Practice in Proteomics (LRR / CVPRO)

Course objectives:
To learn practical methods used in proteomic.
Requirements on student
100% attendance, completion of all experimental tasks
Content
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).
Activities
Guarantors and lecturers
Literature
  • Recommended: Keough T., Youngquist R. S., Lacey M. P. 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, 1999.
  • Recommended: Pingoud A., Urbanke C., Hoggett J., Jeltsch A. Biochemical methods: A concise guide for students and researchers. Wiley-VCH Verlag GmbH, Weinheim, Germany, 2002.
  • Recommended: Keough T., Lacey M. P., Youngquist R. S. 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, 2000.
  • Recommended: Marekov L. N., Steinert P. M. 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, 2003.
  • Recommended: Wang D., Kalb S. R., Cotter R. J. 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, 2004.
  • Recommended: Winter M., Sherman N. E. Protein sequencing and identification using tandem mass spectrometry. John Wiley & Sons, Inc., New York, NY, USA, 2000. ISBN 0471322490.
  • Recommended: Westermeier, R., Naven, T. Proteomics in practice. A laboratory manual of proteome analysis.. Wiley-VCH, Weinheim, Germany, 2002. ISBN 3-527-30354-5.
  • On-line library catalogues
Time requirements

Activities

Time requirements for activity [h]

Homework for Teaching

1

Attendace

20
Total

21

Prerequisites – other information about course preconditions
Basic experiences with laboratory work, knowledge from proteomic lectures.
Competences acquired
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
Teaching methods
  • Laboratory Work
Assessment methods
  • Written exam