In general,
a 1 μL aliquot of an HPLC fraction was mixed
with 0.2 μL of exoglycosidase and 0.8 μL of 100 mM ammonium
acetate solution, pH 5.0, and incubated overnight. Exoglycosidases
employed were: α-galactosidase from green coffee beans (Sigma),
β-galactosidase from either Aspergillus niger(33 (link)) (recombinant, produced in-house) or Aspergillus oryzae (native; Sigma), jack bean α-mannosidase
(Sigma), recombinant Xanthomonas manihotis α1,2/3-mannosidase34 (link) (New England
Biolabs), and α-L-fucosidase from bovine kidney (Sigma). For
removal of α1,2/3-linked fucose or methylfucose, glycan samples
were dried in a SpeedVac, and then incubated with 3 μL of 48%
(w/v) hydrofluoric acid (HF) on ice for 24 h. The HF was immediately
removed in a SpeedVac. Chemically or enzymatically treated glycans
were generally reanalyzed, unless otherwise stated, by MALDI-TOF MS
and MS/MS without further purification. For further details refer
to theSupporting Information .
a 1 μL aliquot of an HPLC fraction was mixed
with 0.2 μL of exoglycosidase and 0.8 μL of 100 mM ammonium
acetate solution, pH 5.0, and incubated overnight. Exoglycosidases
employed were: α-galactosidase from green coffee beans (Sigma),
β-galactosidase from either Aspergillus niger(33 (link)) (recombinant, produced in-house) or Aspergillus oryzae (native; Sigma), jack bean α-mannosidase
(Sigma), recombinant Xanthomonas manihotis α1,2/3-mannosidase34 (link) (New England
Biolabs), and α-L-fucosidase from bovine kidney (Sigma). For
removal of α1,2/3-linked fucose or methylfucose, glycan samples
were dried in a SpeedVac, and then incubated with 3 μL of 48%
(w/v) hydrofluoric acid (HF) on ice for 24 h. The HF was immediately
removed in a SpeedVac. Chemically or enzymatically treated glycans
were generally reanalyzed, unless otherwise stated, by MALDI-TOF MS
and MS/MS without further purification. For further details refer
to the
