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

Abstract

Murine S100A8 (A8) is a major cytoplasmic neutrophil protein and is converted to novel oxidation products containing Cys–ε 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 α 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.