Anal Chem. 2019 Aug 20;91(16):10492-10500. doi: 10.1021/acs.analchem.9b01393. Epub 2019 Jul 31.
Changes of α-2,3-/α-2,6-linked sialic acids (SAs) in sialylglycans have been found to be closely related with some diseases. However, accurate quantification of sialylglycans at the isomeric level remains challenging due to their instability, structural complexity, and low mass spectrometry (MS) detection sensitivity. Herein, we propose an analytical strategy named "glycoqueuing", which allows sequential chromatographic elution and high-sensitivity MS quantification of various sialylglycan isomers based on isotopic labeling followed by analysis via online reversed-phase high performance liquid chromatography coupling with MS (RP-HPLC-MS). The new method was validated by detailed structural identification and quantification of fetal bovine serum (FBS) -linked sialylglycan isomers, during which many branching isomers were successfully differentiated, and 28 sialylglycan compositions with Neu5Gc residues were analyzed. The method was successfully applied to isomer-specific, quantitative comparison of sialylated -glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated -glycan biomarker candidates of hepatocellular carcinoma, during which a 55% increase of α-2,6-sialylated fucosylated -glycans was revealed, demonstrating the great applicability and potential clinical usage of the method.
α-2,3-/α-2,6-连接唾液酸（SAs）的变化与一些疾病密切相关。然而，由于唾液糖蛋白的不稳定性、结构复杂性和低质谱（MS）检测灵敏度，对其异构体水平的准确定量仍然具有挑战性。在此，我们提出了一种名为“糖排序”的分析策略，该策略允许基于同位素标记的各种唾液糖蛋白异构体的顺序色谱洗脱和高灵敏度 MS 定量，然后通过在线反相高效液相色谱与 MS（RP-HPLC-MS）进行分析。该新方法通过详细的结构鉴定和胎牛血清（FBS）连接的唾液糖蛋白异构体的定量得到了验证，在此期间成功区分了许多分支异构体，并分析了 28 种含有 Neu5Gc 残基的唾液糖蛋白组成。该方法成功应用于牛和兔免疫球蛋白 G（IgG）中唾液酸化 -糖的异构体特异性、定量比较，以及寻找肝细胞癌的血清唾液酸化 -糖生物标志物候选物，在此期间，α-2,6-唾液酸化岩藻糖基化 -糖的增加了 55%，证明了该方法的极大适用性和潜在的临床用途。
