Digested glycopeptides were separated on nanoAcquity chromatographic system (Waters, Milford, MA) coupled to Compact mass spectrometer (Bruker, Bremen, Germany) with an electrospray ionization (ESI) source. Samples were loaded either directly after the overnight trypsin digestion (2 μL from 20 μL) or after the enrichment procedure (20 μL). They were loaded onto a PepMap 100 C18 trap column (5 mm x 300 μm, Thermo Fisher Scientific) at a flow rate of 40 μL/min of solvent A (0.1% formic acid) to wash off impurities and salts. Glycopeptides were separated on C18 analytical column (150 mm x 100 μm, 100 Å, Advanced Materials Technology) using a linear gradient from 0% to 80% of solvent B (80% ACN) in solvent A, at a flow rate of 1 μL/min in a 90-minute analytical run.

Fragmentation of glycopeptides was performed by tandem MS/MS by using CaptiveSpray interface, where nanoBooster was used to introduce gaseous acetonitrile into nitrogen flow. The mass spectrometer operated in positive ion mode; capillary voltage was set to 1300 V, nitrogen pressure was set to 0.2 bar, and the drying gas to 4.0 l/min at 150°C. Auto MS/MS method was used by selecting three precursor ions and exclusion criteria after three MS/MS spectra. Mass range was from 50 m/z to 4000 m/z, with a spectra rate of 1 Hz. Transfer time was from 70 μs to 150 μs, and pre-pulse storage was 12 μs.

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