Research Unit Macromolecular Chemistry, Institute of Applied Synthetic Chemistry, TU Wien, 1060, Vienna, Austria; Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090, Vienna, Austria.
Research Unit Macromolecular Chemistry, Institute of Applied Synthetic Chemistry, TU Wien, 1060, Vienna, Austria; Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090, Vienna, Austria.
Talanta. 2022 May 15;242:123326. doi: 10.1016/j.talanta.2022.123326. Epub 2022 Feb 17.
Characterization of serum glycoprotein N-glycans with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in positive-ion mode needs a derivatization step to stabilize and neutralize the negative charge on sialic acids. The acidic sugars are attached to the end of glycoproteins, glycolipids or gangliosides. Here, we present a method for sialic acid stabilization via modification based on derivatization of carboxylic acid group activated with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) with methylamine. DMTMM substitutes in many processes N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) as activation reagent due to its better performance and higher stability in water. Glycosylated proteins are used as solid phase support for glycan derivatization and purification from excess of derivatization reagents. We evaluated our glycan analysis method in murine sera and intestinal lavages. The stabilization of sialic acid enables a complete conservation of the glycan structures, in contrast to other methods where sialic acids are partially lost. In BALB/c mouse sera, we detected predominantly mono- and di-sialylated N-glycans with mostly N-Glycolylneuraminic acid (Neu5Gc) and only trace amounts of N-Acetyl neuraminic acid (Neu5Ac). BALB/c mouse intestinal lavages glycoproteins contained asialo N-glycans. DMTMM-mediated methylamidation of N-glycans for MALDI mass spectrometry analysis is a fast and cheap method for structurally conserved glycan derivatization.
用基质辅助激光解吸/电离质谱（MALDI-MS）在正离子模式下对血清糖蛋白 N-聚糖进行表征需要进行衍生化步骤，以稳定和中和唾液酸上的负电荷。酸性糖通过与 4-(4,6-二甲氧基-1,3,5-三嗪-2-基）-4-甲基吗啉氯化物（DMTMM）的羧酸基团激活的衍生化附着在糖蛋白、糖脂或神经节苷脂的末端。在这里，我们提出了一种通过基于羧酸基团的衍生化来稳定唾液酸的方法，该方法通过 4-(4,6-二甲氧基-1,3,5-三嗪-2-基）-4-甲基吗啉氯化物（DMTMM）与甲胺的活化来取代许多过程中的 N-(3-二甲基氨基丙基）-N'-乙基碳二亚胺盐酸盐和 N-羟基琥珀酰亚胺（EDC/NHS）作为活化试剂，由于其在水中的更好性能和更高稳定性。糖基化蛋白被用作糖基衍生化和从过量衍生化试剂中纯化的固相支持物。我们在鼠血清和肠灌洗液中评估了我们的聚糖分析方法。与其他部分丢失唾液酸的方法相比，唾液酸的稳定能够完全保留聚糖结构。在 BALB/c 鼠血清中，我们检测到主要是单和二唾液酸化的 N-聚糖，主要是 N-糖基神经氨酸（Neu5Gc），只有痕量的 N-乙酰神经氨酸（Neu5Ac）。BALB/c 鼠肠灌洗液糖蛋白含有无唾液酸的 N-聚糖。用于 MALDI 质谱分析的 DMTMM 介导的 N-聚糖甲基酰胺化是一种快速且廉价的结构保守聚糖衍生化方法。
