Differential isotopic mass splitting as a mass spectrometric tool for identifying protease substrates

Abstract

A method is described whereby stable isotopic signatures were partially incorporated into both termini of a peptide sequence giving rise to a characteristic cluster of four peaks in the mass spectral analysis. Cleavage of this peptide by a protease between the labeled positions generates two fragments both displaying their own individual signature peaks. The event of protease cleavage of the peptide was monitored by the changes in clusters within the spectrum. We believe that this technique could be used to aid the discovery of new cleavage substrates for proteases. Additionally, the analysis can be automated with dedicated software designed to select and interpret the data since all peaks of interest contain predefined signatures and can be easily distinguished from background noise.

Application of Differential Isotopic Mass Splitting (DiMaS), a New Mass Spectrometry Methodology for the Analysis of Truncated Peptides, to Amyloid Precursor Protein Cleavage by Human Bleomycin Hydrolase and Neprilysin

“…In order to unambiguously differentiate between Y-type and btype ions, we have recently described a method, DiMaS (differential isotopic mass splitting), based on the incorporation of stable isotopes (for example, D 3 /H 3 acetate and D 2 /H 2 Gly) at both C and N termini. 5 The use of DiMaS ensures that any mass spectral peaks related to the original peptide are accompanied by labelled-ion peaks, thus distinguishing them from spurious singlet signals arising from impurities, background noise etc. that could confuse the interpretation.…”