{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T08:57:09Z","timestamp":1790758629024,"version":"4.1.0"},"reference-count":44,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2021,7,9]],"date-time":"2021-07-09T00:00:00Z","timestamp":1625788800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000066","name":"National Institute of Environmental Health Sciences","doi-asserted-by":"publisher","award":["1R21ES030132-01A1"],"award-info":[{"award-number":["1R21ES030132-01A1"]}],"id":[{"id":"10.13039\/100000066","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","award":["P30CA036727"],"award-info":[{"award-number":["P30CA036727"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]},{"name":"TUBITAK","award":["SBAG-318S259"],"award-info":[{"award-number":["SBAG-318S259"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>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 <jats:italic>Sus scrofa<\/jats:italic> 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 \u226520, 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 \u03bcM 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.<\/jats:p>","DOI":"10.1371\/journal.pone.0254450","type":"journal-article","created":{"date-parts":[[2021,7,9]],"date-time":"2021-07-09T18:28:25Z","timestamp":1625855305000},"page":"e0254450","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":12,"title":["Evaluation of mass spectrometry MS\/MS spectra for the presence of isopeptide crosslinked peptides"],"prefix":"10.1371","volume":"16","author":[{"given":"Lawrence M.","family":"Schopfer","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seda","family":"Onder","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8345-3640","authenticated-orcid":true,"given":"Oksana","family":"Lockridge","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,7,9]]},"reference":[{"issue":"1","key":"pone.0254450.ref001","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1096\/fj.201801544R","article-title":"Transglutaminase diseases: from biochemistry to the bedside","volume":"33","author":"L Lorand","year":"2019","journal-title":"FASEB J"},{"key":"pone.0254450.ref002","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0065-3233(08)60217-X","article-title":"The epsilon-(gamma-glutamyl)lysine crosslink and the catalytic role of transglutaminases.","volume":"31","author":"JE Folk","year":"1977","journal-title":"Advances in protein chemistry"},{"issue":"4","key":"pone.0254450.ref003","doi-asserted-by":"crossref","first-page":"762","DOI":"10.1021\/acs.chemrestox.9b00001","article-title":"Mass Spectrometry Identifies Isopeptide Cross-Links Promoted by Diethylphosphorylated Lysine in Proteins Treated with Chlorpyrifos Oxon","volume":"32","author":"LM Schopfer","year":"2019","journal-title":"Chem Res Toxicol"},{"issue":"35","key":"pone.0254450.ref004","doi-asserted-by":"crossref","first-page":"13566","DOI":"10.1074\/jbc.RA118.004172","article-title":"Chlorpyrifos oxon promotes tubulin aggregation via isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications for neurotoxicity","volume":"293","author":"LM Schopfer","year":"2018","journal-title":"The Journal of biological chemistry"},{"key":"pone.0254450.ref005","doi-asserted-by":"crossref","first-page":"5171","DOI":"10.1007\/s00216-014-7706-y","article-title":"V-type nerve agents phosphonylate ubiquitin at biologically relevant lysine residues and induce intramolecular cyclization by an isopeptide bond","volume":"406","author":"C Schmidt","year":"2014","journal-title":"Anal Bioanal Chem"},{"issue":"6","key":"pone.0254450.ref006","doi-asserted-by":"crossref","first-page":"1369","DOI":"10.1111\/j.1471-4159.2004.02825.x","article-title":"Developmental regulation of tissue transglutaminase in the mouse forebrain","volume":"91","author":"CD Bailey","year":"2004","journal-title":"J Neurochem"},{"key":"pone.0254450.ref007","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.ejphar.2017.12.023","article-title":"Activation of transglutaminase 2 by nerve growth factor in differentiating neuroblastoma cells: A role in cell survival and neurite outgrowth","volume":"820","author":"AS Algarni","year":"2018","journal-title":"Eur J Pharmacol"},{"issue":"2","key":"pone.0254450.ref008","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1016\/S0306-4522(00)00482-6","article-title":"Tissue transglutaminase is essential for neurite outgrowth in human neuroblastoma SH-SY5Y cells","volume":"102","author":"J Tucholski","year":"2001","journal-title":"Neuroscience"},{"issue":"8","key":"pone.0254450.ref009","doi-asserted-by":"crossref","first-page":"1627","DOI":"10.1096\/fj.07-0602ufm","article-title":"Crosslinks in blood: transglutaminase and beyond","volume":"21","author":"L Lorand","year":"2007","journal-title":"FASEB J"},{"issue":"5","key":"pone.0254450.ref010","first-page":"313","article-title":"Transglutaminases in skin epidermis. 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