Université Paris-Saclay, CNRS, CEA, Univ Evry, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, F-91025, Evry, France.
ICOA, CNRS UMR 7311, Université d'Orléans, F-45067, Orléans, France.
Rapid Commun Mass Spectrom. 2017 Dec 15;31(23):2003-2010. doi: 10.1002/rcm.7987.
Chondroitin sulfate (CS) glycosaminoglycans are bioactive sulfated polysaccharides comprising repeating units of uronic acid and N-acetyl galactose sulfated at various positions. The optimal length and sulfation pattern of the CS bioactive sequences remain elusive so that structure-activity relationships cannot be easily established. Development of efficient analytical methods allowing the differentiation of the various sulfation patterns of CS sequences is therefore of particular importance to correlate their biological functions to the sulfation pattern.
Discrimination of different oligomers (dp2 to dp6) of synthetic chondroitin sulfate isomers was evaluated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative-ion mode from deprotonated and alkali adduct species. In addition, ion mobility mass spectrometry (IMS-MS) was used to study the influence of both the degree of polymerization and sulfate group location on the gas-phase conformation of CS oligomers.
ESI-MS/MS spectra of chondroitin sulfate isomers show characteristic product ions exclusively from alkali adduct species (Li, Na, K and Cs). Whatever the alkali adducts studied, MS/MS of chondroitin oligosaccharides sulfated at position 6 yields a specific product ion at m/z 139 while CS oligosaccharides sulfated at position 4 show a specific product ion at m/z 154. Being observed for the different CS oligomers di-, tetra- and hexasaccharides, these fragment ions are considered as diagnostic ions for chondroitin 6-O-sulfate and chondroitin 4-O-sulfate, respectively. IMS-MS experiments reveal that collision cross-sections (CCS) of CS oligomers with low charge states evolved linearly with degrees of polymerization indicating a similar gas-phase conformation.
This study allows the fast and unambiguous differentiation of CS isomers sulfated at position 6 or 4 for both saturated and unsaturated analogues from MS/MS experiments. In addition, the CCS linear evolution of CS oligomers in function of the degree of polymerization indicates that no folding occurs even for hexasaccharides.
硫酸软骨素（CS）糖胺聚糖是由重复的糖醛酸和 N-乙酰半乳糖单元组成的生物活性硫酸化多糖，这些单元在不同位置发生硫酸化。CS 生物活性序列的最佳长度和硫酸化模式仍难以捉摸，因此难以建立结构-活性关系。因此，开发能够区分 CS 序列各种硫酸化模式的有效分析方法对于将其生物学功能与硫酸化模式相关联非常重要。
通过负离子模式下的电喷雾串联质谱（ESI-MS/MS）评估了不同的合成硫酸软骨素异构体的低聚物（dp2 到 dp6）的区分。此外，离子淌度质谱（IMS-MS）用于研究聚合度和硫酸基团位置对 CS 低聚物气相构象的影响。
CS 异构体的 ESI-MS/MS 图谱显示出仅从碱加合物（Li、Na、K 和 Cs）中产生的特征产物离子。无论研究的碱加合物如何，在位置 6 硫酸化的软骨素低聚糖的 MS/MS 产生特定的产物离子在 m/z 139 处，而在位置 4 硫酸化的 CS 低聚糖显示出特定的产物离子在 m/z 154 处。这些片段离子在不同的 CS 低聚物二糖、四糖和六糖中均有观察到，被认为分别是软骨素 6-O-硫酸酯和软骨素 4-O-硫酸酯的诊断离子。IMS-MS 实验表明，具有低电荷态的 CS 低聚物的碰撞截面（CCS）与聚合度呈线性关系，表明具有相似的气相构象。
这项研究允许通过 MS/MS 实验快速且明确地区分在位置 6 或 4 硫酸化的 CS 异构体，无论是饱和的还是不饱和的类似物。此外，CS 低聚物的 CCS 线性演变与聚合度的关系表明，即使是六糖也不会发生折叠。
