Dataset Information

Terminal alkynes react with active-site cysteine nucleophiles

ABSTRACT: Propargyl labeled ubiquitin was immobilized on beads and used for click-chemistry based pull downs. Covalently linked proteins were washed and digested off-bead, or eluted with strong acid and loaded into a SDS-PAGE gel. Digestion with Lys-C, trypsin consequetively and LC-MS/MS. Both dimethyl labeling, as well as qualitative measurements were done. For both the qualitative and the quantitative dataset peak lists were generated from the raw data files using the Proteome Discoverer software package version 1.3.339. Peptide identification was performed by searching the combined peak lists of the entire gel lane (10 bands) against a concatenated target-decoy database containing the human sequences in the Uniprot database (release 2012_06) supplemented with a common contaminants database using the Mascot search engine version 2.3 via the Proteome Discoverer interface. The search parameters included the use of trypsin as proteolytic enzyme allowing up to a maximum of 2 missed cleavages. For the qualitative dataset, carbamidomethylation of cysteines was set as a fixed modification whereas oxidation of methionines was set as variable modification. Precursor mass tolerance was initially set at 50 ppm, while fragment mass tolerance was set at 0.6 Da. Subsequently, the peptide identifications were filtered for true mass accuracy <10 ppm and an ion score of 25. For the quantitative dataset carbamidomethylation of cysteines was set as a fixed modification whereas oxidation of methionines and the dimethyl light and intermediate labels on N-termini and lysine residues were set as variable modifications. Precursor mass tolerance was initially set at 50 ppm, while fragment mass tolerance was set at 0.6 Da for ETD-IT fragmentation and 0.05 Da for HCD and ETD-FT fragmentation. Subsequently, the peptide identifications were filtered for true mass accuracy <10 ppm and an ion score of 25.

INSTRUMENT(S): LTQ Orbitap Discovery, LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Patrick Wijten

LAB HEAD: Patrick Wijten

PROVIDER: PXD000073 | Pride | 2013-08-14

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	OR2_20120305_SSG_AS_C1.RAW	Raw
	OR2_20120305_SSG_AS_C2_1.RAW	Raw
	OR2_20120305_SSG_AS_C2_2.RAW	Raw
	OR2_20120305_SSG_AS_C3.RAW	Raw
	OR2_20120305_SSG_AS_C4.RAW	Raw

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Publications

On terminal alkynes that can react with active-site cysteine nucleophiles in proteases.

Ekkebus Reggy R van Kasteren Sander I SI Kulathu Yogesh Y Scholten Arjen A Berlin Ilana I Geurink Paul P PP de Jong Annemieke A Goerdayal Soenita S Neefjes Jacques J Heck Albert J R AJ Komander David D Ovaa Huib H

Journal of the American Chemical Society 20130215 8

Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound t ...[more]

PMID: 23387960

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