Reversed phase spin columns

Phagosome pellets were dissolved in 25 µl of 5% SDS/50 mM triethylammonium bicarbonate (TEAB) buffer (pH 7.6) and centrifuged. Supernatants were transferred into new tubes, and protein concentrations were determined by BCA assay (Sigma-Aldrich). ∼6 µg of phagosomal proteins from each sample was reduced by 10 mM tris(2-carboxyethyl)phosphine for 30 min at RT and alkylated by 10 mM iodoacetamide for 30 min in dark. The excess of iodoacetamide was quenched by the addition of tris(2-carboxyethyl)phosphine (final concentration 20 mM) for 15 min. Protein digestion was done using S-Trap (ProtiFi) according to the manufacturer’s instructions. Briefly, samples were acidified by phosphoric acid, mixed with 6 vol of 90% methanol (MeOH)/100 mM TEAB, and loaded onto the S-Trap phase. Columns were then washed four times with 90% MeOH/100 mM TEAB, and captured proteins were digested by 1 µg of trypsin (Pierce) in 50 mM TEAB at 37°C overnight. After digestion, peptides were eluted by consecutive washes of 50 mM TEAB, 0.2% formic acid (FA), and 50% acetonitrile/0.2% FA and the combined eluate was evaporated to ∼20 µl. Samples were acidified by TFA, desalted using reversed-phase spin columns (Thermo Fisher Scientific), and dried on Speed-Vac.

Plasma and tissue peptide samples were TMT-10plex labeled according to the manufacturer's instructions. Samples and pool were then grouped in equal amounts, ensuring randomization, and dried using a SpeedVac system (Thermo Fisher Scientific; Savant SPD131DDA). Grouped TMT samples were then reconstituted with 0.1% TFA in H₂O, for peptide fractionation using high pH reversed-phase spin columns, following the manufacturer's instructions (Thermo Fisher Scientific).