The protein composition of 10A EV and 231 EV samples

Extracellular vesicles (EVs) were isolated from 231 and 10A cell lines using differential centrifugation. The harvested EVs were lysed with a lysis buffer containing 8 M urea with ultrasonication, followed by centrifugation at 12,000 × g for 30 min at 4 °C. The protein supernatant was collected, and total protein concentration was determined using a BCA assay.For proteomic analysis, 100 μg of protein was subjected to reduction with 10 mM tris and 100 mM triethylammonium bicarbonate (TEAB) at 37 °C for 60 min, followed by alkylation with 40 mM iodoacetamide (IAM) at room temperature for 40 min. Proteins were precipitated using pre-chilled acetone (6:1, v/v) at −20 °C for 4 h, pelleted by centrifugation at 10,000 g for 20 min, and resuspended in 100 µL of 100 mM TEAB. Digestion was performed with trypsin at a 1:50 enzyme-to-protein ratio overnight at 37 °C.The resulting peptides were labeled using TMT10-plex reagents (Thermo Fisher, Cat# 90111) according to the manufacturer’s instructions. Specifically, TMT10-126 and TMT10-130N were used to label peptides from 231_EVs and 10A_EVs, respectively. Equal amounts of labeled peptides from each group were pooled, dried under vacuum, and reconstituted in UPLC loading buffer (2% acetonitrile, pH 10 adjusted with ammonia water).<br>High-pH reverse-phase liquid chromatography (RPLC) was performed using an ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 mm × 150 mm; Waters) on a Thermo Scientific Vanquish Flex UHPLC system. The separation used a gradient from 0% to 100% buffer B (80% acetonitrile, pH 10) over 48 min at a flow rate of 200 μL/min. A total of 20 fractions were collected based on UV absorption at 214 nm, pooled into 10 fractions, concentrated, and reconstituted in LC-MS loading buffer (2% acetonitrile, 0.1% formic acid).The fractions were analyzed by LC–MS/MS using an EASY-nLC 1200 system coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific). Peptides were separated on a C18 column (75 μm × 25 cm) over a 120-min gradient from 8% to 48% buffer B (80% acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min. MS data were acquired in data-dependent acquisition (DDA) mode with a full scan range of 350–1500 m/z at 60,000 resolution. MS2 spectra were generated using HCD fragmentation at 30,000 resolution with a dynamic exclusion time of 35 s.Raw files were processed using Proteome Discoverer software (v2.4) and searched against a specified protein database. Peptide identifications were filtered at a false discovery rate (FDR) of ≤ 0.01.<br>