Proteomics 101


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Proteomics 101

  1. 1. Why is Sequence Information Important <ul><li>A protein’s amino acid sequence is unique. </li></ul><ul><ul><li>As little as 5 amino acid sequences can ID a protein </li></ul></ul><ul><li>The sequence defines the primary structure of the protein </li></ul><ul><ul><li>the primary structure is fundamental to understanding the structure and function of the protein </li></ul></ul><ul><li>The interrelationship between an amino acid sequence and the corresponding DNA sequence </li></ul><ul><ul><li>Protein sequences access gene sequences and are key to molecular biology </li></ul></ul>
  2. 2. Current Methods for Proteome Research <ul><li>SDS-PAGE </li></ul><ul><ul><li>separates based on molecular weight and/or isoelectric point </li></ul></ul><ul><ul><li>10 fmol - > 10 pmol sensitivity </li></ul></ul><ul><ul><li>Tracks protein expression patterns </li></ul></ul><ul><li>Protein Sequencing </li></ul><ul><ul><li>Edman degradation or internal sequence analysis </li></ul></ul><ul><li>Immunological Methods </li></ul><ul><ul><li>Western Blots </li></ul></ul>
  3. 3. Drawbacks <ul><li>SDS-Page can track the appearance, disappearance or molecular weight shifts of proteins, but can not ID the protein or measure the molecular weight with any accuracy </li></ul><ul><li>Edman degradation requires a large amount of protein and does not work on N-terminal blocked proteins </li></ul><ul><li>Western blotting is presumptive, requires the availability of suitable antibodies and have limited confidence in the ID related to the specificity of the antibody. </li></ul>
  4. 4. Advantageous of Mass Spectrometry <ul><li>Sensitivity in attomole range </li></ul><ul><li>Rapid speed of analysis </li></ul><ul><li>Ability to characterize and locate post-translational modifications </li></ul>
  5. 5. Protein Identification Experiment Separated Proteins Enzymatic Digestion and Extraction MALDI-TOF Nano LC-MS-MS Database Search Sequence Tag Protein Identification Database Search Protein Identification
  6. 6. Enzymes for Proteome Research K-X and R-X except when X = P Trypsin X-M Cyanogen Bromide X-L, X-F, X-Y and X-W Chymotrypsin E-X except when X = P Endoprotease Glu-C X-D Endoprotease Asp-N R-X except when X = P Endoprotease Arg-C K-X except when X = P Endoprotease Lys-C
  7. 7. Protease digestion Protein Sample Peptides m/z MALDI Mass Spectrum 1000 2000 Peptides analyzed by MALDI VVWCAVGPKKQK 337-348 0.209 1400.675 1400.884 CSTSPLLEACAFLTR 667-681 0.301 1725.122 1725.122 VVWCAVGPEEQKK 337-349 0.221 1528.991 1528.991 NLLFNDNTECLAK 630-642 0.246 1550.985 1550.985 GEADALNLDGGYIYTAGK 379-396 0.344 1827.212 1827.212 IPSKVDSALYLGSR 302-315 0.249 1505.073 1505.073 NLRETAEEVKAR 322-333 0.158 1414.752 1414.910 QVLLHQQALFGK 601-612 0.223 1380.787 1381.010 Sequence Residues Error Theoretical (Da) Measured (Da)
  8. 8. Micro-Sequencing by Tandem Mass Spectrometry (MS/MS) <ul><li>Ions of interest are selected in the first mass analyzer </li></ul><ul><li>Collision Induced Dissociation (CID) is used to fragment the selected ions by colliding the ions with gas (typically Argon for low energy CID) </li></ul><ul><li>The second mass analyzer measures the fragment ions </li></ul><ul><li>The types of fragment ions observed in an MS/MS spectrum depend on many factors including primary sequence, the amount of internal energy, how the energy was introduced, charge state, etc. </li></ul><ul><li>Fragmentation of peptides (amino acid chains) typically occurs along the peptide backbone. Each residue of the peptide chain successively fragments off, both in the N->C and C->N direction. </li></ul>
  9. 9. Sequence Nomenclature for Mass Ladder 1598 1424 1295 1166 1052 965 852 723 586 529 401 Q G H E L S N E E R +H Roepstorff, P and Fohlman, J, Proposal for a common nomenclature for sequence ions in mass spectra of peptides . Biomed Mass Spectrom, 11(11) 601 (1984). a 1 a 3 a 2 b 3 b 2 b 1 c 2 c 2 c 1 x 1 x 3 x 2 y 3 y 2 y 1 z 2 z 2 z 1
  10. 10. Protease digestion Protein Sample Peptides GDVE K G KK IFVQ K CAQCHTVE K GG K H K TGPNLHGLFG R K TGQAPGFTYTDAN K N K GITWKEETLMEYLENP KK YIPGT K MIFAGI KKK TEREDLIAYL KK ATNE m/z First Stage Mass Spectrum 300 2200 TGPNLHGFGR Selected Precursor mass and fragments TGPNLHGLFGR R GR FGR GFGR etc Protein Sequence Peptides of precursors molecular weight fragmented Peptides eluted from LC Second Stage (fragmentation) Mass Spectrum m/z 75 2000
  11. 11. References <ul><li>Kinter, M.; Sherman, N. E. Protein Sequencing and Identification Using Tandem Mass Spectrometry ; Wiley-Interscience: New York, 2000. </li></ul><ul><li>Aebersold, R.; Mann, M. Nature 2003 , 422 , 198-207. </li></ul>