Automated Glycan Derivatization and Analysis

The automated sample preparation consists of derivatization, hydrophilic interaction liquid chromatography (HILIC) purification, and MALDI-target plate spotting which was performed using an automated liquid handling platform.⁴⁰ (link) Ethyl esterification, for stabilization and linkage-specific derivation of sialic acids, was performed with freshly prepared chemicals and solutions. Therefore, 2 μL of the released glycan samples was added to 40 μL of ethyl esterification reagent, which consisted of 0.25 M EDC and 0.25 M HOBt dissolved in 100% ethanol, followed by incubation for 1 h at 37°C.⁸⁸ (link) Subsequently, 40 μL of acetonitrile was added. For the purification of the N-glycans, in-house assembled cotton HILIC microtips were used (approx. 3 mm or 180 μg cotton thread per tip). The tips were pre-washed with MQ water and 85% acetonitrile. Then, the glycans were bound to the cotton by pipetting the samples up and down 20 times. The tips were washed with 85% acetonitrile with 1% TFA followed by 85% acetonitrile and eluted in 20 μL MQ water. Subsequently, 7 μL of the purified sample plus 7 μL of sDHB matrix (2.5 mg/mL in 50% ACN with 0.1 mM NaOH)⁸⁹ (link) was premixed in a 384-well plate. Then, 2 μL of the mixture was spotted onto a MALDI target plate (800/384 MTP AnchorChip, Bruker Daltonics, Bremen, Germany), and after air-drying the spots were measured by MALDI-FTICR-MS.