A34808

Post cell surface labeling cells were harvested by centrifugation, resuspended in 400 μL PBS and lysed by sonication. After precipitation overnight in MeOH (−20°C) the pellet was dissolved in Urea (6 M) and 10 μL of 10% (w/v) SDS in PBS. Proteins were reduced(30 mins, 37 °C) with 50 μL 1:1 solution of 20 mM TCEP (PBS) and 600 mM K₂CO₃ (PBS). Samples were treated (RT, 30 mins) in the dark with iodoacetamide followed by 10% SDS (w/v) prior to incubation with streptavidin agarose beads (RT, 1.5 hr). After washing to remove unbound protein, beads were incubated in sequencing grade trypsin solution(Promega, 20 μg) in 100 mM TEAB supplemented with CaCl₂ (overnight, 37 °C). Supernatant was harvested and labelled with tandem mass tags (Thermo Scientific cat# A34808, 1 hr, RT) before quenching with hydroxylamine (15 mins, RT). Samples wereacidified with formic acid, before being dried by vacuum centrifugation. Dry samples were combined, desalted, and dried by vacuum centrifugation and stored at −80 °C until injection.

Following proximity tagging, adherent cells were scraped, resuspended in 400 μL of PBS, and lysed by sonication. After precipitation in cold MeOH (overnight, −20 °C), the resulting pellet was resuspended in 6 M urea in PBS and 10 μL of 10% (w/v) sodium dodecyl sulfate (SDS). Dissolved proteins were reduced by adding 1:1 solution of 20 mM (tris(2-carboxyethyl)phosphine (50 μL, PBS) and 600 mM K₂CO₃ (PBS) and further incubated (shaking, 30 min, 37 °C). Samples were treated with iodoacetamide, followed by 10% SDS before incubation with streptavidin agarose beads (1.5 h, room temperature). After being washed to remove unbound materials, beads were resuspended in 100 mM triethylammonium bicarbonate and sequencing grade porcine trypsin and digested overnight (37 °C). Supernatants were harvested and labeled with a tandem mass tag (Thermo Scientific cat# A34808; 1 h, room temperature). The reaction was quenched with hydroxylamine and acidified with formic acid before being dried by vacuum centrifugation. Dried samples were redissolved in 5% (v/v) MeCN, 0.1% formic acid, combined, desalted, dried by vacuum centrifugation, and stored at −80 °C until ready for injection.