Quantitative analysis of intact glycosaminoglycans in cerebrospinal fluid of stroke patient using LC-Q-TOF MS coupled with the MSALL technique

Glycosaminoglycans (GAGs), linear polysaccharides with varying degrees of sulfation, are key structural components of the endothelial glycocalyx. The endothelial glycocalyx helps maintain the integrity of the blood-brain barrier (BBB); however, its degradation or shedding contributes to BBB dysfunction. Understanding the relationship between GAG levels and BBB dysfunction requires a robust, high-throughput strategy for GAG quantification. In previous studies using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), GAGs were typically enzymatically or chemically depolymerized into disaccharides before analysis. Intact GAGs are characterized by heterogeneous molecular weights, diverse sulfation patterns, and multiple charge states, making direct MS analysis challenging. To overcome these challenges, we employed a MS^ALL-based liquid chromatography-triple-quadrupole/time-of-flight mass spectrometry (LC-Q-TOF MS) technique. To our knowledge, this is the first quantification of intact GAGs by LC-MS/MS without chemical or enzymatic digestion. Additionally, a unique sample preparation method, involving concentration followed by phenol and dichloromethane extraction, was developed to overcome the electrostatic interactions between GAGs and proteins. Method validation demonstrated a linear detection range for GAGs at concentrations of 50-5000 ng/mL with limit of detection (LOD) of 20 ng/mL and lower limit of quantification (LLOQ) of 50 ng/mL. As a proof of principle, analysis of CSF from 83 stroke patients and 42 control subjects revealed elevated GAG levels in the stroke group, corresponding to 83.13% specificity and 76.19% sensitivity in distinguishing stroke-induced BBB dysfunction from controls. These findings suggest that GAG quantification can serve as an effective measure of glycocalyx shedding and that GAG may represent a potential biomarker for BBB dysfunction.