최진기 (충남대학교)

안현주 (충남대학교)

최진기 (충남대학교)

김정원 (충남대학교)

오명진 (충남대학교)

김진우 (충남대학교)

안현주 (충남대학교)

Chondroitin sulfate proteoglycans (CSPGs) are major extracellular matrix components in the central nervous system, and their functions are associated with chondroitin sulfate (CS) sulfation patterns. Selective quantification of CS disaccharide isomers is needed to define sulfation-dependent changes in neural tissues but remains challenging because positional sulfation isomers have identical compositions and similar chromatographic and fragmentation behaviors. Disulfated CS analysis is further limited by in-source sulfate loss and matrix-induced ion suppression. Although our previous LC–MRM/MS approach enabled simultaneous analysis of seven CS isomers, the sensitivity for disulfated species remained insufficient. Here, we optimized a negative-mode LC–MRM/MS platform for disulfated CS disaccharides in mouse brain tissue by integrating matrix cleanup, chromatography, and MS optimization. MWCO filtration, solid-phase extraction, and ethanol precipitation were evaluated to reduce matrix interference. After enzymatic depolymerization and 2-AB labeling, mobile-phase, source, and MRM conditions were optimized to improve separation, precursor-ion stability, and selectivity. The optimized platform increased signal intensity and signal-to-noise ratio by approximately 1.5- and 1.6-fold, respectively,  enabling reliable quantification in mouse brain tissue. Our sensitive platform serves as a key tool for investigating CS sulfation dynamics associated with neuropathological conditions.