Metastable decomposition at the peptide C‐terminus: Possible use in protein identification

Abstract

Rationale:The b n−1 ion of a peptide, as well as a [b n−1 + 18] ion, can be observed not only as normal product ions, but also as prominent metastable ions in a reflectronembedded matrix-assisted laser desorption ionization time-of-flight 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 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 C-terminal amino acids. Results:The b n−1 as well as [b n−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 b n−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 Cterminal amino acids to be more reliably read out than other internal sequences, information that could be efficiently used for protein identification.

Electron-transfer MALDI MS methodology for microalgae/phytoplankton pigments analysis

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DOPAnization of tyrosine in α-synuclein by tyrosine hydroxylase leads to the formation of oligomers

“…Each fraction was spotted onto a stainless steel plate, dried, and mixed with a matrix solution (0.5 µL per spot; 7 mg/mL of α-cyano-4-hydroxycinnamic acid (Sigma) in 50% ACN, 0.1% TFA). MALDI-TOFMS and MS/MS measurements were carried out with a 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems) as previously described 57 . All mass spectra were obtained by averaging 2500 laser shots from each sample well in positive-ion mode.…”