Before MS analysis of each glycan peak, the 2-AB labeled IgG N-glycan pool was fractionated by hydrophilic interaction high performance liquid chromatography (HILIC) on a 100 × 2.1 mm i.d., 1.7 μm BEH particles column using a linear gradient of 75–62% acetonitrile with 100 mM ammonium formate, pH 4.4, as solvent A and acetonitrile as solvent B. UltiMate Dual Gradient LC system (Dionex, Sunnyvale, CA) controlled by Chromeleon software and connected to FP-2020 Plus fluorescence detector (Jasco, Easton, MD) was used. To obtain the same separation as with UPLC system, flow was adjusted to 0.3 ml/min and analytical run time was prolonged to 60 min. Collected fractions were dried by vacuum centrifugation and resuspended in water.
Nano-LC-ESI-MS/MS. MS analysis of the collected glycan fractions was performed using an Ultimate 3000 nano-LC system (Dionex/LC Packings, Amsterdam, The Netherlands) equipped with a reverse phase trap column (C18 PepMap 100Å, 5 μm, 300 μm × 5 mm; Dionex/LC Packings) and a nano column (C18 PepMap 100Å, 3 μm, 75 μm × 150 mm; Dionex/LC Packings).
The column was equilibrated at room temperature with eluent A (0.1% formic acid in water) at a flow rate of 300 nL/min. For fractions with disialylated glycans, extra 0.04% of trifluoroacetic acid was added to the eluent A. After injection of the samples, a gradient was applied to 25% eluent B (95% acetonitrile) in 15 min and to 70% eluent B at 25 min followed by an isocratic elution with 70% eluent B for 5 min. The eluate was monitored by UV absorption at 214 nm. The LC system was coupled via an online nanospray source to an Esquire HCTultra ESI-IT-MS (Bruker Daltonics, Bremen, Germany) operated in the positive ion mode. For electrospray (1100–1250 V), stainless steel capillaries with an inner diameter of 30 μm (Proxeon, Odense, Denmark) were used. The solvent was evaporated at 170 °C employing a nitrogen stream of 7 L/min. Ions from m/z 500 to 1800 were registered. Automatic fragment ion analysis was enabled, resulting in MS/MS spectra of the most abundant ions in the MS spectra. Glycan structures were assigned using GlycoWorkbench (41 (link)).
MALDI-TOF-MS. 2-AB labeled glycan fractions were spotted onto an AnchorChip target plate (Bruker Daltonics, Bremen, Germany). Subsequently 1 μl of 5 mg/ml 2,5-dihydroxybenzoic acid in 50% acetonitrile was applied on top of each sample and allowed to dry at room temperature. MALDI-TOF-MS was performed on an UltrafleX II mass spectrometer (Bruker Daltonics). Calibration was performed on a peptide calibration standard. Spectra were acquired in reflector positive mode over the m/z range from 700 to 3500 Da for a total of 2000 shots. Glycan structures were assigned using GlycoWorkbench (41 (link)).
Nano-LC-ESI-MS/MS. MS analysis of the collected glycan fractions was performed using an Ultimate 3000 nano-LC system (Dionex/LC Packings, Amsterdam, The Netherlands) equipped with a reverse phase trap column (C18 PepMap 100Å, 5 μm, 300 μm × 5 mm; Dionex/LC Packings) and a nano column (C18 PepMap 100Å, 3 μm, 75 μm × 150 mm; Dionex/LC Packings).
The column was equilibrated at room temperature with eluent A (0.1% formic acid in water) at a flow rate of 300 nL/min. For fractions with disialylated glycans, extra 0.04% of trifluoroacetic acid was added to the eluent A. After injection of the samples, a gradient was applied to 25% eluent B (95% acetonitrile) in 15 min and to 70% eluent B at 25 min followed by an isocratic elution with 70% eluent B for 5 min. The eluate was monitored by UV absorption at 214 nm. The LC system was coupled via an online nanospray source to an Esquire HCTultra ESI-IT-MS (Bruker Daltonics, Bremen, Germany) operated in the positive ion mode. For electrospray (1100–1250 V), stainless steel capillaries with an inner diameter of 30 μm (Proxeon, Odense, Denmark) were used. The solvent was evaporated at 170 °C employing a nitrogen stream of 7 L/min. Ions from m/z 500 to 1800 were registered. Automatic fragment ion analysis was enabled, resulting in MS/MS spectra of the most abundant ions in the MS spectra. Glycan structures were assigned using GlycoWorkbench (41 (link)).
MALDI-TOF-MS. 2-AB labeled glycan fractions were spotted onto an AnchorChip target plate (Bruker Daltonics, Bremen, Germany). Subsequently 1 μl of 5 mg/ml 2,5-dihydroxybenzoic acid in 50% acetonitrile was applied on top of each sample and allowed to dry at room temperature. MALDI-TOF-MS was performed on an UltrafleX II mass spectrometer (Bruker Daltonics). Calibration was performed on a peptide calibration standard. Spectra were acquired in reflector positive mode over the m/z range from 700 to 3500 Da for a total of 2000 shots. Glycan structures were assigned using GlycoWorkbench (41 (link)).