In situ digestion of alcohol-fixed cells for quantitative proteomics

• Published in: Journal of biochemistry (Tokyo) Vol. 173; no. 4; pp. 243 - 254
• Main Authors: Hatano, Atsushi, Takami, Tomoyo, Matsumoto, Masaki
• Format: Journal Article
• Language: English
• Published: England 01.04.2023
• Subjects: Animals; Digestion; Ethanol; Mice; Proteins - chemistry; Proteomics - methods; Reproducibility of Results; Trypsin - chemistry; Trypsin - metabolism; 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

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...