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Lecturer(s)
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Zemánková Natálie, Mgr.
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Simerský Radim, Mgr. Ph.D.
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Chamrád Ivo, Mgr. Ph.D.
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Lenobel René, Mgr. Ph.D.
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Course content
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1. Extraction of a soluble fraction of proteins from a biological material; 1D or 2D SDS/PAGE gel electrophoresis; Commassie Briliant Blue gel staining. 2. Cutting of selected protein spots from a gel, in-gel digestion with trypsin and/or other proteases (e.g. chymotrypsin); extraction of digested peptides. 3. Preparation of peptide extracts for mass spectrometry analysis - Zip Tip purification on a C18 sorbent, a chemical modification of peptides with two different reagents; mass spectrometry analysis of purified and modified peptides and protein identification after database search (MALDI-TOF, ESI-QTOF, MASCOT DATABASE).
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Learning activities and teaching methods
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Laboratory Work
- Homework for Teaching
- 1 hour per semester
- Attendace
- 20 hours per semester
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Learning outcomes
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To learn practical methods used in proteomic.
Student should be able to (after attending the course): - apply basic proteomic methods for analysis of proteins in a biological material - analyse collected raw data (protein identification, determination of molecular mass, study of post-translational protein modification and others) - write an experimental protocol and discuss of obtained results
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Prerequisites
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Basic experiences with laboratory work, knowledge from proteomic lectures.
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Assessment methods and criteria
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Written exam
100% attendance, completion of all experimental tasks
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Recommended literature
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Keough T., Lacey M. P., Youngquist R. S. (2000). Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry, Rapid Communication. Mass Spectrom. 14, 2348-2356.
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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. Proc. Natl. Acad. Sci. USA 96, 7131-7136.
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Marekov L. N., Steinert P. M. (2003). Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J. Mass Spectrom. 38, 373-377.
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Pingoud A., Urbanke C., Hoggett J., Jeltsch A. (2002). Biochemical methods: A concise guide for students and researchers. Wiley-VCH Verlag GmbH, Weinheim, Germany.
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Wang D., Kalb S. R., Cotter R. J. (2004). Improved procedures for N-terminal sulfonation of peptides for matrix-assisted laser desorption/ionization post-source decay peptide sequencing, Rapid Communication. Mass Spectrom. 18, 96-102.
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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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