Efficient Glycan Release and Labeling

The samples were immobilized in gels and N-glycans were removed from all samples using PNGase F [20 (link)]. O-glycans were released using microwave-assisted non-reductive β-elimination and labelled with 2-aminobenzamide (2AB) (75%) and with reductive β-elimination (25%) as described in Wilkinson et al [21 (link)].
Microwave-assisted non-reductive β-elimination was performed as described in Wilkinson et al. [21 (link)]. Briefly, the gels containing O-glycans were crushed, lyophilized, and resuspended in 250 μL 40% dimethylamine in water with 0.1 g/mL ammonium carbonate. Samples were incubated in a Monowave 450 microwave reactor at 600W (Anton Paar) under 12-minute microwave radiation at 70°C. Samples were then neutralized with 1M HCl, desalted using a Hypersep Hypercarb SPE cartridge, lyophilized and fluorescently labelled with 2AB [22 (link)]. The label excess was removed by a Hypersep Diol SPE cartridge [23 (link)].
Reductive β-elimination and desalting was performed according to Schulz et al [24 (link)] and modified for an in-gel release [25 (link)].