Identification of alternative splice variants using unique isoform-specific tryptic peptide sequences for database searches

Alternative splicing is a mechanism in eukaryotes by which different forms of messenger RNAs (mRNAs) are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database which contains only proteotypic tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to the peptide sequences that matches the unique amino acid sequence of splice variants to MS data. Furthermore, we combined this database without isoform 1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa), and phosphoproteomics studies were analyzed using these two databases. Several non-isoform 1-specific peptides were found in both cell lines, some of them seemed to be cell line specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several non-isoform 1-specific peptides and some of them showed clear quantitative differences between the two states.