IsoTop‐ABPP samples were prepared as described previously (Weerapana et al, 2010; Backus et al, 2016). Briefly, cells were harvested and lysed by sonication in PBS. Proteomes were adjusted to 1 mg/ml. Samples were labeled for 1 h at ambient temperature with either 10 or 100 µM iodoacetamide alkyne (IA‐alkyne, 5 µl of 1 or 10 mM stock in DMSO). Samples were conjugated by CuAAC to either the light (fragment treated) or heavy (DMSO treated) TEV tags (10 µl of 5 mM stocks in DMSO, final concentration = 100 µM), with TCEP (10 µl of fresh 50 mM stock in water, final concentration = 1 mM), TBTA (30 µl of 1.7 mM stock in DMSO/t‐butanol 1:4, final concentration = 100 µM), and CuSO4 (10 µl of 50 mM stock in water, final concentration = 1 mM). After 1h, the samples were pelleted and the pellets sonicated in ice‐cold methanol (500 µl) and combined pairwise. The pellets were solubilized in PBS containing 1.2% SDS (1 ml) with sonication and heating (5 min, 95°C) and any insoluble material was removed by an additional centrifugation step at ambient temperature (14,000 g, 1 min). Samples were then enriched on streptavidin resin (100 µl slurry) in PBS (10 ml) with rotating for 90 min. Beads were then washed (2× PBS and 2× water), resuspended in 6 M urea reduced (20 mM DTT), and alkylated (40 mM iodoacetamide). Samples were then diluted to 2 M urea and 6 μl (2 µg) reconstituted MS grade trypsin (Promega V5111) was added and the samples were allowed to digest overnight. The beads were then pelleted, washed (3× PBS and 3× water), and then resuspended in 75 µl TEV buffer (50 mM Tris, pH 8, 0.5 mM EDTA, 1 mM DTT). 5 µl TEV protease (80 µM) was added and the reactions were rotated for 7 h at 29°C. The samples were then cleaned using Micro Bio‐Spin columns, desalted using Pierce C18 100 µl bed zip‐tips, concentrated by speedvac and reconstituted in 20 μl 5% ACN and 1% formic acid.

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