Nanobooster

The tryptic IgG glycopeptide separation was performed by an Ultimate 3000 HPLC nano-LC system (Thermo Fisher Scientific) as described previously (23 (link)). (Glyco-)peptides (0.25 µL) were concentrated on the trapping column (Acclaim PepMap100 C18, 5 mm x 300 µm i.d., Thermo Fisher Scientific) and separated on the analytical column (nanoEase M/Z Peptide BEH C18, 100 mm x 75 µm i.d., 1.7 µm, 130 Å, 1/PK, Waters) in a linear gradient from 3% to 50% solvent B (95% acetonitrile) in 5.5 min. The separation system was coupled online by electrospray ionization (ESI) to a Maxis Impact HD quadrupole time-of-flight MS instrument (Bruker Daltonics, Bremen, Germany). The sample was nebulized and ionized in ion positive mode in a CaptiveSpray ESI source assisted by ACN-doped nitrogen gas from the nanoBooster (Bruker Daltonics). Mass spectra were acquired within a mass range m/z 550-1800 at a frequency of 1 Hz.

Sample (preparation mentioned above) was injected into an Ultimate 3000 RSLCnano system (Thermo Scientific, Breda, the Netherlands) coupled to a quadrupole-TOF-MS (MaXis HD; Bruker Daltonics, Bremen, Germany). The LC system was equipped with an Acclaim PepMap 100 trap column (particle size 5 μm, pore size 100 Å, 100 μm × 20 mm, Thermo Scientific) and an Acclaim PepMap C18 nano analytical column (particle size 2 μm, pore size 100 Å, 75 μm × 150 mm, Thermo Scientific). The samples were loaded and washed on the trap column for 2 min at 15 μl/min with 0.1% FA in water. A mixture of solvent A (0.1% FA in water) and solvent B (95% acetonitrile, 0.1% FA) was applied with a constant flow of 0.7 μL/min using a linear gradient: t(min) = 0, %B = 1; t = 5, %B = 1; t = 30, %B = 50; with washing and equilibration starting at t = 31, %B = 70; t = 35, %B = 70; t = 36, %B = 1; t = 70, %B = 1. The sample was ionized in positive-ion mode using a CaptiveSprayer (Bruker Daltonics) electrospray source at 1250 V. A nanoBooster (Bruker Daltonics) was used to enrich the nitrogen gas with acetonitrile to enhance ionization efficiency (0.2 bar). Mass spectra were acquired with a frequency of 1 Hz and the MS ion detection window was set at mass-to-charge ratio (m/z) 550–1,800.