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Lecturer(s)
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Šebela Marek, prof. Mgr. Dr.
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Course content
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Introduction to proteomics. Basic terminology of protein separation (electrophoresis, chromatography), mass spectrometry, imaging. Gene and protein databases. Significance of expression, differential and functional proteomics. Proteomic technology, application. Identification and characterization of proteins in proteomics. Partial separation of protein mixtures (conventional chromatography, affinity chromatography, preparative electrophoresis). One-dimensional (1-D) electrophoresis - SDS/PAGE. Two-dimensional (2-D) electrophoresis. Sample preparation. First dimension, isoelectric focusing (IEF). Ampholytes, IEF in tube gels. Strips with immobilized pH gradient (IPG strips). Methodology and instrumentation of IEF separation. Staining of IPG strips. Second dimension, SDS/PAGE. Buffer composition. Preparation of gels. Instrumentation of SDS-PAGE (horizontal and vertical units). Staining of proteins in SDS/PAGE gels, compatibility of protein staining and mass spectrometry. Fluorescence staining of gels. Imaging, densitometer, software for evaluation of 2-D gels. Comparison of two or more 2-D gels, protein maps, difference of patterns. Excision of protein spots. Electroelution. Chemical cleavage and enzymatic digestion of proteins in gels. Digestion in solution. General principles of mass spectrometry. Ionization techniques utilized in proteomics. Electrospray ionization (ESI), matrix-assisted ionization (MALDI). Ion analyzers, quadruple (Q), ion trap (IT), time-of-flight analyzer (TOF), reflectron. Tandem mass spectrometry (MS/MS) and its instrumentation. Post-source-decay (PSD) on MALDI-TOF mass spectrometers. Peptide mapping (peptide mass fingerprinting), sequencing analysis of peptides (de novo sequencing). Protein identification by database searching. Searching tools Mascot, Sequest and Protein Prospector. Organisms with non-sequenced genomes, EST databases, MS Blast program. Multidimensional LC-MS/MS. Affinity chromatography and LC-MS/MS. Quantification in proteomics. Methods H2O18, ICAT, SILAC, AQUA. Shotgun proteomics, a tool for analysis of complex protein mixtures. SELDI MS, clinical proteomics.
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Learning activities and teaching methods
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Dialogic Lecture (Discussion, Dialog, Brainstorming), Work with Text (with Book, Textbook)
- Preparation for the Exam
- 50 hours per semester
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Learning outcomes
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Students will acquaint themselves in detail with the history and orientation of proteomics, separation methods for proteins, mass spectrometry of proteins and peptides a with methods for identification and quantification of proteins.
Explain up to date novelties in proteomics, compare methods, choose the proper method for a given purpose.
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Prerequisites
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It is necessary to have knowledge at the level of the courses KBC/PROT Proteomics, KBC/BCH Fundamentals of Biochemistry, KBC/UBCH Introduction to Biochemistry for students of Bioinformatics) and KBC/BIME Biochemical Methods.
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Assessment methods and criteria
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Oral exam
the candidate is expected to conduct scientific discussion in broader context of the whole discipline.
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Recommended literature
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Johnstone R. A. W., Rose M. E. (2010). Mass Spectrometry for Chemists and Biochemists. Cambridge University Press.
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Mishra N. C. (2010). Introduction to Proteomics: Principles and Applications. Wiley.
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Westermeier, R., Naven, T. (2002). Proteomics in practice. A laboratory manual of proteome analysis.. Wiley-VCH, Weinheim, Germany.
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