KBC/PROT Proteomics

Lecturer: Ivo Chamrád
Lecture: 2 hours/week
Credits: 3
Summer semester
Form of course completion: course credit, exam

Introduction to proteomics. Proteome. Basic terminology of protein separation and analysis. Branches of proteomics. Significance of expression, differential and functional proteomics. Partial separation of protein mixtures. Conventional chromatography, affinity chromatography, preparative electrophoresis. One-dimensional electrophoresis (1-D) – SDS/PAGE. Two-dimensional (2-D) electrophoresis. Sample preparation. First dimension – isoelectric focusing (IEF). Ampholytes, tube IEF gels. Gel strips with immobilized pH gradient ("IPG strips"). Methodology and instrumentation of IEF separation. Reversible staining of IPG strips. Second dimension – SDS/PAGE. Buffer composition, casting of gels. Instrumentation of SDS/PAGE (horizontal and vertical units). Protein staining in SDS/PAGE gels, compatibility of staining with mass spectrometry. Fluorescence staining of gels. Imaging, densitometry, software for evaluation of 2D gels. Comparison of two or more 2D gels, difference analysis. DIGE method. Excision of protein spots. Electroelution. Chemical and enzymatic digestion of proteins. In-gel digestion. Enzyme reactor, on-membrane digestion. General principles of mass spectrometry. Ionization techniques used in proteomics. Electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI). Mass analyzers, quadrupole (Q), ion trap (IT), time-of-flight analyzer (TOF), reflectron, Fourier transformed ion cyclotron resonance (FT-ICR). Tandem mass spectrometry (MS/MS). Hybrid instruments. Peptide mass fingerprinting (PMF), de novo sequencing of peptides. Sequence analysis using the method of post source decay (PSD). Protein identification by database searching. Searching tools for PMF and MS/MS. Organisms with unsequenced genomes, EST databases, MS Blast program. LC-MS/MS of peptides. Shotgun proteomics – a tool for analysis of complex protein mixtures. Multidimensional liquid chromatography (LC) and capillary electrophoresis (CE). LC-LC, LC-CE and CE-CE combinations. Multidimensional protein identification technology (MuD-PIT). Post-translational modifications (PTM). Affinity chromatography of phosphopeptides. Mass spectrometry of phosphopeptides and glycopeptides. Quantitative analysis in proteomics: H2O18, ICAT, SILAC and AQUA. Clinical proteomics. Biomarkers. Protein chips. SELDI MS. Sample purification on affinity materials. MALDI imaging. Characterization of microorganism by mass spectrometry.