Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy , University of Gothenburg , Medicinaregatan 9A , 40530 Gothenburg , Sweden.
Department of Neuroscience , Georgetown University , 3970 Reservoir Road NW, New Research Building EP20 , Washington, D.C. , United States.
Anal Chem. 2019 Aug 6;91(15):9819-9827. doi: 10.1021/acs.analchem.9b01485. Epub 2019 Jul 11.
Glycosylation is a fundamental post-translational modification, occurring on half of all proteins. Despite its significance, our understanding is limited, in part due to the inherent difficulty in studying these branched, multi-isomer structures. Accessible, detailed, and quantifiable methods for studying glycans, particularly -glycans, are needed. Here we take a multiple reaction monitoring (MRM) approach to differentiate and relatively quantify all detectable glycans, including isomers, on the heavily -glycosylated protein lubricin. Lubricin (proteoglycan 4) is essential for lubrication of the joint and eye. Given the therapeutic potential of lubricin, it is essential to understand its -glycan repertoire in biological and recombinantly produced samples. -Glycans were released by reductive β-elimination and defined, showing a range of 26 neutral, sulfated, sialylated, and both sulfated and sialylated core 1 (Galβ1-3GalNAcα1-) and core 2 (Galβ1-3(GlcNAcβ1-6)GalNAcα1-) structures. Isomer-specific MRM transitions allowed effective differentiation of neutral glycan isomers as well as sulfated isomeric structures, where the sulfate was retained on the fragment ions. This strategy was not as effective with labile sialylated structures; instead, it was observed that the optimal collision energy for the / 290.1 sialic acid B-fragment differed consistently between sialic acid isomers, allowing differentiation between isomers when fragmentation spectra were insufficient. This approach was also effective for purchased Neu5Acα2-3Galβ1-4Glc and Neu5Acα2-6Galβ1-4Glc and for Neu5Acα2-3Galβ1-4GlcNAc and Neu5Acα2-6Galβ1-4GlcNAc linkage isomers with the Neu5Acα2-6 consistently requiring more energy for optimal generation of the / 290.1 fragment. Overall, this method provides an effective and easily accessible approach for the quantification and annotation of complex released -glycan samples.
糖基化是一种基本的翻译后修饰，发生在所有蛋白质的一半上。尽管它意义重大，但我们的理解是有限的，部分原因是研究这些分支的、多异构体结构固有困难。需要可访问、详细和定量的方法来研究聚糖，特别是β-聚糖。在这里，我们采用多重反应监测 (MRM) 方法来区分和相对定量润滑素（蛋白聚糖 4）上所有可检测的聚糖，包括异构体。润滑素对于关节和眼睛的润滑至关重要。鉴于润滑素的治疗潜力，了解其在生物和重组产生的样品中的β-聚糖谱至关重要。β-聚糖通过还原β-消除释放并进行定义，显示出一系列 26 种中性、硫酸化、唾液酸化和同时硫酸化和唾液酸化的核心 1（Galβ1-3GalNAcα1-）和核心 2（Galβ1-3(GlcNAcβ1-6)GalNAcα1-）结构。异构体特异性的 MRM 跃迁允许有效区分中性聚糖异构体以及硫酸化的异构体结构，其中硫酸根保留在片段离子上。这种策略对于不稳定的唾液酸化结构效果不佳；相反，观察到 290.1 个唾液酸 B-片段的最佳碰撞能对于唾液酸异构体始终存在差异，这允许在碎片谱不足时区分异构体。这种方法对于购买的 Neu5Acα2-3Galβ1-4Glc 和 Neu5Acα2-6Galβ1-4Glc 以及 Neu5Acα2-3Galβ1-4GlcNAc 和 Neu5Acα2-6Galβ1-4GlcNAc 连接异构体也有效，其中 Neu5Acα2-6 始终需要更多的能量来最佳生成 290.1 片段。总体而言，该方法为定量和注释复杂释放的β-聚糖样品提供了一种有效且易于访问的方法。
