Quantitative HILIC-Q-TOF-MS analysis of glycosaminoglycans and non-reducing end carbohydrate biomarkers via glycan reductive isotopic labeling

Glycosaminoglycans (GAGs) are linear, heterogeneous polysaccharides expressed on all animal cells. Sulfated GAGs, including heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS), are involved in numerous physiological and pathological processes; therefore, precise and robust analytical methods for their characterization are essential to correlate structure to function. In this study, we developed a method utilizing hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry (HILIC-Q-TOF-MS) and glycan reductive isotopic reducing end labeling (GRIL) for the quantitative compositional analysis of HS and CS/DS polysaccharides. Lyase-generated disaccharides and commercial standards were chemically tagged on the reducing end with aniline stable isotopes, thus enabling absolute quantification of HS and CS/DS disaccharides in complex biological samples. In addition, we adapted this workflow, in conjunction with new synthetic carbohydrate standards, for the quantification of disease-specific non-reducing end (NRE) carbohydrate biomarkers that accumulate in patients with mucopolysaccharidoses (MPS), a subclass of lysosomal storage disorders. As proof of concept, we applied this method to measure NRE biomarkers in patient-derived MPS IIIA and MPS IIID fibroblasts as well as cortex tissue from a murine model of MPS VII. Overall, this method demonstrates improved sensitivity compared to previous GRIL-LC/MS techniques and, importantly, avoids the use of ion pairing reagents, which is undesirable in certain mass spectrometry instrumentation and contexts. By combining the benefits of HILIC separation with isotopic labeling, our approach offers a robust and accessible tool for analysis of GAGs, paving the way for advancements in understanding GAG structure and function.