MassIVE MSV000082160

Partial Public PXD009202

MudPIT analyses of the protein succination after fumarate treatment of whole cell extracts from Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) fumarate hydratase-deficient (FH-/-) cells

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Description

In brief, TCA-precipitated proteins from whole cell extracts treated with fumarate were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. The peptide mixtures were loaded onto microcapillary fused silica columns (100um i.d.), packed with C18 reverse phase (Aqua; Phenomenx), SCX (Luna; Phenomenex) and C18-RP, placed in-line with an Agilent 11000 quaternary pump, and analyzed by a 10-step MudPIT on Orbitrap Velos Pro or Elite. Full MS spectra were recorded on the eluting peptides over a 400 to 1600 m/z range in the Orbitrap at 60K resolution, followed by fragmentation in the ion trap (at 35% collision energy) on the first to fifteenth most intense ions selected from the full MS spectrum. MS/MS datasets searched using ProLuCID (Xu et al., 2015) with a peptide mass tolerance of 10 ppm and 500 ppm for fragment ions. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database combining 36,628 human proteins (NCBI 2016-06-10 release), as well as 193 usual contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database, for a total search space of 73,642 amino acid sequences. Masses of 57.0215 Da and 116.0112 Da were differentially added to cysteine residues to account for alkylation by CAM and succination, respectively, while 15.9949 Da were differentially added to methionine residues. Peptide/spectrum matches were sorted and selected using DTASelect with the following criteria set: spectra/peptide matches were retained only if they had a DeltCn of at least 0.8, and minimum XCorr of 1.8 for singly, 2.0 for doubly, and 3.0 for triply charged spectra. Additionally, the peptides had to be minimum 7 amino acids in length and fully tryptic. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: succination ; fumarate ; oncometabolite ; chemoproteomic

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens

Publications

Kulkarni RA, Bak DW, Wei D, Bergholtz SE, Briney CA, Shrimp JH, Alpsoy A, Thorpe AL, Bavari AE, Crooks DR, Levy M, Florens L, Washburn MP, Frizzell N, Dykhuizen EC, Weerapana E, Linehan WM, Meier JL.
A chemoproteomic portrait of the oncometabolite fumarate.
Nat. Chem. Biol. 2019 Apr;15(4):391-400. Epub 2019 Feb 4.

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