Dataset Information

Quantitative analysis of protein ubiquitination

ABSTRACT: Protein ubiquitination orchestrates nearly all eukaryotic cellular events.1 It starts by attaching ubiquitin through isopeptide bonds to a single or multiple lysine residues of a target protein via a coordinated enzymatic reaction involving activating (E1), conjugating (E2), and ligating (E3) enzymes to form mono- or multi-mono-ubiquitinated products. There is a need for a technique that can facilitate the high-yield production of monoubiquitinated proteins. Here we present an efficient approach that fills this gap.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yue Chen

LAB HEAD: Yue Chen

PROVIDER: PXD037416 | Pride | 2023-05-10

REPOSITORIES: pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	05142022_5ugUb-a-synuclein_NEDD4L_AcGGNHS_GluC_heavyAcNHS_Trial1_rep1_half.raw	Raw
	05142022_5ugUb-a-synuclein_NEDD4L_AcGGNHS_GluC_heavyAcNHS_Trial1_rep2_half.raw	Raw
	05142022_5ugUb-a-synuclein_NEDD4L_AcGGNHS_GluC_heavyAcNHS_Trial1_rep3_half.raw	Raw
	05142022_5ugUb-a-synuclein_NEDD4L_AcGGNHS_GluC_heavyAcNHS_Trial2_rep1_half.raw	Raw
	05142022_5ugUb-a-synuclein_NEDD4L_AcGGNHS_GluC_heavyAcNHS_Trial2_rep2_half.raw	Raw

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Publications

Avidity-Based Method for the Efficient Generation of Monoubiquitinated Recombinant Proteins.

Nelson Spencer L SL Li Yunan Y Chen Yue Y Deshmukh Lalit L

Journal of the American Chemical Society 20230403 14

Monoubiquitination of proteins governs diverse physiological processes, and its dysregulation is implicated in multiple pathologies. The difficulty of preparing sufficient material often complicates the biophysical studies of monoubiquitinated recombinant proteins. Here we describe a robust avidity-based method that overcomes this problem. As a proof-of-concept, we produced milligram quantities of two monoubiquitinated targets, Parkinson's protein α-synuclein and ESCRT-protein ALIX, using NEDD4- ...[more]

PMID: 37010382

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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.