HIGH-COVERAGE PROTEOMIC IDENTIFICATION OF POST-TRANSLATIONALLY MODIFIED PROTEINS

Helena Řehulková, Josef Chmelík, Jitka Žídková, Pavel Řehulka, Janette Bobáĺová

Research Centre for Study of Extract Compounds of Barley and Hop

Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veveří 97, 602 00 Brno, Czech Republic

rehulkova@iach.cz

Proteomics is systematic analysis of proteins for their identity, quantity and function. It is a very important tool for study of life processes in cells, tissues or organisms. In contrast to a cell’s static genome, the proteome is both complex and dynamic. Proteome analysis is most commonly accomplished by the combination of a proper separation technique, mass spectrometry and bioinformatics.

Both top-down (combining protein separation with MS analysis of intact proteins) and bottom-up (MS analysis of digested proteins) proteomic approaches were used for a detailed characterization of various proteins with the aim to obtain the high-coverage of the primary structure including post-translational modifications. The influence of different proteomic protocols (differing in separation technique, enzyme and digestion procedure) on the extent of coverage of protein primary structure was studied. The most successful protocols were in-gel digestion of the alkylated protein with trypsin and in-solution digestions of the nonalkylated protein with trypsin or trypsin/chymotrypsin mixture.

Acknowledgements.

The authors thank the project “Research Centre for Study of Extract Compounds of Barley and Hop“ No. 1M0570 for financial support.