Easy spray pepmap c18 75 μm 250 mm column

Reverse-phase nLC-MS/MS was performed on control and acrylamide-treated HILIC fractions (12 from each seminal vesicle) (supplemental Fig. S1, A and B) using a Q-Exactive Plus hybrid quadrupole-Orbitrap MS coupled to a Dionex Ultimate 3000RSLC nanoflow high-performance LC system (Thermo Fisher Scientific). Samples were loaded onto an Acclaim PepMap 100 C18 75 μm × 20 mm trap column (Thermo Fisher Scientific) for preconcentration and online desalting. Separation was then achieved using an EASY-Spray PepMap C18 75 μm × 250 mm column (Thermo Fisher Scientific), employing a linear gradient of acetonitrile (2–40%, 300 nl/min, 120 min). The MS was operated in data-dependent acquisition (DDA) mode. The Orbitrap mass analyzer was used at a resolution of 35,000, to acquire full MS with an m/z range of 380 to 2000, incorporating a target automatic gain control value of 1 × 10⁶ and maximum fill times of 50 ms. The 20 most intense multiple charged precursors were selected for higher-energy collision dissociation fragmentation with stepped collisional energies of 25%, 28%, and 30%. MS/MS fragments were measured at an Orbitrap resolution of 17,500 using an automatic gain control target of 5 × 10⁵ and maximum fill time of 120 ms.

Phosphopeptides were loaded onto an Acclaim PepMap100 C18 75 μm × 20 mm trap column (Thermo Fisher Scientific) for pre-concentration and online desalting, then separated using EASY-Spray PepMap C18 75 μm × 250 mm column (Thermo Fisher Scientific). An optimized stepped 82 min gradient was employed (5 to 22 to 35%). Each sample was run in data-dependent acquisition mode (described above) to evaluate phosphopeptide enrichment efficiency (93–97%) and loading. Parallel reaction monitoring (PRM) was performed using optimized methods for collision energy, charge state and retention times at a resolution of 17 500, automatic gain control of 2e5, maximum fill times of 90 ms and 1.6 m/z isolation window.