Compact mass spectrometer

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.

IgG Fc glycopeptides were separated on an Acquity M-class chromatographic system (Waters, Milford, MA, USA), which was coupled to a Compact mass spectrometer (Bruker, Bremen, Germany) using a CaptiveSpray source equipped with a nanoBooster. Glycopeptides (6 μL) were loaded onto PepMap 100 C8 (5 mm × 300 μm i.d.; Thermo Fisher Scientific, Waltham, MA, USA) in a mobile phase A (0.1 % TFA) at a flow rate of 40 μL/min. IgG subclasses were separated on a C18 analytical column (150 mm × 100 µm i.d., 100 Å; Advanced Materials Technology, Wilmington, DE, USA) in a 3.5-min-long gradient from 16% to 25% of mobile phase B (80% can in 20% solvent A) at a flow rate of 1 μL/min. The column temperature was maintained at 30 °C. Acetonitrile vapors were introduced directly into the source using a nanoBooster to increase the ionization of glycopeptides. Mass spectra were recorded using an m/z range of 600–1900 with 0.5 Hz and averaging two sequential scans. The collision and quadrupole energies were set to 4 eV, while transfer time and pre-pulse storage were set to 110 μs and 10 μs, respectively. The nanoACQUITY UPLC system was controlled by MassLynx software version 4.1 (Waters), while the mass spectrometer was controlled by HyStar software version 4.1.2 (Bruker).

The biosurfactant sample was identified by electrospray ionization (ESI) mass spectrometry (MS) using a Compact Mass Spectrometer (Bruker Daltonics, Bremen, Germany) in the positive ionization mode, using the following settings: capillary voltage 3500 V, nebulizer 1.5 bar, dry gas 8 L min⁻¹, dry temperature 180 °C. Data were collected for 50–3000 m/z. Next, the data obtained were processed with the Compass DataAnalysis 4.2 software package (Bruker, Bremen, Germany).