PXD026790

PXD026790 is an original dataset announced via ProteomeXchange.

Title	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.
HostingRepository	PRIDE
AnnounceDate	2021-07-12
AnnouncementXML	Submission_2021-07-11_22:09:01.868.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Lawrence Schopfer
SpeciesList	scientific name: Sus scrofa domesticus (domestic pig); NCBI TaxID: 9825;
ModificationList	iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2021-06-18 03:51:11	ID requested
⏵ 1	2021-07-11 22:09:02	announced

Dataset with its publication pending

submitter keyword: transglutaminase, isopeptide, crosslinks, MAP-rich tubulin, dansyl cadaverine, dansyl-aminohexyl QQIV, Protein Prospector, neurodegenerative disease

Oksana Lockridge
contact affiliation	Eppley Institute, University of Nebraska Medical Center, United States
contact email	olockrid@unmc.edu
lab head
Lawrence Schopfer
contact affiliation	University of Nebraska Medical Center
contact email	lmschopf@unmc.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD026790
     1. Label: PRIDE project
     2. Name: Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides