Acquity uplc beh c18 1.7mm column 2 1 x 5 mm

Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2°C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The full details of the automated LC system are described in (Stariha et al., 2021 (link)). The samples were run over one immobilized pepsin column (Waters; Enzymate Protein Pepsin Column, 300Å, 5μm, 2.1 mm X 30 mm) at 200 μL/min for 3 minutes at 2°C. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7mm column (2.1 x 5 mm); Waters 186003975). The trap was subsequently eluted in line with an ACQUITY 1.7 μm particle, 100 × 1 mm² C18 UPLC column (Waters), using a gradient of 3-35% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 11 minutes immediately followed by a gradient of 35-80% over 5 minutes. Mass spectrometry experiments acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200°C and a spray voltage of 4.5 kV.

Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2 °C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The full details of the automated LC system are described in Stariha et al., 2021 (link). The samples were run over one immobilized pepsin column (Waters; Enzymate Protein Pepsin Column, 300 Å, 5 µm, 2.1 mm X 30 mm) at 200 µL/min for 3 min at 2 °C. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7 mm column (2.1x5 mm); Waters 186003975). The trap was subsequently eluted in line with an ACQUITY 1.7 μm particle, 100×1 mm² C18 UPLC column (Waters), using a gradient of 3–35% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 11 min immediately followed by a gradient of 35–80% over 5 min. Mass spectrometry experiments acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200 °C and a spray voltage of 4.5 kV.