Mass spec grade trypsin lys c mix

Individual fresh frozen tissue samples were pulverised in liquid N₂ and thoroughly homogenised in an extraction buffer consisting of 500 mM Tris-Cl [pH 8], 6 M guanidine-HCl in 50 mM ammonium bicarbonate (AMBIC) along with protease and phosphatase inhibitor cocktail. The obtained extracts were then subjected to 4 freeze-thaw cycles, followed by ultrasonic bath for 20 min at 0 °C. The soluble proteins were then reduced with 15 mM dithiothreitol (DTT) for 60 min at 60 °C, alkylated using 50 mM iodoacetamide (IAA) for 30 min at room temperature in the dark, precipitated with a sample to ethanol (99.5%) ratio of 1:9 at −20 °C. The protein precipitates were dissolved in 50 mM AMBIC and digested at 37 °C overnight using Mass Spec Grade Trypsin/Lys-C Mix (Promega, Madison, WI, USA), with an enzyme to protein ratio of 1:100. The digested samples were dried and dissolved in 50 μl 0.1% Formic Acid (mobile phase A), and the concentration was specified using Pierce quantitative colorimetric peptide assay from Thermo Scientific (Rockford, IL, USA). Finally, to enable normalisation and as a control of the chromatographic performance, 25 fmol peptide retention time mixture (PRTC) (Thermo Fisher) consisting of 15 peptides was added to each sample.

The following materials were purchased from Thermo Fisher Scientific (Wilmington, DE): 1 M tris hydrochloride solution pH 7.5, 1 M tris hydrochloride solution pH 8, sodium chloride, ammonium bicarbonate (ABC), Promega Mass Spec Grade trypsin/lys-C mix, porcine pancreatic elastase Type I, LC/MS grade water, LC/MS grade acetonitrile, LC/MS grade formic acid. Urea, dithiothreitol (DTT), and iodoacetamide (IAA) were purchased from Bio-Rad (Hercules, CA). Sodium deoxycholate (SDC) and calcium chloride, were purchased from MilliporeSigma (St Louis, MO). Pall Omega 10 kDa molecular weight cutoff filters were purchased from VWR (Radnor, PA). Collagenase HA was purchased from VitaCyte (Indianapolis, IN).