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A competitive activity-based protein profiling method is reported for quantifying the reactivity of lipid-derived electrophilic compounds against cysteine residues in the human proteome.
growth: MDA-MB-231 and Ramos cells cultured under standard protocols from ATCC.
treatment: Lipid derived electrophiles were treated either in vitro with soluble proteomes or in situ with living cells.
extraction: The treated proteomes were labeled with a cysteine-reactive iodoacetamide-alkyne probe and then conjugated with the isotopically labeled azide-biotin tags by click chemistry. The proteins were extracted by cold methanol precipitation.
separation: Streptavidin enrichment in 0.2% SDS/PBS
digestion: sequential on-bead digestion with trypsin and TEV protease to obtained the final sample of IA-probe labeled peptides
acquisition: MudPIT LC-MS/MS, one MS1 followed by 18 MS2 data dependent scans.
informatics: SEQUEST, DTASelect for peptide identification and CIAMGE for quantitation.
instruments: Thermo Scientific LTQ Orbitrap
species: Human
massModifications: static: C+57.021464, variable: C+464.29596, C+470.29977
v. 4.14
