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Available peptide fragmentation interpretation software is focused on sequence database–driven protein identification, rather than on primary structure elucidation. Searching for posttranslational modification (PTM) or sequence errors currently needs time-critical manual intervention and evaluation. Here we describe the results and performance of a novel interpretation software, which was used to characterize more than 50 recombinant serine/threonine kinases, receptor tyrosine kinases, or cytoplasmatic tyrosine kinases.
LC-MS/MS data was acquired after tryptic digestion of the recombinantly produced kinases. The datasets have been imported to the proteome bioinformatics platform ProteinScape. The spectra were screened for a set of modifications, amino acid substitutions, unsuspected large measurement errors, enzyme no-specificity, and unknown mass shifts. The software restricts the search space by testing only sequences of interest. In widely used sequence database searches, testing all modifications and possible non-specific cleavages is not feasable.
Besides the increase in sequence coverage basically caused by detection of one side non-specifically cleaved peptides, numerous modifications were found—namely, phosphorylation. methylation, pyroglutamate formation, methinonine oxidation, and N-terminal acetylation. As spectra of phosphorylated peptides are almost always in the minority compared to their unmodified counterparts, their detection is a challenge, but internal significance analysis revealed a substantial amount of phosphorylation.The phenomenon of auto-phosphorylation of kinase proteins was successfully monitored. The phosphorylation sites are categorized according to their sequence motive, and additionally their distribution is compared to phosphoryation sites described in public databases. Using this software triggered by the proteome database software proteinscape, searches were performed in a highly automated manner. Manual analysis could be reduced to minutes for the LC-MS/MS datasets containing more than 1000 spectra. Integrated result presentation strategies, which use clustering of spectra results on the amino acid level to annotate the protein sequence of interest, avoided the the possibility of seeing excess PTM contained in the large amount of acquired spectra.