Glycosidic linkage analysis

To determine the glycosidic linkages, yeast samples and commercial polysaccharides were subjected to methylation [46], TFA-hydrolysis, reduction and acetylation to generate PMAAs, similar to a previously described method [29] with the following modifications. To start, 100 µL of AKI/EM solutions or 1 mg/mL polysaccharide standards were dried in a glass tube and mixed overnight in 200 µL DMSO, pre-dried using molecular sieves. Solubilized polymers were methylated by using 200 µL of a NaOH/DMSO slurry and 100 µL of methyl iodide, under N₂ atmosphere for 2–3 h. Reactions were quenched by adding 2 mL of water, and N₂ was gently bubbled into each tube until the solution became clear. After adding 2 mL of dichloromethane, ~ 1.5 mL of the organic phase was transferred to a new tube, dried and the methylated polymers were hydrolyzed into monomers using 2 M TFA. Dried monosaccharides, with myo-inositol added as internal standard, were reduced using 200 µL of fresh 10 mg/mL sodium borodeuteride in 1 M ammonium hydroxide for 60 min at room temperature. After neutralization with acetic acid and extensive methanol washes, samples were dried and acetylated with 50 μL acetic anhydride and 50 μL pyridine for 20 min at 120 °C. The resulting PMAAs were then dried, washed twice with 200 μL of toluene, and finally cleaned using 1.2 mL ethyl acetate and 5 mL of water. The organic molecules were dried in new tubes, resuspended in 300 μL acetone, and 2 µL of each PMAA sample was automatically injected for GC–MS using an Agilent Technologies 6890 N GC system equipped with a Supelco SP-2380 column (30 m × 0.25 mm × 0.2 µm) and coupled to an Agilent 5975 quadrupole EI detector. The GC oven started at 80 °C for 3 min, increased to 170 °C (at a rate of 30 °C/min), followed by second ramp to 240 °C (rate of 4 °C/min) and a 15 min hold time per run. PMAAs were semi-automatically quantified in Agilent MSD Chemstation Classic Data Analysis (G1701FA) based on the retention time of the glycosidic linkage peaks from polysaccharide standards, and their relative ion spectra or available data in the CCRC Spectral Database for PMAAs (https://www.ccrc.uga.edu/specdb/ms/pmaa/pframe.html).