Acquity hsst3 c18 stationary phase

The peptide mixtures were desalted using C18 resin (The Nest Group) according to the manufacturer’s protocol. LC-MS/MS analyses were performed on a Q-Exactive Plus high-resolution mass spectrometer (Thermo Scientific, Inc.) with a nanoAcquity UPLC system (Waters Corp.) and a nano-electrospray ionization source. Samples were trapped on a Symmetry C18 300 mm × 180 μm trapping column for 3 min at 5 μL/min (99.9/0.1 v/v water/acetonitrile 0.1% formic acid), and separated on a 75 μm × 250 mm column packed with 1.7 μm Acquity HSST3 C18 stationary phase (Waters Corp.). Peptides were separated using a gradient of 3 to 30% acetonitrile with 0.1% formic acid over 90 min at a flow rate of 0.4 μL/min. Data collection was performed in a data-dependent acquisition (DDA) mode with a resolution of 70,000 (at m/z 200) for full MS scan from m/z 375 ~ 1600 with a target AGC value of 1 × 10⁶ ions, followed by 20 product ion scans at a resolution of 17,500 (at m/z 200), using an AGC target value of 1 × 10⁵ ions, a max fill time of 60 ms, and normalized collision energy of 30 V. Each sample was analyzed in triplicate.

The peptide mixtures were desalted using C18 resin (The Nest Group) according to the manufacturer’s protocol. LC-MS/MS analyses were performed on a Q-Exactive Plus high-resolution mass spectrometer (Thermo Scientific, Inc.) with a nanoAcquity UPLC system (Waters Corp.) and a nano-electrospray ionization source. Samples were trapped on a Symmetry C18 300 mm × 180 µm trapping column for 3 min at 5 µL/min (99.9/0.1 v/v water/acetonitrile 0.1% formic acid), and separated on a 75 µm × 250 mm column packed with 1.7 µm Acquity HSST3 C18 stationary phase (Waters Corp.). Peptides were separated using a gradient of 3 to 30% acetonitrile with 0.1% formic acid over 90 min at a flow rate of 0.4 µL/min. Data collection was performed in a data-dependent acquisition (DDA) mode with a resolution of 70,000 (at m/z 200) for full MS scan from m/z 375–1600 with a target AGC value of 1 × 10⁶ ions, followed by 20 product ion scans at a resolution of 17,500 (at m/z 200), using an AGC target value of 1 × 10⁵ ions, a max fill time of 60 ms, and normalized collision energy of 30 V.