Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, 30-348, Krakow, Poland.
Universität zu Lübeck, Institut für Physiologie, Ratzeburger Allee 160, Gebäude 61, D-23562, Lübeck, Germany.
Talanta. 2022 Feb 1;238(Pt 1):123008. doi: 10.1016/j.talanta.2021.123008. Epub 2021 Oct 28.
Glycosaminoglycans (GAGs) constitute the main building blocks of the endothelial glycocalyx (GLX), and disruption of GLX initiates and promotes endothelial dysfunction. Here, we aimed to develop a novel, specific and accurate LC-SRM/MS-based method for glycosaminoglycans (GAGs) profiling. The method involved butanolysis derivatization to facilitate GAG-specific disaccharide generation and its subsequent retention in LC-reversed-phase mode followed by mass spectrometric detection performed in positive ion-selected reaction monitoring (SRM) mode. GAG contents were measured in media of endothelial cells (EA.hy926) subjected to various GAG-degrading enzymes, as well as in murine plasma and urine in apolipoprotein E/low-density lipoprotein receptor-deficient (ApoE/LDLR -/-) mice and age-matched wild-type C57BL/6 mice. Alternatively, GLX disruption was verified by atomic force microscopy (AFM)-based analysis of GLX thickness. The proposed assay to quantify GAG-specific disaccharides presented high sensitivity for each of the analytes (LLOQ: 0.05-0.1 μg/mL) as well as accuracy and precision (86.8-114.9% and 2.0-14.3%, respectively). In medium of EA.hy926 cells subjected to GAG-degrading enzymes various GAG-specific disaccharides indicating the degradation of keratan sulphate (KS), heparan sulphate (HS), chondroitin sulphate (CHS) or hyaluronan (HA) were detected as predicted based on the characteristics of individual enzyme activity. In turn, AFM-based assessment of GLX thickness was reduced to a similar extent by all single enzyme treatments, whereas the most prominent reduction of GLX thickness was detected following the enzyme mixture. Plasma measurements of GAGs revealed age- and hypercholesterolemia-dependent decrease in GAGs concentration. In summary, a novel LC-SRM/MS-based method for GAG profiling was proposed that may inform on GLX status in cell culture for both in vitro and in vivo conditions.
糖胺聚糖（GAGs）是内皮糖萼（GLX）的主要结构单元，GLX 的破坏会引发并促进内皮功能障碍。在这里，我们旨在开发一种新的、特异的和准确的基于 LC-SRM/MS 的糖胺聚糖（GAG）分析方法。该方法涉及丁醇解衍生化，以促进 GAG 特异性二糖的生成，并随后在 LC 反相模式下保留，随后在正离子选择反应监测（SRM）模式下进行质谱检测。我们在经各种 GAG 降解酶处理的内皮细胞（EA.hy926）的培养基中以及载脂蛋白 E/低密度脂蛋白受体缺陷（ApoE/LDLR -/-）小鼠和年龄匹配的野生型 C57BL/6 小鼠的血浆和尿液中测量了 GAG 含量。或者，通过 GLX 厚度的原子力显微镜（AFM）分析来验证 GLX 破坏。用于定量 GAG 特异性二糖的测定方法对每种分析物均具有高灵敏度（LLOQ：0.05-0.1μg/mL）以及准确性和精密度（86.8-114.9%和 2.0-14.3%）。在经 GAG 降解酶处理的 EA.hy926 细胞培养基中，根据各种 GAG 特异性二糖的特征，检测到预测的角蛋白硫酸盐（KS）、肝素硫酸盐（HS）、软骨素硫酸盐（CHS）或透明质酸（HA）降解。相反，基于 AFM 的 GLX 厚度评估在受到所有单一酶处理后都以相似的程度减少，而 GLX 厚度的最显著减少则是在混合酶处理后检测到的。血浆 GAG 测量结果表明，GAG 浓度随年龄和高胆固醇血症的增加而降低。总之，我们提出了一种新的基于 LC-SRM/MS 的 GAG 分析方法，该方法可用于体外和体内条件下细胞培养中的 GLX 状态。
