Beh c18 1.7 m

Reversed phase chromatography was performed using Waters AQUITY Ultra Performance LC system (Milford, MA, USA), with an analytical column BEH C18 1.7 µm, (2.1 × 100 mm, beds diameter: 1.7 µm). The column was heated to 40 °C and eluted with a gradient of solvents from 99% A and 1% B to 1% A and 99% B, where: A: water, with 0.1% formic acid; B: acetonitrile, with 0.1% formic acid.
The flow rate of the mobile phase was 0.4 mL/min. The sample volume was 5–10 µL. Samples were dissolved in 50% acetonitrile and were injected using AQUITY autosampler.
ESI-MS spectrometry was performed with SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) operating in positive ion mode. Acquisition of the data were performed at a range of 100–2000 m/z, using MassLynx software, version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). Mass spectra were assigned with a multi-point external calibration using sodium iodide (Sigma-Aldrich, St. Louis, MO, USA). Mass spectrometer conditions were as follows: capillary voltage: 3.00 kV, sampling cone: 40 V, source offset: 80 V. Ion source temperature was established at 100 °C and desolvation temperature: 200 °C. Cone gas flow was set at 100 L/h and desolvation gas flow—800 L/h.

Chromatographic separation of tryptic peptides (~3.5 µg) was carried out on an UltiMate3000 Rapid Separation system (U3000 RSLC, Thermo Fisher Scientific, Germering, Germany) using a C18 column (BEH C18 1.7 µm, 2.1 × 150 mm; Waters, Manchester, UK) and the eluate online analyzed with an Orbitrap Fusion electrospray mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The chromatography was carried out using a gradient from 1 to 40% solvent B in 110 min at a flow rate of 0.3 mL/min and temperature of 65 °C. UV absorption was monitored at a wavelength of 220 nm.
Data parameters for MS and MS/MS detection were adjusted according to general experience available from peptide analysis of recombinant antibodies. MS/MS experiments were performed on-line on an Orbitrap Fusion instrument (Thermo) performing a full scan acquisition (in the Orbitrap), followed by a MS/MS scan of the top five most intense ions of each full scan (in the Ion Trap) using helium as collision gas (low-energy CID). The collision energy was adjusted according to stability and mass of the parent ion. MS/MS-data were analyzed manually using PEAKS software for mass detection and data interpretation.