1. To choose and select appropriate method for extraction of proteins and their subsequent analysis.
2. To apply and utilize proteomic techniques to solve complex biological and biochemical processes regardless of the type of organism which is the model for the research.
3. To apply and utilize proteomic techniques in the research of only one protein of interest or of the whole proteome.
4. To find adequate approach for resolution of the scientific problem.
5. To analyse results obtained by above mentioned techniques and to estimate their quality.
6. To compile results obtained by different techniques, to explain them and to carry out conclusions.
7. To analyse, summarise and interpret information obtained from the scientific articles and compile them with their own results.
8. To provide a good basis for research work in the team and acquire competencies for work in interdisciplinary teams.
It is an introductory course where students get a basic knowledge on techniques of proteome research and their possible application.
1. Short history and basic strategies in proteomics.
2. Gel-electrophoresis techniques (SDS-PAGE, Tricine-PAGE, Blue Native PAGE, 2-D electrophoresis).
3. Western blotting and immunodetection.
4. Capillary electrophoresis analysis of peptides and proteins.
5. Liquid chromatography analysis of peptides and proteins.
6. Chemical and enzymatic protein fragmentation.
7. Amino acid sequence analysis.
8. Mass spectrometry in proteomics and description of mass spectrometer (ion source, analyser, and detector).
9. Examples of the most commonly used mass spectrometers in proteomics.
10. Basics of peptide ion fragmentation and fragment nomenclature.
11. From peptide fragment to peptide and protein sequence, Mass spectra analysis and database search.
12. Complex peptide mixture analysis: liquid chromatography coupled to mass spectrometer (LC-MS).
13. De novo protein sequencing by mass spectrometry.
14. Proteomic approach to posttranslational modification analysis.
15. Selected examples of proteomics application in biological research with overview on proteomics development and perspective.
1. Protein extraction for two-dimensional electrophoresis (2-DE)
2. Bradford protein assay for estimation of protein concentration
3. Preparation of protein extracts for 2-DE
4. Performance of the 1st dimension: isoelectric focusing (IEF)
5. Performance of the 2nd dimension: SDS-PAGE
6. Protein visualisation by Commassie Brilliant Blue dye
7. Analysis of obtained results by computer programme and selection of proteins for further analysis
8. Protein excision from the gel and destaining
9. Tryptic digestion
10. Peptide extraction from the gel pieces
11. Peptide purification by Zip-Tip
12. Protein analysis by MALDI mass spectrometry
13. Protein analysis by liquid chromatography coupled to mass spectrometer (LC-MS)
14. Protein database search and protein identification
15. Analysis of obtained results
Seminar part of the course is linked to lectures and for each student includes preparation and writing of seminar based on scientific article from the field of proteomics.
- Balen, B., Pavoković, D., Peharec Štefanić, P., Krsnik-Rasol, M. Elektroforetske tehnike istraživanja proteina, sveučilišni priručnik, Hrvatska sveučilišna naklada, Zagreb 2011.
- Alberts, Bruce i sur. Molecular Biology of the Cell. 5th ed. New York: Garland Publishing; 2008.
- Kellner R., Lottspeich F, Meyer H.E. Microcharacterization of Proteins, WILEY-VCH second edition, 1999
- N. Galić, M. Cindrić 2008 Analiza proteina spektrometrijom masa, Kem. Ind. 57 (5) 231-243.