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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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Brain

SUBMITTER: Lawrence Schopfer

LAB HEAD: Oksana Lockridge

PROVIDER: PXD026790 | Pride | 2021-07-12

REPOSITORIES: Pride

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Project description:Due to the distinctive features of oral mucosa, determination of the proteins involved in the formation of “oral protein pellicle” is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides (bPRPs) named P-H, P-D, P-F, P-J, and II-2 as substrates of transglutaminase-2. The reactivity of P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated at diverse temperatures with two different transglutaminases (type 2; guinea pig and human), in the presence or in the absence of monodansyl-cadaverine. High resolution HPLC-ESI-MS and MS/MS analyses of the reaction products highlighted that P-H, and P-D were active substrates of transglutaminase-2, II-2 was less reactive and the reactivity of P-F and P-J was negligible. All the peptides formed cyclo-derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine. Q29 for P-H, Q37 for P-D, and Q21 for II-2 were the principal reactive residues. One or two secondary glutamine residues were hierarchically susceptible to reaction with dansyl-cadaverine. The main consensus sequence recognized by transglutaminase-2 in basic proline-rich peptides was GNPQ, and, despite the great similarity of P-F and P-J with the other salivary peptides, their reactivity was negligible, probably because the GNPQ consensus sequence was missing in their sequence. The reactivity of P-D A32 variant was lower than that of P-D P32 peptide suggesting that the more rigid peptide structure was a better substrates for transglutaminase-2. The reactivity of P-C was high, but followed rules different from the other basic peptides under study, Q41 being the principal reactive residue, confirming that P-C is not strictly related to the basic proline-rich proteins. The reactivity of statherin was very high too, and the manual inspection of the MS/MS fragmentation patterns of the reaction products confirmed, as determined in previous studies, that Q37 is the pivotal residue, Q39 and Q40 the secondary residues recognized by transglutaminase-2. Moreover, Q39 and Q40 residues were more reactive in cyclo-statherin Q37 than in statherin, suggesting that cycle formation increases the reactivity of the secondary glutamine residues. Finally, the physiological temperature (37°C) was the best for the reaction and no significant qualitative differences were observed using human or guinea pig transglutaminase-2.

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

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