Glycan Characterization by Enzymatic Digestion

Glycans were treated, prior to re-analysis by MALDI-TOF–MS, with α-fucosidase (bovine kidney from Sigma-Aldrich), α-mannosidase (jack bean from Sigma), β-glucuronidases (E. coli from Megazyme or Helix pomatia from Sigma; desalted and concentrated ten-fold with a centrifugal device with a 10 kDa molecular weight cut-off before use) or β-N-acetylhexosaminidases (jack bean from Sigma-Aldrich, Xanthomonas manihotis from New England Biolabs, Streptomyces plicatus chitinase from New England Biolabs or in-house-produced recombinant forms of Caenorhabditis elegans HEX-4 specific for β1,4-GalNAc-linked residues or Apis mellifera FDL specific for the β1,2-linked product of GlcNAc-transferase I^{19 (link)}) in 50 mM ammonium acetate, pH 5, at 37 °C overnight (except for pH 6.5 in the case of HEX-4, or pH 7 in the case of E. coli β-glucuronidase or an incubation time of only 3 h in the case of FDL or <2 h for H. pomatia β-glucuronidase). Hydrofluoric acid was used for removal of core or antennal α1,3-fucose or of phosphorylcholine^{17 (link)}. As appropriate, treated glycans were re-chromatographed by RP-amide HPLC to ascertain retention time shifts prior to MALDI-TOF–MS. See also Supplementary Note 2 for discussion of glycosidase specificities and Supplementary Figure 23 for the HEX-4 and chitinase sensitivity of defined disaccharide conjugates.