Electrospray low energy CID and MALDI PSD Fragmentations of protonated sulfinamide cross-linked peptides

Murine S100A8 (AS) is a major cytoplasmic neutrophil protein and is converted to novel oxidation products containing Cys-epsilon amino-Lys sulfinamide cross-links and Met-sulfoxide by the neutrophil oxidant HOCl. Seven products were separated using RP-HPLC, with electrospray ionization mass spectrometry (ESI-MS) masses after deconvolution of 10,354, 10,388, +/-1, and 20,707, +/-3 Da, and all were resistant to reduction by dithiothreitol. The major products with masses of 10,354 Da contained Cys(41)-Lys(34/35) intramolecular cross-links. Additional isomeric products with identical masses (10,354 Da) were isolated and peptide mapping and ESI/MS indicated that Cys(41) forms covalent sulfinamide cross-links with either Lys(6), Lys(76), Lys(83), or Lys(87) present in A8. Electrospray low energy collisionally induced (CID) spectra of multiply-charged AspN digest peptides with sulfinamide cross-links contained characteristic fragmentations that corresponded to simple cleavage of the nitrogen-sulfur bond with charge retention on either of the fragment ions, allowing conformation of cross-linked peptides. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) post source decay spectra of [M + H](+) ions of the same sulfinamide-containing cross-linked peptides fragment similarly, but additional facile fragmentation reactions corresponding to formation of a protonated peptide containing de-hydroalanine were attributed to cleavage of the carbon-sulfur bond. In addition, lose of methanesulfenic acid from Met-sulfoxide was observed. A sulfinamide-containing adduct was isolated after incubation of the A8/HOCl reaction mixture with Lys or a N-acetyl Lys with masses of 10,500 or 10,542 Da. ESI/MS/MS and MALDI/ post decay source (PSD) analysis of A8(32-57)-sulfinamide showed the same characteristic fragmentations as those in the sulfinamide cross-linked peptides, confirming the Cys(41)-Lys sulfinamide cross-link and suggesting that peptide-peptide sulfinamides may all fragment similarly, allowing ready identification of these cross-links in proteins from more complex biological materials. (C) 2002 American Society for Mass Spectrometry.