Bond elut cn e

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).

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).