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 with 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 the 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 a proof of concept, we applied this method to measure NRE biomarkers
in patient-derived MPS IIIA and MPS IIID fibroblasts, as well as in
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 are 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 the
analysis of GAGs, paving the way for advancements in understanding
GAG structure and function.

糖胺聚糖（Glycosaminoglycans, GAGs）是一类线性、异质性多糖，广泛表达于所有动物细胞表面。硫酸化GAGs包括硫酸乙酰肝素（heparan sulfate, HS）与硫酸软骨素/硫酸皮肤素（chondroitin/dermatan sulfate, CS/DS），参与众多生理与病理过程；因此，建立精准可靠的分析方法以表征其结构并关联结构与功能至关重要。本研究开发了一种结合亲水作用液相色谱-飞行时间质谱（hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry, HILIC-Q-TOF-MS）与聚糖还原型同位素还原端标记（glycan reductive isotopic reducing end labeling, GRIL）的方法，用于HS与CS/DS多糖的定量组成分析。研究人员将裂解酶酶解产生的二糖与商业标准品的还原端用苯胺稳定同位素进行化学标记，从而实现复杂生物样品中HS与CS/DS二糖的绝对定量。此外，本研究结合新型合成碳水化合物标准品，适配该实验流程以定量检测黏多糖贮积症（mucopolysaccharidoses, MPS，一类溶酶体贮积症）患者体内积累的疾病特异性非还原端（non-reducing end, NRE）碳水化合物生物标志物。作为概念验证，我们将该方法应用于检测患者来源的MPS IIIA、MPS IIID成纤维细胞，以及MPS VII小鼠模型的皮层组织中的NRE生物标志物。总体而言，相较于此前的GRIL-LC/MS技术，本方法展现出更优的灵敏度，且尤为重要的是，无需使用离子对试剂——这类试剂在部分质谱仪器与实验场景中并不适用。通过结合亲水作用色谱分离与同位素标记的优势，本方法为GAGs的分析提供了一种可靠且易于实现的工具，为推动GAGs结构与功能的研究奠定了基础。