Quantitative profiling of the detergent-resistant membrane proteome of Iota-b toxin induced Vero cells

Enzyme-mediated ¹⁸O/¹⁶O differential labeling of proteome samples often suffers from incomplete exchange of the carboxy-terminus oxygen atoms, resulting in ambiguity in the measurable abundance differences. In this study, an ¹⁸CV¹⁶O labeling strategy was optimized for and applied to the solution-based comparative analysis of the detergent-resistant membrane proteome (DRMP) of untreated and lota-b (Ib)-induced Vero cells. Solubilization and tryptic digestion of the DRMP was conducted in a buffer containing 60% methanol. Unfortunately, the activity of trypsin is attenuated at this methanol concentration hampering the ability to obtain complete oxygen atom turnover. Therefore, the incorporation of the ¹⁸O atoms was decoupled from the protein digestion step by carrying out the trypsin-mediated heavy atom incorporation in a buffer containing 20% methanol; a concentration at which trypsin activity is enhanced compared to purely aqueous conditions. After isotopic labeling, the samples were combined, fractionated by strong cation exchange and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. In total, over 1400 unique peptides, corresponding to almost 600 proteins, were identified and quantitated, including all known caveolar and lipid raft marker proteins. The quantitative profiling of Ib-induced DRMP from Vero cells revealed several proteins with altered expression levels suggesting their possible role in Ib binding/uptake.