Xselect hss t3 xp

LC-MS experiments were performed using an Ultimate 3000 HPLC system fitted to a Velos Pro Ion Trap LC-MSⁿ system (Thermo Fisher Scientific). PSA from LNCaP was digested with thermolysin and peptides were purified as described above. For analysis of non-glycosylated peptides, samples were dissolved in 100 μL water in a 250 μL auto sampler vial. The LC-MS/MS analysis was performed using a C18 column, Xselect HSS T3 XP (2.1 × 100 mm, 2.5 µm, Waters). After equilibration with 20 mM ammonium formate, isocratic elution was performed with 0.1% formic acid, 100 µL/min and a 5 min linear gradient with 0–100% acetonitrile and a 5 min isocratic elution with 100% acetonitrile were performed to elute residual peptides. The temperature of the heated capillary was set at 150 °C and the ion spray voltage at 3.5 kV.

Qualitative and quantitative analyses were carried out by LC–MS/MS using a LC system Surveyor MS PUMP PLUS (Thermo Fisher Scientific, Monza, Italy) coupled with an LTQ ion-trap mass spectrometer (Thermo Fisher Scientific, Monza, Italy) equipped with an ESI source operating in the negative mode. The column used was a XSelect HSS T3 XP (100 Å, 2.5 μm, 2.1 × 100 mm; Waters, Sesto San Giovanni, Italy) with a flow rate of 0.15 mL/min. A gradient elution was performed, and the mobile phase was a mixture of water containing 0.1% formic acid (A) and acetonitrile (B). The elution gradient was set as follows: 0–1 min (5% B), 1–10 min (5–100% B), 10–15 min (100% B), 15–25 min (100–5% B). The operating conditions for MS analysis were as follows: spray voltage, −5 kV; capillary temperature, 250 °C; sheath gas and auxiliary gas flow, 60 and 5 arbitrary units, respectively; tube lens, −110 V; total ion current (TIC); base peak-dependent mass range, m/z 230–1500; collision energy, 20 eV.
Compounds in the extract (injection: 25 μg) were identified by comparison of the retention times and mass spectra with those of authentic compounds (injection: 5 or 10 ng) commercially available at a high grade of purity.