Enzymatically hydrolysed AIR samples (with or without SEC) were desalted by dialysis (as described above), lyophilised and then further desalted by HyperSep Hypercarb cartridges (Thermo-Fisher, http://www.thermofisher.com ). The purified oligosaccharides were reductively aminated with 2-aminobenzoic acid (2-AA; Sigma) using optimised labelling conditions described previously (Ridlova et al., 2008 (link)). Capillary HILIC was carried out using an LC-Packings Ultimate system (Dionex, http://www.dionex.com ) equipped with an amide-80 column (300 μm × 25 cm; 3 μm particle size; Dionex) as previously described (Tryfona et al., 2012 (link)).
2 aminobenzoic acid 2 aa
Manufactured by Merck Group
Sourced in Germany
2-aminobenzoic acid (2-AA) is a chemical compound with the molecular formula C7H7NO2. It is a white crystalline solid that is soluble in water and organic solvents. 2-AA is commonly used as a starting material in the synthesis of various pharmaceutical and agrochemical products.
Automatically generated - may contain errors
Lab products found in correlation
Acetic acid, by Merck Group (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
2 picoline borane, by Merck Group (2 mentions)
2475 fluorescence detector, by Waters Corporation (2 mentions)
Bond elut cn e, by Agilent Technologies (2 mentions)
Hypersep hypercarb cartridges, by Thermo Fisher Scientific (1 mentions)
Lc packings ultimate system, by Thermo Fisher Scientific (1 mentions)
Amide 80 column, by Thermo Fisher Scientific (1 mentions)
Picoline borane, by Merck Group (1 mentions)
Human serum, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
N glycosidase f pngase f, by New England Biolabs (1 mentions)
Pure water, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Endoglycosidase buffer pack, by New England Biolabs (1 mentions)
Methanol, by Merck Group (1 mentions)
Sodium chloride (nacl), by Sinopharm (1 mentions)
Sodium acetate, by Sinopharm (1 mentions)
9 protocols using 2 aminobenzoic acid 2 aa
1
Oligosaccharide Purification and Labeling
2
Glycan Profiling with Mass Spectrometry
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Polyethylene-glycol (200, 600, 1000), acetonitrile, ammonium-hydroxide, acetic acid, formic acid, picoline-borane, 2-aminobenzoic acid (2-AA), and human serum were purchased from Sigma-Aldrich (St. Louis, MO, USA). Iron (II)-oxalate dihydrate (FeC2O4 ·2H2O) was provided by Alfa Aesar (Haverhill, MA, USA). PNGase F was purchased from New England Biolabs (Ipswich, MA, USA). CU (clean-up) cartridges were obtained from Prozyme (Agilent Technologies, Inc. Santa Clara, CA, USA) and normal phase tips were provided by Phynexus (San Jose, CA, USA).
3
Glycoprotein Extraction and Analysis
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Sodium hydroxide beads (20–40 mesh), porous-graphitized carbon (PGC), 2,5-dihydroxybenzoic acid (DHB) and 2-aminobenzoic acid (2-AA) were purchased from Sigma-Aldrich (MO, U.S.A.). N-glycosidase F (PNGase F) and endoglycosidase buffer pack (EBP) were purchased from New England Biolabs (MA, U.S.A.). Acetonitrile, (ACN) and methanol were from Merck KGaA (Darmstadt, Germany). Formic acid (FA) and pure water were obtained from Thermo Fisher Scientific (MA, U.S.A.). Trifluoroacetic acid (TFA), chloroform, sodium acetate and sodium chloride were from Sinopharm Chemical Reagent (Shanghai). Dimethyl sulfoxide (DMSO) was purchased from Aladdin Industrial Corporation (Shanghai). Iodomethane was obtained from Ai Keda Chemical Technology (Chengdu). Empty spin column and sample tube were purchased from Harvard Apparatus (U.S.A.). Human serum samples, including 29 HCC cases and 17 healthy controls, were donated by Tongji Hospital (Tongji Medical College, Huazhong University of Science and Technology). The study was carried out in accordance with the Helsinki declaration and informed consents were obtained from the participants in accordance with the study protocols approved by the Ethics Committee of Huazhong University of Science and Technology.
4
Oligosaccharide Labeling and Purification
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For labelling, aliquots containing ~125 μg of carbohydrates for mixtures or ~0.3 μmol for pure oligosaccharides were dried under vacuum and labelled as described elsewhere [38 (link)]. The labelling was performed in a solution of 30% (v/v) acetic acid in DMSO containing 0.35 M 2-aminobenzoic acid (2-AA; Sigma-Aldrich Chemie, GmbH, Steinheim, Germany) and 1 M 2-picoline borane (Sigma-Aldrich). The mixture was incubated at 65 °C for 2 h.
Excess label and reducing agent were removed using a modification of a cleaning procedure described elsewhere [39 ]. The sample was cooled to room temperature, mixed with 50 μL water and 900 μL acetonitrile, then loaded onto a Bond-Elut cellulose SPE column (Agilent, Santa Clara, CA, USA) that was preconditioned with 4 mL water and washed with 6 mL 90%(v/v) acetonitrile. After sample loading, the column was washed with 6 mL 90% (v/v) acetonitrile to remove excess label. Labelled oligosaccharides were eluted with three aliquots of 500 μL water, dried under vacuum and dissolved in 100 μL water. The sample was diluted ten times before analysis with UHPLC-MSn.
Excess label and reducing agent were removed using a modification of a cleaning procedure described elsewhere [39 ]. The sample was cooled to room temperature, mixed with 50 μL water and 900 μL acetonitrile, then loaded onto a Bond-Elut cellulose SPE column (Agilent, Santa Clara, CA, USA) that was preconditioned with 4 mL water and washed with 6 mL 90%(v/v) acetonitrile. After sample loading, the column was washed with 6 mL 90% (v/v) acetonitrile to remove excess label. Labelled oligosaccharides were eluted with three aliquots of 500 μL water, dried under vacuum and dissolved in 100 μL water. The sample was diluted ten times before analysis with UHPLC-MSn.
5
Structural Analysis of N-Glycans from Cryptococcus
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cwMPs were isolated from C. neoformans cells as described previously (30 (link), 53 (link)). N-linked glycans were released from the purified cwMPs using PNGase F (New England Biolabs) and were purified over a Carbograph Extract-Clean (Grace) column. The N-glycans were labeled with 2-aminobenzoic acid (2-AA; Sigma) and purified using a Cyano Base cartridge (Bond Elut-CN-E; Agilent) (100 mg) to remove excess 2-AA. Purified N-glycans were reacted with α-1,2 mannosidase (α-1,2 MNS; Prozyme) and subsequently with α-1,6 mannosidase (α-1,6 MNS; New England Biolabs). To remove enzymes, the N-glycans were purified using a 30K Microcon device (Millipore). 2-AA-labeled oligosaccharides were analyzed with a Waters 2690 HPLC system and a 2475 fluorescence detector with excitation and emission wavelengths of 360 nm and 425 nm, respectively. Data were collected using Empower 2 software (Waters). For MALDI-TOF analysis, the matrix solution was prepared as previously described (53 (link)) and was mixed with samples of equal volume. Mixed glycan samples were spotted on a MSP 96 polished-steel target (Bruker Daltonics). Crystallized samples were analyzed using a Microflex mass spectrometer (Bruker Daltonics).
6
O-Glycan Release and Analysis from Cell Wall Mannoproteins
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For release of the O-glycans, the cell wall mannoproteins (cwMPs) were prepared as described in previous studies (31 (link), 59 (link)). Briefly, the completely dried cwMPs (50 μg) were resuspended in 100 μL of hydrazine monohydrate (Tokyo Chemical Industry), and the mixture was incubated at 60°C for 4 to 6 h. The reactants were dried to remove the hydrazine monohydrate, and the pellets were dissolved in 100 μL of saturated NaHCO3 (Sigma), mixed with 10 μL of (CH3CO)2O, and incubated on ice for 30 min without shaking. The O-glycans were purified by using Dowex 50WX8-400 resins (H+ form; Sigma), and the isolated O-glycans were labeled with 2-aminobenzoic acid (2-AA; Sigma) and purified using a cyano base cartridge (Bond Elut-CN-E; Agilent) (100 mg). The HPLC analysis of the 2-AA-labeled O-glycan was conducted on a TSKgel Amide-80 column (0.46 by 25 cm, 5 μm; Tosoh, Tokyo, Japan) at a flow rate of 1.0 mL/min. 2-AA-Oligosaccharides were detected with a 2475 fluorescence detector (Waters) at excitation and emission wavelengths of 360 and 425 nm, respectively. Data were collected using Empower 2 chromatography data software (Waters).
7
Glycoprotein Sample Preparation Protocol
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Ammonium formate was purchased from Fluka
(Steinheim, Germany). Acetic acid, 2-aminobenzoic acid (2-AA), 2-picoline
borane (2-PB), ammonium acetate, ammonium bicarbonate (ABC), bovine
serum albumin (BSA), cesium iodide, cytochrome C (cyt C), dimethyl
sulfoxide (DMSO), disodium hydrogen phosphate dihydrate, Tris (2-carboxyethyl)phosphine
hydrochloride (TCEP), chloroacetamide (CAA), dithiothreitol (DTT),
iodoacetamide (IAA), nonidet P-40 substitute (NP-40), potassium dihydrogen
phosphate, sodium chloride, super-DHB, and urea were obtained from
Sigma-Aldrich (Steinheim, Germany). Formic acid (FA) and 2-propanol
(IPA) were supplied by Riedel-De Haen (Seelze, Germany). The HPLC-grade
acetonitrile (ACN) was purchased from Biosolve (Valkenswaard, The
Netherlands). TrifluoroAcetic acid (TFA) was obtained from Merck (Darmstadt,
Germany). Recombinant Peptide-N-Glycosidase F (PNGase F) was purchased
from Roche Diagnostics or Promega. Milli-Q water was provided by Purelab
ultra (ELGA Labwater, Ede, The Netherlands). EndoPro purified fromAspergillus nigerwas provided by DSM formulated in
50% glycerol.
(Steinheim, Germany). Acetic acid, 2-aminobenzoic acid (2-AA), 2-picoline
borane (2-PB), ammonium acetate, ammonium bicarbonate (ABC), bovine
serum albumin (BSA), cesium iodide, cytochrome C (cyt C), dimethyl
sulfoxide (DMSO), disodium hydrogen phosphate dihydrate, Tris (2-carboxyethyl)phosphine
hydrochloride (TCEP), chloroacetamide (CAA), dithiothreitol (DTT),
iodoacetamide (IAA), nonidet P-40 substitute (NP-40), potassium dihydrogen
phosphate, sodium chloride, super-DHB, and urea were obtained from
Sigma-Aldrich (Steinheim, Germany). Formic acid (FA) and 2-propanol
(IPA) were supplied by Riedel-De Haen (Seelze, Germany). The HPLC-grade
acetonitrile (ACN) was purchased from Biosolve (Valkenswaard, The
Netherlands). TrifluoroAcetic acid (TFA) was obtained from Merck (Darmstadt,
Germany). Recombinant Peptide-N-Glycosidase F (PNGase F) was purchased
from Roche Diagnostics or Promega. Milli-Q water was provided by Purelab
ultra (ELGA Labwater, Ede, The Netherlands). EndoPro purified fromAspergillus nigerwas provided by DSM formulated in
50% glycerol.
8
Glycan Labeling and Purification
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The AG oligosaccharides were reductively aminated with 2-aminobenzoic acid (2-AA, Sigma) or 12C6- and 13C6-aniline (Sigma) using optimised labelling conditions described previously [29] (link). The saccharides were then purified from the reductive amination reagents using a Glyko Clean S cartridge (Prozyme, San Leandro, CA) as described previously [48] (link). When isotopic labelling was performed samples labelled with the two aniline isotopes were mixed equally prior to purification from the reductive amination reagents.
9
N-Glycan Analysis by HILIC-UHPLC-MS
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N-linked glycans (N-glycans) were evaluated by glycan map analysis using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. N-glycans were released by treatment with PNGase F. The reducing termini of the released glycans were then labeled through reductive amination with a fluorescent tag, 2 aminobenzoic acid (2-AA, Sigma Aldrich). Following incubation at 80°C, precipitated proteins were pelleted and the supernatant, containing released and labeled glycans, was transferred to a new vial. The labeled glycans were separated by HILIC-UHPLC on a BEH Glycan column (2.1 × 150 mm, 1.7 μm; Waters Corporation), using a 100 mM ammonium formate, pH 3.0 and acetonitrile gradient. The gradient was 22% 100 mM ammonium formate to 40% over 111 min. Peak identification was performed using MS by coupling the HILIC UHPLC with an ion-trap MS for verification against the expected glycan mass.
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