Dataset Information

SILAC IP-Mass spectrometry of TTLL-transfected HEK cells

ABSTRACT: Identification of the non-tubulin substrates of tubulin glycylases, TTLL3/TTLL8 and tubulin glutamylases TTLL4/TTLL5 using SILAC-IP-MS/MS quantitative mass spectrometry approach. HEK cells transfected with the respective enzymes were lysed and the proteins modified by the enzymes enriched by immunopulldown using modification specific antibodies and subsequently, the potential substrates identified using quantitative mass spectrometry. The identity of the proteins was further validated by cellular and in vitro modification assays

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Vanessa Masson

LAB HEAD: Damarys Loew

PROVIDER: PXD065251 | Pride | 2026-05-18

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 41

Similar Datasets

Project 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.

			Action	DRS
	Design_PRIDE.xlsx	Xlsx
	X2325FD.msf	Msf
	X2325FD.raw	Raw
	X2326FD.msf	Msf
	X2326FD.raw	Raw

Dataset Information

SILAC IP-Mass spectrometry of TTLL-transfected HEK cells

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets