Electrospray Tandem Mass Spectrometry Analysis of S- and N-Nitrosopeptides: Facile Loss of NO and Radical-Induced Fragmentation

The covalent addition of nitric oxide (NO) to protein thiols, a posttranslational modification termed S-nitrosation, is a ubiquitous event that modulates diverse cellular processes. The in vivo addition of NO to protein amines (N-nitrosation) has also been described and may similarly modify protein structure and function. While mass spectrometry has been employed for identification of nitrosoproteins, little is known about how S- and N-nitrosopeptides fragment. Such knowledge is important for its potential to inform on sites of protein nitrosation. Here we used electrospray tandem mass spectrometry to elucidate collision-induced dissociation (CID) features of S- and N-nitrosopeptide ions. We show that S- and N-nitrosopeptide ions readily lose NO, giving rise to species that contain thiyl and aminyl radicals, respectively. Fragmentation (MS3) of these radical peptide ions revealed an atypical pattern, characterized by the cleavage of select αCC and NαC bonds, rather than the more usual cleavage of amide bonds that result in b- and y-ions. These unanticipated fragmentation patterns are reconciled by radical-mediated abstraction of hydrogen from β-carbon followed by β-fragmentation. For thiyl radical peptides, we also observed dominant loss of SH and CH2SH from the Cys side-chain. Our findings provide new insights into the gas-phase chemistry of NO-modified peptide ions and suggest an unusual fragmentation pattern that may aid in future MS-based attempts to define the nitrosoproteome.