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Lecturer(s)
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Šebela Marek, prof. Mgr. Dr.
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Lenobel René, Mgr. Ph.D.
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Course content
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Introduction to Proteomics. Basic terms in the field of protein separation (electrophoresis, chromatography), mass spectrometry, image processing ("imaging"), use of gene and protein databases. Importance of expression, differential and functional proteomics. Proteomics technology, applications. 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. The first dimension - isoelectric focusing (IEF). Ampholytes, tubular IEF gels. Strips with an immobilized pH gradient ("IPG strips"). Methodology and instrumentation of IEF separation. Staining of IPG strips. Second dimension - SDS/PAGE. Composition of buffers. Preparation of gels. Instrumentation of SDS/PAGE separation (horizontal and vertical apparatus). Protein staining in SDS/PAGE gels, staining compatibility and mass spectrometry. Fluorescence staining of gels. Image analysis, densitometer, 2-D gel evaluation software. Comparison of two or more 2-D gels, difference. Excision of protein spots. Electroelution. Chemical and enzymatic cleavage of proteins in gel. Fission in solution. General principles of mass spectrometry. Ionization techniques used in proteomics. Electrospray ionization (ESI), matrix assisted ionization (MALDI). Ion analyzers, quadrupole (Q), ion trap (IT), time-of-flight analyzer (TOF), reflectron. Tandem mass spectrometry (MS/MS) and its instrumentation. MALDI-TOF post source decay (PSD) technique. Peptide mapping (peptide mass fingerprinting), sequence analysis of peptides (de novo sequencing). Protein identification by searching databases. Search programs Mascot, Sequest and Protein Prospector. Organisms with unsequenced genome, EST database, MS Blast program. Multidimensional LC-MS/MS. Affinity chromatography and LC-MS/MS. Quantitative analysis in proteomics. H2O18, ICAT, SILAC, AQUA methods. Shotgun proteomics - a tool for the analysis of complex protein mixtures. SELDI MS, Clinical Proteomics.
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Learning activities and teaching methods
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Lecture
- Attendace
- 26 hours per semester
- Preparation for the Exam
- 60 hours per semester
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Learning outcomes
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Students will acquaint themselves 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.
After completing the course, the student should be able to: - describe the basic proteomic methods of protein analysis in biological material - explain the theoretical basis of individual proteomic procedures that can be used in the analysis of proteins in biology - choose the appropriate method for the analysis of the given sample based on the desired results and the type of sample - analyze the proteins from the obtained results
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Prerequisites
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Basic knowledge of biochemistry and analytical chemistry is assumed.
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Assessment methods and criteria
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Oral exam
Oral xam: 2 questions + 5 clear concepts from the lectures
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Recommended literature
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Keough T., Youngquist R. S., Lacey M. P. (1999). A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spektrometry.. roc. Natl. Acad. Sci. USA 96, 7131-7136.
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Hillenka M. P. F., Peter-Katalinic, J. (2007). MALDI MS: A Practical Guide to Instrumentation, Methods and Application. Wiley-VCH, Weinheim.
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Pingoud A., Urbanke C., Hoggett J., Keltech A. (2002). Biochemical methods: A concise guide for students and researchers. Weinheim Německo.
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Simson, R. J. (ed.). (2004). Purifying proteins for Proteomics: a laboratory manual.. Cold Spring Harbor Laboratory Press, New York.
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Tramontano, A. (2006). Protein Structure Prediction. Wiley-VCH, Weinheim.
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Westermeier, R.. (2001). Electrophoresis in Practice. Wiley-VCH, Weinheim.
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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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