Quantification of CS/DS Disaccharides in Mouse Brain

CS/DS disaccharides were isolated and quantified according to our previously published methods (Alonge et al., 2019 (link), 2020 (link); Logsdon et al., 2021 (link)). Five PFA-fixed coronal brain sections (30 μm) from each mouse were chosen for CS/DS disaccharide extraction and quantification. Sections were washed 3× in Optima LC/MS-grade water and 1× in 50 mM ammonium bicarbonate (pH 7.6) at RT. Chondroitinase ABC enzyme (ChABC, Sigma, C3667), a combinatorial endo- and exo-lyase that selectively degrades all CS/DS-GAG chains into their individual disaccharide units (Yamagata et al., 1968 (link); Hamai et al., 1997 (link); Prabhakar et al., 2009 (link); Spliid et al., 2021 (link)), was reconstituted (500 mU/ml) in 50 mM ammonium bicarbonate (pH 7.6) to digest CS/DS-GAGs at 37°C in a Thermo Scientific MaxQ4000 orbital shaker for 24 h. Supernatants were collected in sterile 1.7 ml microcentrifuge tubes and spun for 10 m at 14k× g to pellet any debris. The supernatant was then dehydrated using a SpeedVac Concentrator, and the lyophilized product was reconstituted in 30 μl of LC/MS-grade water.