Metastable Decomposition at the Peptide C‐ Terminus ‐Possible Use in Protein Identification‐

RATIONALE: The bn-1 ion of a peptide, as well as a [bn-1 +18] ion, could be observed not only as normal product ions, but also as prominent metastable ions in a reflectron- embedded MALDI-TOF spectrometer. The m/z values for the peaks are slightly shifted compared with the ordinary product ions and appear as relatively broad peaks, which permits them to be discriminated from other ions. METHODS: A standard protein mixture and gel-derived proteins digested with LysN protease, which cleaves peptide linkages in proteins at the N-terminal side of Lys residues, were examined. The collected data were used for protein identification by using in-house software, "iD-plus" (http://coco.protein.osaka-u.ac.jp/id-plus/), which was developed for searching for proteins in the peptide database, based on enzyme-specificity (N-terminal Lys in this study), peptide masses, and the C-terminal AAs. RESULTS: The bn-1 as well as [bn-1 +18] ions were observed as broad ion peaks for all of the peptides (86 peptides) examined in this study. In-silico calculations using the database of LysN digested peptides (11,969,470), created from 553,941 protein sequences (SwissProt: 2017_03), indicate that the use of no less than four peptides permits a protein to be identified without the need of any probability-based scoring. CONCLUSIONS: The preference for bn-1 ion formation is probably due to the higher propensity of the C-terminal peptide bond to be cleaved than other internal bonds. The fact that such C-terminal fragmentation takes place for most of the peptides examined suggests that the use of an N-terminal specific enzyme would allow the C-terminal amino acids to be more reliably read out than other internal sequences, information for which could be efficiently used for protein identification.