Normal human liver shotgun protein database search results and SRM analysis results

Liver tissue was homogenized in the lysis buffer containing: 2M Urea, 1% Na deoxycholate, 147 mM NaCl, 100 mM PBS pH 7.2, 15% AcN, 5 mM TCEP. The homogenized tissue was sonicated for 10 cycles on ice. The solution was clarified by centrifugation 10000 g for 5 min. Cysteines were reduced by TCEP at 60 C for 30 min. After cooling the solution cysteines were alkylated by 2-CAA 50 mM for 30 min. The resulted solution was diluted 10 times by 100 mM TEA pH 8.0 to concentration 1 mg/ml of total protein. Trypsin was added to the solution in a 1:100 ratio and incubated for 4 hours at 38 C. After that one more aliqote of trypsin was added and incubated for 4 hours at 38 C. The digestion was stopped by the addition of concentrated formic acid to a final concentration of 3%. The digest was centrifuged at 4 C to clarify the solution. The peptide samples were evaporated in a vacuum concentrator and reconstituted in 0.5% formic acid solution.Peptide mixture was analyzed on Q Exactive HFX mass spectrometer coupled to UHPLC chromatography system. Two dimensional fractionation of peptides included one more separation on Agilent 1200 Series RP chromatography in alkaline conditions with fractions collected and also analyzed by MS. The fractions were also analyzed by SRM SIS analysis to detect proteins encoded by the 18th human chromosome. The analysis was carried out at QQQ LC/MC system (Agilent). Stable isotope labelled peptides were used as internal standard. The Shotgun data was processed with MaxQuant software v1.6.3.4 by target/decoy method. Cleavage by trypsin, max number of missed cleavages - 2. Oxidation of M and Acetylation of protein N-term were set as variable modifications. Carbamidomethylaion was set as fixed modification.