Profiling of LN5P45-Reactive Cysteines by SLC-ABPP

SLC-ABPP profiling was performed as described before.²⁴ In short, HeLa cells, transfected with GFP or GFP-OTUB2 WT with PEI reagent for 24 h, were grown to 80% confluence and incubated with either DMSO or 10 μM LN5P45 for 4 h in complete medium. Cells were harvested, lysed by sonication in ice-cold PBS, and centrifuged at 15,000 RPM for 2 min to remove cell debris. Protein concentrations were then determined by a BCA Gold protein assay. Proteomes were normalized to 1 mg/mL in 100 μL for each sample.

Each DMSO- and LN5P45-treated proteome was labeled with the 500 μM DBIA probe for 1 h in the dark at room temperature (RT). Excess DBIA, along with disulfide bonds, was quenched and reduced using 5 mM dithiothreitol for 30 min in the dark at RT. Reduced disulfide bonds were alkylated using 20 mM iodoacetamide for 30 min in the dark at RT. Proteins were precipitated using chloroform/methanol, re-solubilized in 40 mM HEPES pH 8.4, and digested using TPCK-treated trypsin and endoLysC (1:12.5 enzyme/protein ratio) overnight at 37 °C. Digested peptides were labeled using TMTpro16-plex reagents in a 1:4 ratio by mass (peptides:TMT reagents) for 1 h at RT. Excess TMT reagent was quenched with 5 μL of 6% hydroxylamine for 15 min at RT. Next, samples were mixed 1:1 across all TMT channels, and the pooled sample was dried using a Speedvac.

Dried samples were reconstituted in 1 mL of PBS and enriched by Pierce streptavidin magnetic beads (Catalog number: 88816, Thermo Scientific) by rotating end-over-end overnight at 4 °C. Nonspecific binding peptides were washed using the following procedure: 3 × 1 mL of PBS pH 7.4, 2 × 1 mL of PBS with 0.1% SDS pH 7.4, and 3 × 1 mL of HPLC-grade water. DBIA probe-containing peptides were eluted using 700 μL of 50% acetonitrile with 0.1% TFA, dried using a Speedvac, and desalted using a mini-SPE column (10% sorbent material of a Waters, OASIS 1 cc HLB 30 mg cartridge). The column was washed with 200 μL of 90% acetonitrile and 3× 200 μL of 10 mM NH₄HCO₃ pH 8.4. Dried samples were dissolved in 200 μL of 10 mM NH₄HCO₃ pH 8.4, loaded onto the column, washed 3× with 200 μL of 10 mM NH₄HCO₃ pH 8.4, and eluted into three fractions with 150 μL each of 10, 20, and 50% acetonitrile (in 10 mM NH₄HCO₃). Samples were re-dried using a Speedvac.