Proteomics Course outline
Medical University of Bialystok (Poland)
Theoretical classes
Theme 1.- Introduction. Proteomics concept. Historical and technological perspective. First and second
generation Proteomics. Protein separation. Protein analysis. Data analysis and interpretation of results. (EMI)
Theme 2: Protein Chemistry and Proteomics. The basics of protein chemistry reactivity. Chemical properties
of amino acids. Protein isoelectric point. Reactions of interest in Proteomics. (JV)
Theme 3.- Experimental approaches in first generation Proteomics. SDS-PAGE. Two-dimensional
electrophoresis. The DIGE technique. Two-dimensional electrophoresis data analysis. (IJ)
Theme 4.- Interpretation of mass spectra. Resolution. Accuracy. Isotopic envelope. Charge deconvolution.
(EN)
Theme 5.- The basics of mass spectrometry. Soft ionization methods: MALDI and ESI. Mass analyzers: TOF,
quadrupole, ion trap, linear trap, FT, Orbitrap. Combinations of ionization sources and analysers. (ECA)
Theme 6.- Identification of gel-separated proteins. In-gel protein digestion techniques. MALDI peptide mass
fingerprinting and peptide fragmentation fingerprinting. Search engines and interpretation of results. (EMI)
Theme 7. Molecular mechanisms of peptide fragmentation by mass spectrometry. Ion pathways of peptide
fragmentation. Roepstorf-Fohlman nomenclature. Interpretation of peptide MS/MS spectra. Peptide
identification on protein databases based on MS/MS spectra. Search engines, scores and discovery rates.
Characterization of post-translational modifications. (JV)
Theme 8: Tandem mass spectrometry (MS/MS). Triple quadrupole. Quadrupole-TOF. Peptide fragmentation
in the ion trap and the linear trap. TOF/TOF mass spectrometers. Fragmentation methods: CID, HCD and ETD.
Multiple fragmentation. Scanning modes in a mass spectrometer. Parent scan. Neutral loss scan. Selected
reaction monitoring. (ECA)
Theme 9.- Second Generation Proteomics. Peptide separation by liquid chromatography. Off-line and on-line
liquid chromatography-mass spectrometry analysis. Micro- and nano-flows. Large-scale analysis of proteomes
by second generation proteomics approaches. Second generation quantitative proteomics approaches. Stable
isotopic labeling. Chemical (ICAT, iTRAQ), metabolic (SILAC) and enzymatic (18O) labeling methods.
Analysis of protein abundance changes. (IJ) (EN).
Practical Classes
Practice 1.- MS/MS spectra interpretation (JV, EMI).
Practice 2.- Protein identification by PMF and MS/MS (EMI, IJ).
Practice 3.- Protein quantitation by stable isotopic labelling (EN, IJ).
Instructors:
Dr. Estefanía Núñez (estefania.nunez@externo.cnic.es), EN
Dr. Emilio Camafeita (ecamafeita@cnic.es), EMI
Dra. Inmaculada Jorge (ijorge@cnic.es), IJ
Prof. Jesús Vázquez (jvazquez@cbm.uam.es), JV
Chronogram:
Time
9:00-12:00h
12:00-13:00h
13:00-15:30h
Day 1
Day 2
Day 3
Theme 1
Theme 4
Theme 7
Theme 2
Theme 3
Lunch
Practice 1
(ECA, JV)
Theme 5
Theme 6
Lunch
Practice 2
(EMI, JV)
Theme 8
Theme 9
Lunch
Practice 3
(EMI, IJ)
Bibliography:
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Proteins and Proteomics, a laboratory manual. Ed. Richard J. Simpson. Cold Spring Harbor
Laboratories, 2003
Protein Sequencing and identification using tandem mass spectrometry. Michael Kinter y Nicholas E.
Sherman, Wiley, 2000.
Chemistry of Protein Conjugation and Crosslinking. Shan S. Wong. CRC Press 1993.
Mass Spectrometry in the Biological Sciences. A.L. Burlingame and Steven A. Carr eds. Humana Press,
1996.
Advances in Mass Spectrometry. E. Gelpi Ed. Wiley 2001
Gel electrophoresis of proteins. A practical approach. B.D. Hames and D. Rickwood. Oxford University
Press, 1990
Electrophoresis in practice. R. Westermeier. WCH-Wiley 1997
Mechanisms in Protein Chemistry. J. Kyte. Garland Publishin inc. 1995
Structure in Protein Chemistry. J. Kyte. Garland Publishing inc. 1995
The Protein protocols handbook. J. M. Walker. Humana Press. 1996
2D Proteome Analysis protocols. A.J. Link (Methods in Molecular Biology). Humana Press 1999
Molecular biologist guide to Proteomics. P. Graves and J. Haystead. Microbiology and Molecular
Biology Reviews 2002, 66, 39-63.
Mass spectrometry-based Proteomics. R. Aebersold and M. Mann. Nature 2003, 422, 198-207.
Human body fluid proteome analysis. S. Hu, J. Loo and D.T. Wong. Proteomics 2006, 6, 6326-6353
The ABC’S (and XYZ’s) of peptide sequencing. A. Steen and M. Mann. Nature Reviews 2004. 5, 699711
Proteomic analysis of phosphorylation, oxidation and nitrosylation in signal transduction. C. M.
Spickett, A.R. Pitt, N. Morrice, W. Kolch. Biochimica et Biophysica Acta 2006, 1764 1823–1841
Target-decoy search strategy for increased confidence in large-scale protein identifications by mass
spectrometry. J.E. Elias & S.P. Gygi. Nature Methods 2007, 4, 207-214
Proteomic analysis of postranslational modifications. M. Mann and O. Jensen. Nature Biotech. 21, 255261.
Mass-spectrometric-based approaches in quantitative proteomics S.E. Ong, L.J. Foster and M.Mann.
Methods 2003, 29, 124-130