In situ digestion of alcohol-fixed cells for quantitative proteomics

Abstract

Currently, the bottom-up approach, in which proteins are digested by enzymes such as trypsin prior to mass spectrometry, is the mainstream approach in mass spectrometer-based proteomics. In this approach, the enzymatic digestion process strongly affects the reproducibility of protein identification and quantification. Here, we quantitatively evaluated the enzymatic digestion of proteins under various conditions by quantitative proteomics using data-independent acquisition and found that proteins precipitated with acetone after solubilization with SDS were fully digestible without re-solubilization. This result implies that organic solvent treatment makes cells amenable to trypsin digestion. Direct trypsin digestion of methanol-fixed cells achieved the same digestion efficiency and quantitative reproducibility as the conventional method. Furthermore, this method was found to be equally applicable to mouse liver samples. The establishment of this method indicates that the sample preparation process in bottom-up proteomics can be simplified while maintaining high digestion efficiency and is expected to become a general method for sample preparation in bottom-up proteomics in the future.

Keywords: mass spectrometry; proteomics; sample preparation; trypsin.Abbreviations: AGC, auto gain control; Ambic, ammonium bicarbonate; BCA, bicinchoninic acid; CVs, coefficients of variation; DDA, data-dependent acquisition; DIA, data-independent acquisition; DMEM, Dulbecco’s modified Eagle’s medium; FBS, foetal bovine serum; GdnHCl, guanidine hydrochloride; IAA, iodoacetamide; iSDAC, in situ digestion of alcohol-fixed cells; IT, injection times; lys-C, lysyl endopeptidase; LC, liquid chromatography; MS, mass spectrometry; PBS, phosphate-buffered saline; SDC, sodium deoxycholate; SLS, sodium deoxycholate-N-lauroylsarcosinate; TCA, trichloroacetic acid; TCEP, tris (2-carboxyethyl) phosphine hydrochloride.