Easy column sc001

The peptides were analyzed using LC-MS/MS according to previously described protocols (Li et al., 2015 (link)). Briefly, peptides passed through a reverse-phase analytical column were loaded into the reverse-phase precolumn (EASY column SC001; Thermo Scientific, USA) that was connected to a reverse-phase column (EASY column SC200; Thermo Scientific, USA), separated with a linear gradient of solvent B (0.1% formic acid in 80% acetonitrile) at a constant flow rate of 300 nL/min on an EASY-nLC 1000 UPLC system. The separated peptides were analyzed with the Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Intact peptides were detected on the Orbitrap at a resolution of 70,000. Peptides of the top 20 most abundant precursor ions from the survey scan (300–1,800 m/z) were selected for HCD fragmentation. The ion fragments were detected on the Orbitrap at a resolution of 17,500. Determination of the target value was based on predictive automatic gain control.

Exosomes were lysed in SDT buffer and ultrasonicated, and protein concentrations were subjected to SDS-PAGE. Proteins were then digested using the FASP protocol [30 (link)]. Two micrograms of enzymatic hydrolysis lysate (obtained by filter-aided sample preparation) were analysed using 150 μm × 20 mm Thermo EASY column SC001 traps (Thermo, USA). Liquid chromatography-mass spectrometry (LC-MS/MS) was performed using the QExactive platform in positive ion mode with a scanning range of 300-1800 m/z. Twenty fragment spectra (MS2 scan) were collected after each full scan. The resolution for MS1 and MS2 at m/z 200 was set to 70000 and 17500, respectively. The raw data were analysed using MaxQuant software (1.3.0.5). Label-free quantitation (LFQ) analysis [31 ] was performed, and the proteins with a certain LFQ intensity and that overlapped between two runs were selected and processed for statistical analysis using Perseus software (1.3.0.4).

The peptides were analyzed by nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Q Exactive plus mass spectrometer (Thermo Fisher Scientific, Waltham, USA). The LC system was equipped with analytical EASY column SC200 (150 μm × 100 mm, RP-C₁₈; Thermo Fisher Scientific) and a trap EASY column SC001 (150 µm × 20 mm, RP-C₁₈; Thermo Fisher Scientific). Five μg of sample was loaded through an automatic sampler. For peptide separation, a gradient protocol of liquid chromatographic conditions was implemented. Solvent A was 2% acetonitrile in water containing 0.1% formic acid. Solvent B was 84% acetonitrile in water containing 0.1% formic acid. The gradient was used in a single run: first the column was balanced with 100% solvent A, followed by a linear gradient of 0–45% solvent B (100 min), and then 45–100% solvent B (8 min), finally 100% solvent B (12 min) at constant flow rate of 0.3 ml/min. The entire instrument was operated in positive mode for 120 min with the scan range of 300–1800 m/z and nominal mass resolving power of 70,000. Rapid, automated data-dependent capabilities enabled real-time acquisition of high-mass accuracy MS/MS spectra per 40 ms.