Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides

Abstract

Isopeptide crosslinked proteins can be the product of transglutaminase or of exposure to organophosphorus toxicants (OP). Transglutaminase links glutamine to lysine with loss of ammonia. OP toxicants induce a link between glutamic acid and lysine with loss of water. Our goal was to establish criteria to distinguish real from false isopeptide crosslinks reported by software searches of mass spectrometry data. We used fragmentation spectra of tryptic peptides from MAP-rich tubulin Sus scrofa as a test system for detection of naturally-occurring isopeptide crosslinks. Data were analyzed with Protein Prospector. Criteria for the assignments included the presence of at least 1 crosslink specific product ion, fragment ions from both peptides, Protein Prospector scores ≥20, and best fit of the MS/MS data to the crosslinked peptide as opposed to a linear peptide. Out of 301,364 spectra, 15 potential transglutaminase-type crosslinked peptide candidates were identified. Manual evaluation of these MS/MS spectra reduced the number to 1 valid crosslink between Q112 of NFH and K368 of Tau. Immunopurification with anti-isopeptide 81D1C2 confirmed that MAP-rich tubulin contained only one isopeptide. Support for this isopeptide bond was obtained by showing that transglutaminase was capable of incorporating dansyl-aminohexyl -QQIV into K368. A model of the KIETHK-QLEAHNR isopeptide was synthesized with the aid of transglutaminase. MS/MS spectra of the model validated our interpretation of the native isopeptide. An OP-induced isopeptide bond between K163 of tubulin alpha-1A and E158 of tubulin beta-4B was induced by treating MAP-rich tubulin with 100 μM chlorpyrifos oxon. This crosslink was supported by the criteria described above and by the presence of diethoxyphospho-lysine 163 in the tubulin alpha-1A peptide. The information obtained in this work is valuable for future studies that aim to understand why exposure to OP is associated with increased risk of neurodegenerative disease.

Organophosphorus Pesticides Promote Protein Cross-Linking

“…A diethylphospho-adduct was found on lysine 163, supporting its involvement in cross-linking. The MSMS spectrum for this latter cross-linked peptide pair was previously published . A cross-link between K336 in peptide DVNAAIATIK 336 TK from tubulin α1A and E158 in peptide EE 158 YPDR from tubulin β4B appeared two times (in charge state 3, from gel slices A and B).…”

“…The MSMS spectrum for this latter cross-linked peptide pair was previously published. 22 A cross-link between K336 in peptide DVNAAIATIK 336 TK from tubulin α1A and E158 in peptide EE 158 YPDR from tubulin β4B appeared two times (in charge state 3, from gel slices A and B). K336 in peptide DVNAAIATIK 336 TK from tubulin α1A was also cross-linked to D98 in peptide ED 98 AANNYAR from tubulin α1A two times (in charge state 3 from gel slices A and B).…”

Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified Proteins in Cultured Cells and Immunopurifies Modified Peptides for Mass Spectrometry

“…In Figure B (lane 7), CPO-treated tubulin has been transformed from its original 50 kDa mass to high-molecular-weight aggregates. In previous reports, , we showed that CPO catalyzes the formation of covalent isopeptide cross-links between diethoxyphospholysine and glutamic acid or diethoxyphospholysine and aspartic acid, with the release of diethoxyphosphate. The cross-linking reaction results in high-molecular-weight protein aggregates.…”

“…Not all diethoxyphospholysine adducts participate in a cross-linking reaction; those that retain the adduct are detectable by mass spectrometry. 13 To date, we have not observed a cross-linking reaction involving diethoxyphosphotyrosine adducts.…”

Mass Spectrometric Identification of a Novel Factor XIIIa Cross-Linking Site in Fibrinogen

“…Although this result demonstrates the ability of the method to correctly identify transglutaminase cross-linked peptides, the optimal MS conditions and software identification parameters have not been discussed. A study conducted by Schopfer et al [ 48 ] demonstrates how a naturally occurring TG cross-link can be successfully identified by comparing its fragmentation spectrum with that of an identical synthetic peptide by using Protein Prospector [ 49 ]. However, this study focuses rather on selecting reliable identifications based on predetermined quality criteria for the MS/MS spectra than finding optimal fragmentation conditions.…”