass spectometry (MS)

 (trypsin)
                 Mass spectrometry – method of separating
                 molecules based on mass/charge ratio




eg. MALDI-TOF
                                  Compare peptide m/z
                                  with protein databases
     Protein Identification by MS
                                      Spectrum of
Spot removed        Fragmented
                                       fragments
  from gel          using trypsin
          Library                      generated




                     MATCH




                                      Database of
  Artificial         Artificially
                                       sequences
spectra built       trypsinated
                                    (i.e. SwissProt)
  ISOTOPE-CODED AFFINITY TAG
   (ICAT): a quantitative method
• Label protein samples with heavy and light
  reagent
• Reagent contains affinity tag and heavy or light
  isotopes
   Chemically reactive group: forms a
   covalent bond to the protein or peptide

   Isotope-labeled linker: heavy or light,
   depending on which isotope is used

  Affinity tag: enables the protein or
  peptide bearing an ICAT to be isolated by
  affinity chromatography in a single step
     Example of an ICAT Reagent
Biotin Affinity tag:   Reactive group: Thiol-
Binds tightly to       reactive group will bind to Cys
streptavidin-agarose
resin
      O
                           Linker: Heavy version will
NH                         have deuteriums at *
            NH             Light version will have
                           hydrogens at *
                       H                                 H
                       N     *       O       O       *   N
                                                 *
                                                                 I
                                 *       O
                                                             O
      S            O
                   How ICAT works?
                      Affinity isolation
                      on streptavidin
                            beads

          Lyse &        Quantification              Identification
           Label            MS                         MS/MS

                                                   NH2-EACDPLR-COOH
                                  Light
                      100
                                                  100
          MIX                             Heavy

Proteolysis
(eg trypsin)
                        0                           0
                            550     570    590          200    400   600
                                   m/z                        m/z
 Advantages vs. Disadvantages
• Estimates relative       • Yield and non specificity
  protein levels between   • Slight chromatography
  samples with a             differences
  reasonable level of      • Expensive
  accuracy (within 10%)
                           • Tag fragmentation
• Can be used on
  complex mixtures of      • Meaning of relative
  proteins                   quantification information
• Cys-specific label       • No presence of cysteine
  reduces sample             residues or not accessible
  complexity                 by ICAT reagent
• Peptides can be
  sequenced directly if
  tandem MS-MS is used

								
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