Proteomics

1. Proteomics What is a proteome? Proteome Characterization Proteomic Projects Use of the Proteome Reading: Ch 15.2 BIO520 Bioinformatics Jim Lund

2. Protein Sequences from Mastodon and Tyrannosaurus Rex Revealed by Mass SpectrometryJohn M. Asara,Mary H. Schweitzer, Lisa M. Freimark,Matthew Phillips, Lewis C. CantleyScience 13 April 2007: Vol. 316. no. 5822, pp. 280 - 285

3. Gene vs. Protein diversity • Human Genome = 25,000 genes • Human Proteome = 300,000 to 1,200,000 protein variants

4. 1 gene-> many proteins • Alternative splicing: ~60% of genes in the human genome, several splice forms are typical. • International Human Genome Sequencing Consortium, Nature 2001 • Proteolytic cleavage • Covalent modifications • Phosphorylation, Acylation, Methylation, Glycosylation,Sulfation, Prenylation, lipid linkage and many more…

5. mRNA-protein Correlation • YPD: should have relevant data • will yeast be typical? • Electrophoresis 18:533 • 23 proteins on 2D gels • r=0.48 for mRNA=protein • Post-transcriptional and post translational regulation important!

6. Branches of Proteomics • Protein separation. Basic to all proteomic technologies are protein separation; the separation of a complex mixture so that individual proteins are more easily processed with other techniques. • Protein identification. Low-throughput sequencing through Edman degradation; High throughput proteomic techniques based on mass spectrometry, commonly peptide mass fingerprinting on simpler instruments, or de novo sequencing with tandem mass spectrometry. • Protein quantification. Gel-based methods such as differential staining of gels with fluorescent dyes (difference gel electrophoresis). Gel-free methods include various tagging or chemical modification methods, such as isotope-coded affinity tags (ICATs) or combined fractional diagnoal chromatography (COFRADIC). Mass spec methods are now giving quantification data. • Protein sequence analysis. Bioinformatic branch, search databases for possible protein or peptide matches. • Structural proteomics. High-throughput determination of protein structures in three-dimensional space using x-ray crystallography and NMR spectroscopy. • Interaction proteomics. Investigation of protein interactions using IP then MS, 2-hydrid screens, protein chips. • Protein modification. Almost all proteins are modified from their pure translated amino-acid sequence, so-called post-translational modification. Specialized methods have been developed to study phosporylation (phosphoproteomics) and glycosylation (glycoproteomics). MS methods are also used.

7. Components of Proteomics Protein Separation Mass Spectroscopy Bioinformatics

8. Protein separation • 2D-PAGE • Separate proteins based on size and charge • Types of 2D-PAGE gels: • IEF/SDS • NEPHGE/SDS • HPLC

9. 2D-PAGE

10. Detection Methods • Stains • Fluorescence, Coomassie blue, silver stain • ~500 spots • Radiolabeling • Coupled detection • Mass spectrometry • MALDI/TOF • ESI • Trypsin digestion/MS

11. Instrumentation • 2D gels • Simple • MALDI-TOF and variants • $200,000+, benchtop

12. 2D Gel Results • SwissProt • www.expasy.ch/ch2d/ • 2DWG Meta-database of 2D-gels • http://www-lecb.ncifcrf.gov/2dwgDB/

13. Basic Proteomic Analysis Scheme Separation Protein Mixture Individual Proteins 2D-SDS-PAGE Spot Cutting Digestion Trypsin Mass Spectroscopy Peptide Mass Peptides MALDI-TOF Database Search Protein Identification

14. 2D-PAGE -> MALDI-MS->PMF

15. 2D-PAGE -> MS-MS->PMF

16. MS-MS

17. Principles of MALDI-TOF Mass Spectroscopy

18. MALDI-TOF Results

19. NOVEL A vs B

20. Analytical Approach to Peptide Mass Fingerprinting: Effect of Mass Tolerance Search m/z Mass Tolerance (Da) # Hits Database

21. Analytical Approach to Peptide Mass Fingerprinting: Effect of Multiple Peptide Masses Search m/z Mass Tolerance # Hits Database

22. Find peptide fragments from MS spectra. Charge state deconvolution. Make peptide fragment list. Check versus list of all possible polypeptides. Need to have Protein database! Peptide Mass Fingerprinting

23. Methanol m/z spectra

24. Peptide Mass Fingerprinting

25. Protein Sequences from Mastodon and Tyrannosaurus Rex Revealed by Mass Spectrometry

26. Mascot http://www.matrixscience.com/ SEQUEST http://fields.scripps.edu/sequest/ (Thermo Scientific) X!Tandem http://thegpm.org/ Free and Open Source PeptIdent Uses Swiss-Prot protein database http://au.expasy.org/tools/peptident.html Free and Open Source ProteinProspector Searches user supplied protein database. http://falcon.ludwig.ucl.ac.uk/mshome3.2.htm Free and Open Source GFS Uses raw genome sequence. http://gfs.unc.edu/cgi-bin/WebObjects/GFSWeb Free and Open Source Peptide Search Programs

27. MS varieties • Ionization Methods • EI (Electron Impact) • CI (Chemical Ionization) • MALDI • ESI • Fast Atom Bombardment (FAB) • Mass Analyzers • Ion Trap • Time-of-Flight • Theoretically, no limitation for m/z maximum, high throughput • Magnetic Sector • High resolution, exact mass • Ion Cyclotron Resonance (FTMS) • Very high resolution, exact mass, perform ion chemistry • Quadrupole • Unit mass resolution, fast scan, low cost Technology is developing quickly! • UK has a Proteomics Facility! • http://www.rgs.uky.edu/ukmsf/proteomics.html

28. Searching parameters • Modifications (e.g. cysteine residues, etc.) • Number of allowable missed cleavages • Data properties: monoisotopic/average, charge state, amino acid composition • MS/MS data

29. Protein identification approaches

30. Limitations-2D gel, MS • Protein preparation/electrophoresis • hydrophobic proteins insoluble • Sensitivity • stains, ~1 ng (1/10,000) • MS (1 fmol, ~40 pg for “average”) • Protein modificationsunclear • Data analysis/comparison • Scimagix (www.scimagix.com) • Nature of data • quaternary info lost, localization lost

31. Clinical Diagnostics Proteomics: Protein Profiling

32. Value of Proteome Data • Contains info not in mRNA! • [mRNA] != [protein] • Covalent modification of proteins critical to regulation, often with constant expression • Association state of proteins critical • How can we use this information?