1. PROTEOMICS De novo sequence prediction for: nsi78_11.1803.1806.2.dta SequenceAbsoluteRelative Probability CRGSVNFP[PL]FK 3.9%36.3% CRGSVN[DE][PL]FK 2.3%24.7% CRGSVPFN[PN]FK 6.1%17.2% CRGSV[SR]D[PL]FK 3.1%6.5% CRGSVPFNWGDK< 0.1%2.7%

2. Genomics DNA (Gene) Functional Genomics TranscriptomicsRNA Proteomics PROTEIN Metabolomics METABOLITE Transcription Translation Enzymatic reaction The “omics” nomenclature…

3. Gen Transcript Prote Metabol ~ome Sequence of a complete set of Genes Transcripts Proteins Metabolites = Gen Prote ~omics = Analysis of the Genome Proteome A few definitions…

4. Current -omics

5. The proteome is defined as the set of all expressed proteins in a cell, tissue or organism (Wilkins et al., 1997). Proteomics can be defined as the systematic analysis of proteins for their identity, quantity and function.

6. ProteomeGenome dynamicstatic No amplification possible Amplification possible Hetergenous molecules Homogenous molecules Large variability of the amount No variability of the amount

7. Complexity of the proteome

8. Applications of Proteomics Mining: identification of proteins (catalog the proteins) Quantitative proteomics: defining the relative or absolute amount of a protein 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.

9. Proteins classes for Analysis Membrane Soluble proteins Organelle-specific Chromosome-associated Phosphorylated Glycosylated Multi-protein complexes

10. General flow for proteomics analysis SEPARATION IDENTIFICATION

11. 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) - SILAC (stable isotopic labeling of amino acids)

12. 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) 2D-PAGE gel

13. Isoelectric point (pI) Separation by charge: 4 5 6 7 8 9 10 Stable pH gradient High pH: protein is negatively charged Low pH: Protein is positively charged At the isolectric point the protein has no net charge and therefore no longer migrates in the electric field.

14. 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

16. 2D-gel technique example

18. Some limitations of 2DE: Limited dynamic range of detection - bias towards high abundant proteins Co-migration of proteins Separation of proteins –Basic proteins (IP > 10) –Hydrophobic proteins –Small and large proteins ( 150 kDa)

19. Methods for protein identification

20. 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 Vacuum System SamplesHPLC Detector Data System Mass Analyser Ionisation Method MALDI ESI

21. Aebersold, R. and Mann, M. (2003) Mass spectrometry-based proteomics. Nature, 422, 198-207. Mass spectrometers used in proteomic research

22. Principles of MALDI-TOF Mass Spectrometry Mann, M., Hendrickson, R.C. and Pandey, A. (2001) Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem, 70, 437-473.

23. Electro-spray ionisation ESI M + RH + MH + + R (in solution)

24. Methods for protein identification

25. 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.

26. Mass spectrometry – method of separating molecules based on mass/charge ratio Compare peptide m/z with protein databases eg. MALDI-TOF (trypsin) Mass spectometry (MS)

27. Protein Identification by MS Artificial spectra built Artificially trypsinated Database of sequences (i.e. SwissProt) Spot removed from gel Fragmented using trypsin Spectrum of fragments generated MATCH Library

28. MALDI peptide map and identification of a protein. A 116-kDa band was excised and subjected to tryptic digestion in gel. Mann, M., Hendrickson, R.C. and Pandey, A. (2001) Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem, 70, 437-473.

29. Advantages vs. Disadvantages Determination of MW High-throughput capability Relative low costs Ambiguous results difficult to interpret Requires sequence databases for analysis Limitations can be overcome by peptide sequencing using tandem mass spectrometry

30. How the protein sequencing works? Use Tandem MS: two mass analyzer in series with a collision cell in between Collision cell: a region where the ions collide with a gas (He, Ne, Ar) resulting in fragmentation of the ion Fragmentation of the peptides in the collision cell occur in a predictable fashion, mainly at the peptide bonds (also phosphoester bonds) The resulting daughter ions have masses that are consistent with known molecular weights of dipeptides, tripeptides, tetrapeptides… Ser-Glu-Leu-Ile-Arg-Trp Collision Cell Ser-Glu-Leu-Ile-Arg Ser-Glu-Leu Ser-Glu-Leu-Ile Etc…

31. Peng, J. and Gygi, S.P. (2001) Proteomics: the move to mixtures. J Mass Spectrom, 36, 1083-1091.

32. Schematic of a quadrupole TOF instrument After traversing a countercurrent gas stream (curtain gas), the ions enter the vacuum system and are focused into the first quadrupole section (q0). They can be mass- separated in Q1 and dissociated in q2. Ions enter the time-of-flight analyzer through a grid and are pulsed into the reflector and onto the detector, where they are recorded. There are 14,000 pulsing events per second. Mann, M., Hendrickson, R.C. and Pandey, A. (2001) Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem, 70, 437-473.

33. Peptide Fragmentation

35. Isolates individual peptide fragments for 2 nd mass spec – can obtain peptide sequence Compare peptide sequence with protein databases (trypsin) Tandem Mass Spectrometry

36. Advantages vs. Disadvantages Determination of MW and aa. Sequence Detection of posttranslational modifications High-throughput capability High capital costs Requires sequence databases for analysis

37. LC Ion trap MS 75 µm RP 200 nL to MS Peptide: 1.MW 2.Sequence 3.Modification Tryptic digested proteins Coupling of LC and tandem MS

38. 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 Reverse Phase column

39. Data acquired - Chromatogram

40. Triple Play

41. + c Full ms [ 400.00-2000.00] 400600800100012001400160018002000 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 626.3 835.5 982.4 610.2 1054.4 1156.2 852.2 1157.5 703.2 885.0 578.8 503.9765.9 1217.7 445.1 1469.7 1259.8 1 2 Triple Play Dynamic Exclusion Scan 4501 Scan 4502 Scan 4503

42. + c d Full ms2 852.26@35.00 [ 220.00-2000.00] 400600800100012001400160018002000 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 721.2 471.0 1261.0 697.1 636.8 1141.9 1076.2 787.5 611.5 1029.1 1558.2 515.2 340.0 830.01648.0 930.3 1 + d Z ms [ 848.00-858.00] 848849850851852853854855856857858 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 852.2 853.1 2 3 Triple Play Dynamic Exclusion Scan 4504 Scan 4505 Scan 4506

43. 2D - LC/MS Peng, J. and Gygi, S.P. (2001) Proteomics: the move to mixtures. J Mass Spectrom, 36, 1083-1091.

45. Multidimensional Protein Identification Technology (MudPIT). Whitelegge JP (2002) Plant proteomics: BLASTing out of a MudPIT. Proc Natl Acad Sci U S A 99: 11564-6.

46. Koller A, Washburn MP, Lange BM, Andon NL, Deciu C, Haynes PA, Hays L, Schieltz D, Ulaszek R, Wei J, Wolters D, Yates JR, 3rd (2002) Proteomic survey of metabolic pathways in rice. Proc Natl Acad Sci U S A 5: 5.