Nanoacquity uplc beh130 column

The peptide mixtures were analyzed by full-scan LC-MS on a nanoAcquity system (Waters, Milford, MA) coupled to an LTQ-Orbitrap spectrometer (Thermo Scientific) provided with a PicoView nanospray interface. Peptide mixtures were loaded into a 75-μm I.D. ×250-mm nanoACQUITY UPLC BEH130 column packed with C18 resin (Waters). The prepared peptide mixtures were separated by gradient buffer (buffer A: 0.1% formic acid in water; buffer B: 0.1% formic acid in ACN) with 10 to 35% solvent B for forty min, ensued by a sharp increase to 90% B in five min; then, a 300 nL/min flow rate was maintained for five minutes. The effluent from the HPLC column was electrosprayed directly into the MS. The LTQ-Orbitrap apparatus was ran in full-scan MS acquisition mode (m/z 300–2000). Targeted LC-MS/MS analysis was performed, and the possible phosphopeptide signals (m/z values) obtained from the iPhos process (see the next paragraph in the Methods section) were set in the parent mass list for LTQ-Orbitrap MS.

Digested peptide mixtures of total testicular proteins were subjected to NanoLC-nanoESI-MS/MS analysis performed at the Core Facilities for Proteomics Research (Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan). The mass spectrometry analysis was processed on a nanoAcquity system (Waters, Milford, MA, USA) coupled to a LTQ-Orbitrap Elite hybrid ion trap-orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a PicoView nanospray interface (New Objective, Woburn, MA, USA). The resulting peptide mixtures were loaded onto a nanoAcquity UPLC BEH130 column (75 μm internal diameter, 25 cm length; Waters) packed with a C₁₈ resin containing 1.7 μm particles (pore size, 130 Å) in 0.1% FA in water (solvent A). Peptides were separated using a segmented gradient of 5–40% solvent B (ACN with 0.1% FA) at 300 nl/min. The mass spectrometer was operated in a data-dependent acquisition mode. Full survey mass spectra scans of mass/charge ratio (m/z) 350–1600 were acquired with a resolving power of 120,000 at 400 m/z and an automatic gain control (AGC) target of 10⁶. The twenty most intense ions were sequentially selected for collision-induced dissociation MS/MS fragmentation in a linear ion trap (AGC target value: 10,000) with previously preferred ions dynamically excluded for 60 s. Ions with single and unrecognized charge states were also excluded.