D/E-rich peptides are less suitable than D/E-deficient peptides for identification by negative-ion HCD due to scarce production of sequencing ions from multiply charged precursors

Abstract

For peptides that are refractory to positive-ion-mode mass spectrometry (MS) analysis, the negative-ion-mode MS analysis is an attractive but insufficiently explored alternative. Based on high-resolution and accurate-mass MS analysis of 115 synthetic peptides of 5-28 amino acids, we confirmed that higher-energy collisional dissociation (HCD) of deprotonated peptides induced abundant neutral losses (NL) from the backbone and the side chains, and updated the ranking list of NLs by abundance. The most abundant fragment ion types are y- > x-, z- > c- if the NL ions are included, or c- > y- > z- > b- if not. The most frequent side-chain NLs include among others a loss of CH2O, C2H4O, and CH2N2 from Ser, Thr, and Arg, respectively. Although NL of CO2 is common for all peptides, it is markedly enhanced in Asp/Glu-containing peptides. Long peptides and Asp/Glu-rich peptides are prone to carrying multiple negative charges. HCD spectra produced from multiply deprotonated peptides have a lower fraction of sequencing ions (i.e., a-, b-, c-, x-, y-, z- ions) than those produced from 1- and 2- precursors. With this knowledge, we predict that for negative-ion-mode MS analysis of highly acidic proteins such as pepsin, which is difficult for positive-ion-mode analysis, Glu-C is a better choice than trypsin in sample digestion.