Elute uplc

Modification of intact glycoprotein targets was determined by LC-MS by injection of 5 µl (or about 5 pmol) of His-tag purified, dialyzed glycoprotein into a Bruker Elute UPLC equipped with an ACQUITY UPLC Peptide BEH C4 Column, 300 Å, 1.7 µm, 2.1 mm × 50 mm (186004495 Waters Corp.) with a 10 mm guard column of identical packing (186004495 Waters Corp.) coupled to an Impact-II UHR TOF Mass Spectrometer (Bruker Daltonics, Inc.). Before injection, Fc samples were reduced with 50 mM DTT. Liquid chromatography was performed using 100% H₂O and 0.1% formic acid as Solvent A and 100% acetonitrile and 0.1% formic acid as Solvent B at a flow rate of 0.5 mL/min and a 50 °C column temperature. An initial condition of 20% B was held for 1 min before elution of the proteins of interest during a 4 min gradient from 20 to 50% B. The column was washed and equilibrated by 0.5 min at 71.4% B, 0.1 min gradient to 100% B, 2 min wash at 100% B, 0.1 min gradient to 20% B, and then a 2.2 min hold at 20% B, giving a total 10 min run time. An MS scan range of 100–3000 m/z with a spectral rate of 2 Hz was used. External calibration was performed prior to data collection.

For metabolite analysis, UPLC/MS was performed using an Impact II qTOF mass spectrometer (Bruker, Billerica, MA, USA) coupled to an Elute UPLC (Bruker) chromatographic system. Chromatographic separation was carried out on a Phenomenex Kinetex column XB-C18 (100 × 2.10 mm 2.6 μm particle size) kept at 40 °C and the binary solvent system consisted of solvent A (H₂O + 0.1% formic acid) and solvent B (acetonitrile + 0.1% formic acid). Flow rate was 600 μL/min. The column was equilibrated with 99% A and 1% B. During the first minute of chromatography, solvent B reached 5%. Then a linear gradient from 5% B to 40% B in 5 min allowed the separation of the compounds of interest. The column was then washed at 100% B for 1.5 min and re-equilibrated to 1% B. Injection volume was 2 μL. Mass spectrometry was performed in negative ion mode with a scan range m/z 100–1000. The mass spectrometer was calibrated using sodium formate. The source settings were the following: capillary voltage 3.5 kV, nebulizer 2.5 Bar, dry gas 11.0 L/min, dry temperature 250 °C. Data analysis was performed using the Bruker Data Analysis Metaboscape (Version 2021-B) software.