All assays were performed in Dulbecco's phosphatase buffered saline. GSH solution was prepared with 1 mM TCEP for a final GSH concentration of 2 mM after compound treatment. For each compound, GSH solution (396 µL) was treated with 100 µM compound (4 µL of 10 mM stock in DMSO). Reaction progress was monitored by reverse-phase high performance liquid chromatography (RP-HPLC) using an Agilent 1200 series, monitoring absorbance at 280 nm or 260 nm. At each time point, 50 μL of the reaction mixture was injected onto a C18 column (ZORBAX 300SB-C18, 5 μm, 4.6 x 150 mm). The samples were eluted using water with 0.1% trifluoroacetic acid (solvent A) and acetonitrile with 0.1% trifluoroacetic acid (solvent B) over the following linear gradients: 0-2 minutes: 5% B, 2-12 minutes: 5-95% B, 12-13 minutes: 95% B, 13-14 minutes: 95-5% B, and 14-18 minutes: 5% B. For each reaction, six time points were analyzed over the course of 6-12 hours. Control reactions were run with 100 µM compound in 1 mM TCEP without GSH to identify peaks corresponding to degradation products (Figure ). The peak areas corresponding to the compound, the compound + GSH adduct, and the degradation product were calculated using the Agilent OpenLAB ChemStation software. The fractional peak areas were used to determine the concentrations of HePTP was diluted in a buffer containing 50 mM Tris, pH 7.5, and 150 mM NaCl. For each sample, HePTP was treated with compound in DMSO stock for a final concentration of 1 µM HePTP and 100 µM compound (6 µL stock in 594 µL enzyme, final DMSO concentration: 1% v/v) and incubated for 1 hour at room temperature. Then, the reactions were quenched with 10 mM DTT, and the samples were precipitated with methanol:chloroform:water (4:1:4). The resulting pellets were solubilized in a buffer containing 50 mM Tris, pH 7.5, 9 M urea, and 10 mM DTT, and incubated for 30 minutes while shaking at room temperature. Free cysteines were subsequently alkylated with 20 mM iodoacetamide in the dark for 45 minutes while shaking at room temperature. The samples were diluted to 1 M urea in 50 mM Tris pH 7.5 and digested with Promega sequencing grade modified trypsin (2 µg). After overnight digestion, samples were acidified with formic acid to a final concentration of 1% (v/v), desalted on C18 StageTips according to Rappsilber et al., evaporated to dryness in a vacuum concentrator, and reconstituted in 15 µL of 3% acetonitrile / 0.2% formic acid.