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Lecturer(s)
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Course content
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Proteomic introduction. Fundamental terms from protein separation field (electrophoresis, chromatography), mass spectrometry, image analysis ("imaging"), use of gene and protein databases. Expressional, differential and functional proteomics. Proteomic techniques and applications. Identification and characterization of proteins in the proteomics. Partial separation of a protein mixture (chromatography, affinity chromatography, preparative electrophoresis). Single-dimensional (1D) electrophoresis - SDS/PAGE. Two-dimensional (2D) electrophoresis. Sample preparation. First dimension - isoelectric focusing (IEF). Ampholites, tube-IEF-gels. IPG strips - gel strip with immobilized pH gradient. Techniques and instrumentation of IEF separation. Protein staining into IPG strips. Second dimension - SDS/PAGE electrophoresis. Buffer composition, gel preparation. SDS/PAGE electrophoresis instrumentation (horizontal and vertical devices). Protein staining into SDS//PAGE gels, MS compatibility of staining and mass spectrometry. Fluorescent staining of gels. Image analysis, densitometry, software overview for image analysis of 2D-gels. Comparison of two or more 2D-gels, differential analysis. Cutting of protein spots from gels. Electro-elution of proteins. Chemical and enzymatic digestion of proteins in a gel (in-gel digestion) and in a solution (In solution digestion). General principles of mass spectrometry. Ionisation techniques used in proteomics. Electrospray ionization (ESI), laser ionization with matrix desorption (MALDI). Mass instrumentation (iont traps (IT), single and multiple quadrupols (Q, QqQ), time of flight instrumentation (TOF), reflectron). Tandem mass spectrometry and instrumentation (MS/MS). MALDI-TOF post source decay (PSD). Peptide mass fingerprinting (PMF), sequential peptide analysis (de novo sequencing). Protein identification with database search. Database search algorithms (MASCOT, SEQUEST, PROTEIN PROSPECTOR). Not-sequenced organisms - EST databases, MS Blast software. Multidimensional liquid chromatography (2D(3D)-LC-MS/MS). Affinity chromatography and LC-MS/MS. Quantitative proteomics (techniques: H218O/ H216O, ICAT, SILAC, AQUA). Shotgun proteomics - 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
- 40 hours per semester
- Homework for Teaching
- 1 hour per semester
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Learning outcomes
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Basic introduction of proteomic field. Introduction, basic terms, proteomic techniques and applications.
Student should be able to (after attending the course): - recall the basic proteomic methods of protein analysis in a biological material - explain the theoretical background of particular proteomic procedures suitable for the analysis of proteins in the biology - select suitable a metod for analysis of a sample according to expected results and type of a sample - perform basic analysis of proteins from gained results
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Prerequisites
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It is expected basic knowledge from biochemistry and analytical chemistry.
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Assessment methods and criteria
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Mark, Written exam
Examination: Written examination, 5 questions + 5 overview terms from lectures
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Recommended literature
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Daniel Liebler. (2002). Introduction to Proteomics - tools for the new biology. Humana Press.
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Marie-Isabel Aguilar (eds.). (2004). [Methods in Molecular Biology 251] - HPLC of Peptides and Proteins_ Methods and Protocols. Humana Press.
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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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Westermeier, R., Naven, T. (2002). Proteomics in practice. A laboratory manual of proteome analysis.. Wiley-VCH, Weinheim, Germany.
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Winter M., Sherman N. E. (2000). Protein sequencing and identification using tandem mass spectrometry. John Wiley & Sons, Inc., New York, NY, USA.
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