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

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 criteri...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Lawrence M Schopfer, Seda Onder, Oksana Lockridge
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f71f9d78cba4493881187d548b22dde3
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:f71f9d78cba4493881187d548b22dde3
record_format	dspace
spelling	oai:doaj.org-article:f71f9d78cba4493881187d548b22dde32021-12-02T20:15:32ZEvaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides.1932-620310.1371/journal.pone.0254450https://doaj.org/article/f71f9d78cba4493881187d548b22dde32021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0254450https://doaj.org/toc/1932-6203Isopeptide 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.Lawrence M SchopferSeda OnderOksana LockridgePublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 7, p e0254450 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lawrence M Schopfer Seda Onder Oksana Lockridge Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides.
description	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.
format	article
author	Lawrence M Schopfer Seda Onder Oksana Lockridge
author_facet	Lawrence M Schopfer Seda Onder Oksana Lockridge
author_sort	Lawrence M Schopfer
title	Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides.
title_short	Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides.
title_full	Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides.
title_fullStr	Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides.
title_full_unstemmed	Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides.
title_sort	evaluation of mass spectrometry ms/ms spectra for the presence of isopeptide crosslinked peptides.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/f71f9d78cba4493881187d548b22dde3
work_keys_str_mv	AT lawrencemschopfer evaluationofmassspectrometrymsmsspectraforthepresenceofisopeptidecrosslinkedpeptides AT sedaonder evaluationofmassspectrometrymsmsspectraforthepresenceofisopeptidecrosslinkedpeptides AT oksanalockridge evaluationofmassspectrometrymsmsspectraforthepresenceofisopeptidecrosslinkedpeptides
_version_	1718374621299867648

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

Ejemplares similares