Gomez Toledo Alejandro, Nilsson Jonas, Noborn Fredrik, Sihlbom Carina, Larson Göran
From the ‡Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Sweden;
§The Proteomics Core Facility, Core Facilities, Sahlgrenska Academy at the University of Gothenburg, Sweden.
Mol Cell Proteomics. 2015 Dec;14(12):3118-31. doi: 10.1074/mcp.M115.051136. Epub 2015 Sep 25.
The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)(n), to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.
α-胰蛋白酶抑制剂复合物是一种大分子结构，由结构相关的重链蛋白与蛋白聚糖比昆宁的硫酸软骨素（CS）链共价交联而成。α-胰蛋白酶抑制剂复合物在血浆中含量丰富，与炎症、肾脏疾病、癌症和糖尿病有关。比昆宁在丝氨酸-10处被一条由23-55个单糖组成的单条低硫酸化CS链修饰，该链带有4-9个硫酸基团。最内侧的四个单糖（GlcAβ3Galβ3Galβ4Xylβ-O-）构成连接区域，据信该区域是均匀的，外侧半乳糖有4-O-硫酸化修饰。比昆宁CS链的交联区域位于非硫酸化的非还原端（GalNAcβ4GlcAβ3）(n)，重链（H1-H3）可能通过GalNAc与天冬氨酸的酯键与之结合。在本研究中，我们采用了一种糖蛋白质组学方法来富集和分析源自人血浆、尿液和脑脊液样本的αI复合物的轻链和重链连接及交联区域的CS糖肽。对样本进行胰蛋白酶消化，通过强阴离子交换色谱法富集，用硫酸软骨素酶ABC进行部分解聚，然后使用高能碰撞解离通过液相色谱-串联质谱法进行分析。分析表明，比昆宁的CS连接区域高度异质。除了半乳糖残基的硫酸化外，还观察到木糖磷酸化，不过仅在尿液样本中出现。我们还鉴定出连接区域新的Neu5Ac和Fuc修饰，以及苏氨酸-17上存在单唾液酸化和双唾液酸化的核心1 O-连接聚糖。鉴定出重链H1和H2与相隔一个或两个葡萄糖醛酸残基的N-乙酰半乳糖胺残基交联，并且发现H1与末端或亚末端的N-乙酰半乳糖胺残基相连。CS糖肽在可变的高能碰撞解离条件下的裂解行为表现出能量依赖性，这可用于获得互补的结构细节。最后，我们表明对钠加合物的分析提供了有关聚糖取代基位置的确认信息。
