Quantifying Serine Hydrolases by DIA-MS

We used an established LC-MS/MS-based data-independent acquisition (DIA) proteomics approach [23 (link),24 (link)] to quantify the relative abundance of serine hydrolases enriched by ActivX™ desthiobiotin-FP probe from HlungS9 in the ABPP experiment. The analysis was carried out on a TripleTOF 5600 plus mass spectrometer (AB Sciex, Framingham, MA, USA) coupled with an Eksigent 2D plus LC system (Eksigent Technologies, Dublin, CA, USA). Briefly, we used a trapping column (ChromXP C18-CL, 120 Å, 5 mm, 0.3 mm cartridge; Eksigent Technologies) to load the samples and an analytical column (ChromXP C18-CL, 120 Å, 150 × 0.3 mm², 5 mm; Eksigent Technologies) to separate peptides. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile (ACN) containing 0.1% formic acid (B). Mobile phase A was delivered at a flow rate of 10 μL/min for 3 min to load 1~2 µg proteins to the trapping column. A gradient elution at a flow rate of 5 μL/min was used to separate the injected peptides on the analytical column. The gradient parameters are summarized in Table S2. A blank sample (30% ACN, v/v) was injected between each run to minimize carryover. MS data were collected in a positive mode with the source temperature set at 280 °C and the ion spray voltage of 3000 V for ionization.