Spntm protein assay kit

The sample collected at the end of ultrafiltration and before cryoprotectant addition (1 mL) were concentrated to 50 µL using a SpeedVac system (Savant Instruments, Farmingdale, NY, USA) and treated with RapiGest SF (Waters Corporation, Milford, MA, USA) at the final concentration of 0.25% (w/v). After incubation at 100 °C for 20 min, the samples were cooled at room temperature and centrifuged at 2200× g for 10 min. Subsequently, protein concentration was assayed using the SPNTM Protein Assay kit (G-Biosciences, St. Louis, MO, USA) and 50 ± 0.5 μg of protein from each sample was digested with Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA) using a 1:50 (w/w) enzyme/substrate ratio at 37 °C overnight. The next morning, an additional aliquot of enzyme was added (enzyme/substrate ratio of 1:100 (w/w)). The enzymatic reaction was chemically stopped after 4 h by acidification with TFA 0.5% (Sigma-Aldrich Inc., St. Louis, MO, USA), incubation at 37 °C for 45 min and centrifugation at 13,000× g for 10 min in order to remove hydrolytic RapiGest SF by-products. Finally, the samples were desalted by PierceTM C-18 spin columns (Thermo Fisher Scientific, Waltham, MA, USA), concentrated in a SpeedVac (Savant Instruments, Farmingdale, NY, USA) at 60 °C and resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) at a concentration of 0.1 µg/µL.