In general, a 1 μL aliquot of
a HPLC fraction was mixed with
0.2 μL of exoglycosidase and 0.8 μL of 100 mM ammonium
acetate solution, pH 5.0 (except pH 6.5 in the case of the microbial
α1,2-fucosidase); after an overnight incubation at 37 °C,
0.5 μL aliquot of the mixture was analyzed by MALDI-TOF MS.
Exoglycosidases employed were: α-galactosidase from green coffee
beans (Sigma, 11 mU), recombinant β-galactosidase from Aspergillus niger [144 μU28 (link)], recombinant FDL β1,2-N-acetyl-glucosaminidase
[0.2 μU; specific for the nonreducing terminal GlcNAc on the
α1,3-arm29 (link)], jack bean α-mannosidase
(Sigma-Aldrich, 6.25 mU), and recombinant Xanthomonas manihotis α1,2/3- and α1,6-specific mannosidases [New England
Biolabs, 6–8 U30 (link)]. Also, digestions
were attempted with α-l -fucosidases from bovine kidney
(Sigma-Aldrich, 10 mU), Xanthomonas (α1,2-specific;
New England Biolabs, 4 mU), Corynebacterium (α1,2-specific;
Takara, 4 μU), or microbial (α1,2-specific E-FUCM; kind
gift of Megazyme). For the removal of α1,2/3-linked fucose or
methylfucose, glycan samples were dried in a Speed-Vac and then incubated
with 3 μL of 48% (w/v) hydrofluoric acid (HF) on ice for 24
h. The HF was allowed to evaporate overnight. Chemically or enzymatically
treated glycans were reanalyzed by MALDI-TOF MS and MS/MS without
further purification.
a HPLC fraction was mixed with
0.2 μL of exoglycosidase and 0.8 μL of 100 mM ammonium
acetate solution, pH 5.0 (except pH 6.5 in the case of the microbial
α1,2-fucosidase); after an overnight incubation at 37 °C,
0.5 μL aliquot of the mixture was analyzed by MALDI-TOF MS.
Exoglycosidases employed were: α-galactosidase from green coffee
beans (Sigma, 11 mU), recombinant β-galactosidase from Aspergillus niger [144 μU28 (link)], recombinant FDL β1,2-N-acetyl-glucosaminidase
[0.2 μU; specific for the nonreducing terminal GlcNAc on the
α1,3-arm29 (link)], jack bean α-mannosidase
(Sigma-Aldrich, 6.25 mU), and recombinant Xanthomonas manihotis α1,2/3- and α1,6-specific mannosidases [New England
Biolabs, 6–8 U30 (link)]. Also, digestions
were attempted with α-
(Sigma-Aldrich, 10 mU), Xanthomonas (α1,2-specific;
New England Biolabs, 4 mU), Corynebacterium (α1,2-specific;
Takara, 4 μU), or microbial (α1,2-specific E-FUCM; kind
gift of Megazyme). For the removal of α1,2/3-linked fucose or
methylfucose, glycan samples were dried in a Speed-Vac and then incubated
with 3 μL of 48% (w/v) hydrofluoric acid (HF) on ice for 24
h. The HF was allowed to evaporate overnight. Chemically or enzymatically
treated glycans were reanalyzed by MALDI-TOF MS and MS/MS without
further purification.
