Department of Laboratory Medicine, Sahlgrenska Academy at the University of Gothenburg, Sweden.
Department of Laboratory Medicine, Sahlgrenska Academy at the University of Gothenburg, Sweden.
Mol Cell Proteomics. 2021;20:100074. doi: 10.1016/j.mcpro.2021.100074. Epub 2021 Mar 20.
Of all posttranslational modifications known, glycosaminoglycans (GAGs) remain one of the most challenging to study, and despite the recent years of advancement in MS technologies and bioinformatics, detailed knowledge about the complete structures of GAGs as part of proteoglycans (PGs) is limited. To address this issue, we have developed a protocol to study PG-derived GAGs. Chondroitin/dermatan sulfate conjugates from the rat insulinoma cell line, INS-1832/13, known to produce primarily the PG chromogranin-A, were enriched by anion-exchange chromatography after pronase digestion. Following benzonase and hyaluronidase digestions, included in the sample preparation due to the apparent interference from oligonucleotides and hyaluronic acid in the analysis, the GAGs were orthogonally depolymerized and analyzed using nano-flow reversed-phase LC-MS/MS in negative mode. To facilitate the data interpretation, we applied an automated LC-MS peak detection and intensity measurement via the Proteome Discoverer software. This approach effectively provided a detailed structural description of the nonreducing end, internal, and linkage region domains of the CS/DS of chromogranin-A. The copolymeric CS/DS GAGs constituted primarily consecutive glucuronic-acid-containing disaccharide units, or CS motifs, of which the N-acetylgalactosamine residues were 4-O-sulfated, interspersed by single iduronic-acid-containing disaccharide units. Our data suggest a certain heterogeneity of the GAGs due to the identification of not only CS/DS GAGs but also of GAGs entirely of CS character. The presented protocol allows for the detailed characterization of PG-derived GAGs, which may greatly increase the knowledge about GAG structures in general and eventually lead to better understanding of how GAG structures are related to biological functions.
在所有已知的翻译后修饰中，糖胺聚糖 (GAGs) 仍然是最难研究的一种，尽管近年来在 MS 技术和生物信息学方面取得了进展，但关于 GAGs 作为蛋白聚糖 (PGs) 一部分的完整结构的详细知识仍然有限。为了解决这个问题，我们开发了一种研究 PG 衍生 GAGs 的方案。从已知主要产生 PG 嗜铬粒蛋白 A 的大鼠胰岛素瘤细胞系 INS-1832/13 中分离出的软骨素/硫酸皮肤素缀合物，在经过蛋白酶消化后，通过阴离子交换色谱进行富集。在苯甲酸钠酶和透明质酸酶消化后，由于寡核苷酸和透明质酸在分析中明显存在干扰，包括在样品制备中，GAGs 被正交解聚，并在负模式下使用纳流反相 LC-MS/MS 进行分析。为了便于数据解释，我们通过 Proteome Discoverer 软件应用了自动 LC-MS 峰检测和强度测量。这种方法有效地提供了嗜铬粒蛋白 A 的 CS/DS 非还原端、内部和连接区域结构域的详细结构描述。共聚 CS/DS GAGs 主要由连续的含有葡萄糖醛酸的二糖单元或 CS 基序组成，其中 N-乙酰半乳糖胺残基被 4-O-硫酸化，由单个含有艾杜糖醛酸的二糖单元间隔开。我们的数据表明，由于不仅鉴定了 CS/DS GAGs，而且还鉴定了完全为 CS 特征的 GAGs，因此 GAGs 存在一定的异质性。所提出的方案允许对 PG 衍生 GAGs 进行详细表征，这可能会大大增加对 GAG 结构的一般认识，并最终有助于更好地理解 GAG 结构与生物学功能的关系。
