Beh glycan column

Released glycans were fluorescently labeled via reductive amination with 2-aminobenzoic acid (2-AB) and sodium cyanoborohydride in 30% v/v acetic acid in DMSO at 65° C for 3 hours. Excess labeling reagents and reducing agent were removed from the samples using GlycoClean S solid-phase extraction cartridges according to the manufacturer’s instructions. Hydrophilic interaction chromatography (HILIC) separation of 2-AB labeled glycans was carried out using an Agilent 1100 HPLC system coupled to an Agilent HPLC fluorescence (FLD) detector. Separations were performed using Waters BEH Glycan column, 100 mm × 2.1 mm i.d., 2.5 μm amide sorbent, with the column heated to 60° C. The injection volume was 20 μl. All separations were performed using 100 mM ammonium formate, pH 4.5, as solvent A and 100% acetonitrile as solvent B. The gradient conditions were as follows: 0–5 min, 85–75% B, 0.3 ml min^-1; 5–35 min, 75–64% B, 0.3 ml min^-1; 35–40 min, 64–50% B, 0.3 ml min^-1; 40–42 min, 50–50% B, 0.3–0.1 ml min^-1; 42–43 min, 50–10% B, 0.1 ml min^-1; 43–48 min, 10–10% B, 0.1 ml min^-1; 48–50 min, 10–85% B, 0.1 ml min^-1; 50–60 min, 85–85% B, 0.1–0.3 ml min^-1. The fluorescence detector excitation and emission wavelengths were set at 260 and 430 nm, respectively.

Online coupled fluorescence (FLD)-mass spectrometry detection was performed using a Waters Xevo G2 QTof with Acquity UPLC and BEH Glycan column (1.0×150 mm, 1.7 µm particle size). MS data was acquired in negative mode with the following conditions: 2500 V capillary voltage, 50 V cone voltage, 280°C desolvation temperature, 600 L/hour desolvation gas and 100°C source temperature. The analyzer was set to sensitivity mode. The fluorescence data rate was 1 pts/second and a PMT gain = 10 with altering excitation and emission wavelengths that varied based on the experiment and glycan label used. Sample injection volumes were 10 µl and the flow rate was 0.150 µl/min. Solvent A was 50 mM ammonium formate pH 4.4 and solvent B was acetonitrile. A 40 minute linear gradient was used and was as follows: 28–43% A for 32 minutes, 70% A for 4 minutes and 28% solvent A for 4 minutes. Samples were diluted in 65% acetonitrile prior to analysis.

Lyophilised cell line secretome samples, were reconstituted in 3 μl of water and 9 μl acetonitrile. Online coupled fluorescence (FLR)-mass spectrometry detection was performed using a Waters Xevo G2 QTof with Acquity® UPLC (Waters Corporation, Milford, MA, USA) and BEH Glycan column (1.0 × 150 mm, 1.7 μm particle size). For MS acquisition data, the instrument was operated in negative-sensitivity mode with a capillary voltage of 1.80 kV. The ion source block and nitrogen desolvation gas temperatures were set at 120 and 400°C, respectively. The desolvation gas was set to a flow rate of 600 L/h. The cone voltage was maintained at 50V. Fullscan data for glycans were acquired over m/z range of 450–2,500. Data collection and processing were controlled by MassLynx 4.1 software (Waters Corporation, Milford, MA, USA). The fluorescence detector settings were as follows ex = 330 nm, em = 420 nm. Sample injection volume was 8 μL. The flow rate was 0.150 mL/min and column temperature was maintained at 60°C; solvent A was 50 mM ammonium formate in water (pH 4.4) and solvent B was acetonitrile. A 40 min linear gradient was used, and was as follows: 28% (v/v) A for 1 min, 28–43% (v/v) A for 30 min, 43–70% (v/v) A for 1 min, 70% (v/v) A for 3 min, 70–28% (v/v) solvent A for 1 min and finally 28% (v/v) A for 4 min.