COVALENT MODIFICATION OF PROTEINS FOR THE INSTANTANEOUS VISUALISATION THEREOF AND SUBSEQUENT CHARACTERISATION THROUGH MASS SPECTROMETRY

摘要

The invention described discloses the covalent modification of proteins for the instantaneous visualisation and subsequent characterisation thereof. The chemical mechanism employed is based on nucleophilic addition. The mechanism of the chemical reaction is explained, together with the bonding of the latter to specific aminoacid residues. Identification and characterisation of stained proteins employing mass spectrometry may be carried out by virtue of changes in mass of the residues modified by Uniblue A. A significant advantage of the staining employed in this invention lies in it having shown high compatibility with mass spectrometry analysis, it being unnecessary in addition to remove the colourant from proteins following staining as in the case of Coomassie or silver staining. Moreover, the derivatisation of amino acids gives rise to a change in the proteolytic hydrolysis patterns which may increase peptide sequencing coverage. Furthermore, improvement in the sequencing coverage of MS/MS analysis may be achieved by taking into account the changes in mass of the terminal amino groups of the derivatised amino acids. Additionally, a shorter procedure for protein identification is presented. Using the methods proposed for staining and analysis of proteins, the time required for protein analysis may be substantially reduced whilst the quality of the analysis may be improved.