2-AB labeled N-glycans, or native N-glycans enriched in H10N2-H12N2 species, were treated with purified [21 ] jack bean α-mannosidase (Sigma-Aldrich), as described previously [6 (link)].
Jack bean α mannosidase
Manufactured by Merck Group
Jack bean α-mannosidase is an enzyme that catalyzes the hydrolysis of terminal, non-reducing α-D-mannose residues in α-D-mannosides. This enzyme is commonly used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
α galactosidase from green coffee beans, by Merck Group (3 mentions)
Recombinant β galactosidase, by Merck Group (2 mentions)
Recombinant xanthomonas manihotis α1 2 3 mannosidase, by New England Biolabs (2 mentions)
α l fucosidase from bovine kidney, by Merck Group (2 mentions)
Xanthomonas, by New England Biolabs (1 mentions)
Maldi tof ms, by Merck Group (1 mentions)
Endoh, by Roche (1 mentions)
β galactosidase from bovine testes, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
β galactosidase, by Merck Group (1 mentions)
Orbitrap exactive instrument, by Thermo Fisher Scientific (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Avance 3 hd 400, by Bruker (1 mentions)
P 2000 polarimeter, by Jasco (1 mentions)
11 protocols using jack bean α mannosidase
1
Enzymatic N-glycan Demannosylation
2
Enzyme Characterization Protocol
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Jack bean α-mannosidase and green coffee beans α-galactosidase were obtained from Sigma Aldrich, β-galactosidase from Calbiochem, Xanthomonas manihotis α-1,6-mannosidase from New England Biolabs and Aspergillus saitoi α-1,2-mannosidase from Prozyme. Protocols for their use can be found in the SI, along with details of acid and phosphatase reactions used to assess phosphate esters/diesters.
3
Glycan Characterization by Mass Spectrometry
4
Exoglycosidase-Mediated Glycan Analysis
5
Deglycosylation of Pancreatic Membrane Proteins
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Proteins in pancreatic membrane fractions were denatured by incubation at 80°C for 5 minutes in the presence of 0.5% sodium dodecyl sulfate, and then treated with peptide:N-glycosidase (PNGase) F from Flavobacterium meningosepticum (New England BioLabs, Ipswich, MA) or with endonuclease H from Streptomyces plicatus (Glyko, ProZyme, Hayward, CA) at 37°C for 1 or 3 hours, respectively, according to the manufacturers’ protocols. Incubation with Jack bean α-mannosidase (Sigma-Aldrich) was performed in 25 mM sodium acetate buffer (pH 5.0) containing 1% Triton X-100 at 37°C for 3 hours.
6
Exoglycosidase-Assisted Glycan Analysis
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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.
7
Deglycosylation of Pancreatic Membrane Proteins
8
Insect N-Glycome Analysis by MALDI-TOF MS
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Sand fly salivary glycans were released according to previous procedures and labelled with PA (aminopyridine) as described elsewhere75 (link), prior to RP-HPLC and analysis by MALDI-TOF MS using a Bruker Daltonics Autoflex Speed instrument (Hykollari). Aliquots of samples were treated with Jack bean α-mannosidase (Sigma), α-1,3 mannosidase and 48% aqueous hydrofluoric acid (aq.HF); the latter under control conditions releases phospho(di)esters, phosphonate, α1,3-fucose and galactofuranose groups. Dried glycan fractions were redissolved in 3 μL of aq.HF on ice (in the cold room) for 36 h prior to repeated evaporation. The digests were re-analysed using MALDI-TOF MS and MS/MS. Spectra were annotated by comparison to previous data on insect N-glycomes in terms of monosaccharide composition (Fx Hy Nz), using retention time, manual interpretation, exoglycosidase treatment results and LIFT fragmentation analysis.
9
Enzymatic Modification of EV Glycans
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Five to fifty units (determined empirically for each enzyme) of exo-glycosidase (Jack bean α-mannosidase, Sigma-Aldrich; β-galactosidase from bovine testes, Sigma-Aldrich), endo-glycosidase (endo-β-N-acetylglucosaminidases Endo Tv [21 ] and Endo H, Roche), and glyco-amidase (N-glycoamidase F, PNGase F) were used to modify the surface glycosylation of the EVs. Each enzyme was added to 1 μg of PKH26-labelled EVs (total protein content) in 200 mM sodium phosphate buffer including protease inhibitors (Roche) at pH 4.5 for α-mannosidase and β-galactosidase, pH 5.5 for endoglycosidases and pH 7.4 for glyco-amidase treatment. The reactions, and enzyme-free negative controls, were incubated in the dark for 14 h at 37°C. In parallel, the activity of β-galactosidase and PNGase F were verified by digestion of bovine fetuin under the same conditions used for the EVs. Similarly, the activity of Endo H, Endo Tv and α-mannosidase were verified by digestion with bovine RNAse B.
10
Glycan Analysis via Exoglycosidase Digestion
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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
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