Targeted mass spectrometry detection of citrulline

For intracellular citrulline measurement, cells (231/Ctrl and 231/DDAH1 KO) were harvested from 15-cm dishes after trypsinization and centrifuged (300 × g, 5 min). Supernatants were discarded, and intact cell pellets were retained for subsequent citrulline analysis. To measure the enzymatic activity of EV-derived DDAH1, extracellular vesicles (EVs) were purified from MCF-10A and MDA-MB-231 cells via differential centrifugation. Purified EVs were lysed in buffer containing 1% Triton X-100, 5 mM 1,10-phenanthroline, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) (pH 8.5). The EV lysate was then incubated with 10 mM ADMA in 50 mM Tris buffer at 37°C for 1 hour prior to mass spectrometry (MS) analysis.Samples were mixed with 1 mL of ice-cold methanol/acetonitrile/H₂O (2:2:1, v/v/v) for metabolite extraction. After homogenization, samples underwent three sonication-freeze-thaw cycles using liquid nitrogen. Proteins were precipitated at -20°C for 1 h, followed by centrifugation at 14,000×g for 15 min (4°C). The supernatant was collected and dried in a vacuum concentrator. For LC-MS analysis, dried extracts were reconstituted in 100 μL of acetonitrile/water (1:1, v/v).The dried extract was resuspended in 50% acetonitrile and analyzed by ultraperformance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) conducted on a Waters Acquity UPLC-system coupled with 5500 QTRAP system (SCIEX). For the analysis of citrulline, Chromatographic separation was achieved on a Waters Acquity UPLC HSS T3 Column (2.1 mm× 100 mm, 1.7 μm, Waters) using a flow rate of 0.3 mL/min at 40°C during a 10 min gradient (0–2 min from 95% B, 2–4 min from 95% B to 50% B, 4–7 min 50% B, 7–7.1 min from 50% B to 95% B, 7.1–10 min 95% B), using buffer A (20 mM ammonium acetate, 20 mM ammonia solution and 5% (v/v) acetonitrile in water) and buffer B (acetonitrile). Mass spectrometry was operated in positive mode using an ESI source. The citrulline was monitored in multiple reaction monitoring (MRM) mode using the optimized precursor-to-product ion transitions. Peak determination and area integration were performed using Analyst 1.7.1 and SCIEX OS 1.4.0 software