Q tof xevo g2s mass spectrometer

For the top-down technique, the LC-ESI-MS/MS experiments were carried out with the same Waters Q-TOF Xevo G2S mass spectrometer but an RP-UPLC Acquity column. Elution of the reduced/alkylated peptide sample was performed at a flow rate of 0.8 mL/min with a similar gradient as before and identical buffers. Peptide sample acquisition and analyses were performed in the positive mode using a m/z 50–2000 mass range for the Agilent MassLynx software version 4.1 (Waters, Guyancourt, France). Settings for MS analyses and external calibration remained unchanged. Acquisition of MS/MS data was performed by fragmenting the parent ions in the CID activation mode. The resulting MS/MS data were subject to manual interpretation.

The peptides were analysed by HPLC and high-resolution ESI-MS mass spectrometry. HPLC analyses were carried out on an Agilent system equipped with a Chromolith^® High Resolution RP-18e column (150 Å, 100 × 4.6 mm) at a flow rate of 3 mL/min, with a XSelect Peptide CSH C18 column (130 Å, 2.5 μm, 150 × 4.6 mm) or with an Agilent AdvanceBio Peptide column (2.7 μm, 100 × 2.1 mm or 250 × 2.1 mm) at a flow rate of 1 mL/min and 214 nm. Solvents A and B were 0.1% TFA in H₂O and 0.1% TFA in MeCN. The acquisition of the LC-ESI-MS data was carried out on a Waters Q-TOF Xevo G2S mass spectrometer with an Acquity UHPLC system and Lockspray source equipped with a Waters Acquity UPLC BEH300 C18 column (1.7 μm, 2.1 × 150 mm). Peptide elution was performed at a flow rate of 0.4 mL/min with a 10–70% gradient of buffer B over 10 min. Solvents A and B were 0.1% formic acid (FA) in H₂O and 0.1% FA in MeCN. All calculated and found masses were monoisotopic. Purification of the peptides was performed by HPLC on a preparative Agilent 1260 system equipped with a Phenomenex Jupiter column (4 µm Proteo 90 Å, C12, 250 × 4.6mm) at a flow rate of 20 mL/min or a semi-preparative Agilent 1260 system equipped with an Agilent Poroshell EC-C18 column (120 Å, 4 µm, 250 × 9.4 mm) at a flow rate of 4.5 mL/min.