Tris hcl ph 8

The proteins were dissolved in 20 μL of 0.2% Protease Max Surfactant (Promega, Madison, WI, USA) in 50 mM NH4HCO3 and 15 μL of UREA 8 M (Sigma-Aldrich, St. Louis, MO, USA). The equivalent to 200 μg of protein was reduced with 10 mM dithiothreitol (Sigma-Aldrich) at 37 °C for 60 min and alkylated with 20 mM iodoacetamide (Sigma-Aldrich), for 30 min at room temperature under the dark, then Tris-HCl pH 8.6 (Promega) was added to reach 10 mM. Digestion was made with trypsin (Promega) 1:35 at 37 °C overnight and then peptides were fractionated with HyperSep SCX cartridges (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Five fractions were obtained from each sample which were desalted with Sep-Pak tC18 cartridges (Waters, Milford, MA, USA), dried in a SpeedVac concentrator (Eppendorf, Hamburg, DE), and kept at −80 °C. The samples were reconstituted in 30 µL of 0.1% formic acid and 5% acetonitrile, centrifuged at 20,000× g at 4 °C for 5 min and injected on a C18 Nano HPLC column for separation of peptides [9 (link)].

The proteins were dissolved in 20 μL of 0.2% Protease Max Surfactant (Promega) in 50 mM NH4HCO3 and 15 μL of UREA 8 M (Sigma-Aldrich). The equivalent to 200 μg of protein was reduced with 10 mM dithiothreitol (Sigma-Aldrich) at 37 °C for 60 min and alkylated with 20 mM iodoacetamide (Sigma-Aldrich), for 30 min at room temperature under the dark, then Tris–HCl pH 8.6 (Promega) was added to reach 10 mM. Digestion was made with trypsin (Promega) 1:35 at 37 °C overnight and then peptides were fractionated with HyperSep SCX cartridges (Thermo Scientific) following the manufacturer's instructions. Five fractions were obtained from each sample which were desalted with Sep-Pak tC18 cartridges (Waters), dried in a SpeedVac concentrator (Eppendorf), and kept at − 80 °C. The samples were reconstituted in 30 µl of 0.1% formic acid and 5% acetonitrile, centrifuged at 20,000 g at 4 °C for 5 min and injected on a C18 Nano HPLC column for separation of peptides [16 ].