title: 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-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 Cys41-Lys34/35 intramolecular cross-links. Additional isomeric products with identical masses (10,354 Da) were isolated and peptide mapping and ESI/MS indicated that Cys41 forms covalent sulfinamide cross-links with either Lys6, Lys76, Lys83, or Lys87 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 alpha 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 Cys41-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.
authors.affiliation: Cytokine Research Unit, School of Medical Sciences, University of New South Wales, Kensington, Australia. m.raftery@unsw.edu.au
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Raftery
authors.forename: Carolyn L
authors.initials: CL
authors.lastname: Geczy
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D003432:Cross-Linking Reagents; D006997:Hypochlorous Acid / Q000737:chemistry; D007202:Indicators and Reagents; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D011522:Protons; D009418:S100 Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013457:Sulfur Compounds / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D010455:Peptides; D011522:Protons; D009418:S100 Proteins; D013457:Sulfur Compounds; D006997:Hypochlorous Acid