Sep pak spe cartridge

The labeled peptides were firstly subjected to Sep-Pak SPE cartridges (Waters) to remove salt ions. The hpRP chromatography was performed with Dionex UltiMate 3000 model on an Xterra MS C18 column (3.5 um, 2.1 × 150 mm, Waters). Then the sample were dissolved in buffer A (20 mM ammonium formate, pH 9.5) and eluted with a gradient of 10 to 45% buffer B (80% acetonitrile (ACN)/20% 20 mM NH₄HCO₂) in 30 min, followed by 45% to 90% buffer B in 10 min, and a 5-min hold at 90% buffer B. Forty-eight fractions collected at 1 min intervals were merged into 12 fractions.

Proteins were extracted as described in [48 (link)]; samples were ground using a mortar and pestle while kept frozen using liquid nitrogen. A quantity of 1 mL of 10% TCA-acetone, 2% β-mercaptoethanol was added per sample. Samples were then incubated for 16 h at −20°C, then centrifuged at 5,000g, 4°C, 30 minutes. Pellets were saved and washed 3 times with cold acetone, dried, and resuspended in 8 M urea in 100 mM ammonium bicarbonate (ABC).
Protein was quantified using a Bradford assay; protein integrity was examined by running 5 µg from each sample on 1D gel with bovine serum albumin as a control, and a Coomassie Brilliant Blue staining.
Three biological replicates were chosen per population. Protein samples then proceeded to reduction, cystein blocking, and trypsin digestion; 50 µg of protein was added to a final volume of 10 mM of dithiothreitol in 100 mM ABC; samples were then incubated at 30°C for 1 h. A final volume of 30 mM of methyl methanethiosulfonate (MMTS) in 100 mM ABC was added and samples were incubated for 1 h at room temperature.
Samples were then diluted to ∼1 M urea with 100 mM ABC and trypsin was added in a 1:50 ratio (trypsin: protein). Samples were incubated for 16 h at 37°C, desalted using Waters Sep Pak SPE cartridges (according to manufacturer's protocol), dried, and kept at −80°C till MS analyses.