Crude NVs and all the gUC fractions were analyzed by shotgun proteomics as it was described before [2 (link)]. Briefly, peptides were purified and enriched using Pierce C18 Spin Columns according to the manufacturer’s description. Eluted samples were dried using SpeedVac and kept at −20 °C. Prior analysis, samples were dissolved in 2% acetonitrile (v/v), containing 0.1% (v/v)formic acid (FA). A Waters Acquity I-Class ultra-performance liquid chromatography (UPLC) system equipped with a Waters Acquity CSH Peptide C18 UPLC column (1 mm × 150 mm, 1.7 µm) was employed to separate the peptides. Gradient elution was used with the following parameters: eluent A: 0.1% FA, eluent B: 0.1% FA in acetonitrile; flow rate: 20 µL/min; column temperature: 45 °C; gradient: 1 min: 5% B, 45 min: 35% B, 46 min: 85% B. MS experiments were performed according to the method described in [2 (link)].
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