Oasis hlb 30 mg sorbent cartridges

Protein samples were precipitated with a ProteoExtract Protein Precipitation Kit (539180) from MilliporeSigma as per the manufacturer’s protocol. Samples were resuspended in 50 mM triethylammonium bicarbonate. Total protein concentration was determined with a BCA kit (23227) from Thermo Fisher Scientific. Aliquots of each sample containing ∼100 μg of protein were brought to equal volumes with 50 mM triethylammonium bicarbonate buffer at pH 8. The mixtures were reduced with 20 mM DTT (37 °C for 1 h) and then alkylated with 40 mM iodoacetamide (30 min at RT in the dark). Samples were diluted 10-fold with 50 mM triethylammonium bicarbonate buffer at pH 8 and incubated ON at 37 °C with sequencing grade trypsin (Promega) at a 1:50 enzyme:substrate ratio (wt:wt). Peptide supernatants were collected and desalted with Oasis HLB 30-mg Sorbent Cartridges (Waters; 186003908), concentrated, and resuspended in a solution containing mass spectrometric “Hyper Reaction Monitoring” retention time peptide standards (Biognosys; Kit-3003) and 0.2% formic acid in water.