Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD
Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD
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DOI：
10.1074/mcp.m111.013649
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发表时间：
2012-04-01
影响因子：
7
通讯作者：
Larson, Goran
中科院分区：
文献类型：
--
作者：
Halim, Adnan;Nilsson, Jonas;Larson, Goran
尿液是蛋白质和废物的复杂混合物，并且是生物标志物发现的具有挑战性的生物流体。以前的蛋白质组学研究已经确定了超过2800尿蛋白，但分析旨在解开尿糖蛋白的聚糖结构和糖基化位点是缺乏的。由于主要在天冬酰胺（N-连接）或丝氨酸/苏氨酸（O-连接）残基上发现的聚糖结构的复杂性，糖蛋白组学表征仍然很困难。我们已经开发了一种糖蛋白质组学方法，结合了有效的纯化尿糖蛋白/糖肽与互补的MS-片段化技术的糖肽分析。从临床样本量出发，我们通过透析去除干扰尿中化合物，并通过冷冻干燥浓缩纯化的尿蛋白。通过酰肼化学和胰蛋白酶消化将唾液酸化尿糖蛋白缀合至固体支持物。去唾液酸化糖肽，通过温和的酸水解释放，其特征在于串联MS实验，利用碰撞诱导解离（CID）和电子捕获解离碎片技术。在CID-MS 2中，通常观察到Hex（5）HexNAc（4）-N-Asn和HexHexNAc-O-Ser/Thr，分别与已知的N-连接双触角复合物类型和O-连接核心1样结构一致。还鉴定了特异性N-和O-连接糖肽的其他糖型，例如。G.四触角N-聚糖和岩藻糖基化核心2-样O-聚糖。CID-MS 2分析中选定碎片离子的后续CID-MS 3生成肽特异性B-和y-离子，用于肽鉴定。总共，58个N-和63个O-连接的糖肽从53糖蛋白的特征在于相对于聚糖和肽序列。CID和电子捕获解离技术的组合允许精确鉴定57个推定的O-糖基化位点中的40个的Ser/Thr附着位点。我们定义了29个O-糖基化位点，据我们所知，以前没有报道过。这是第一次对人尿糖蛋白进行研究，其中研究了“完整的”糖肽，即毫无疑问地证明了聚糖及其连接位点的存在。Molecular & Cellular Proteomics 11：10.1074/mcp. M111.013649，1-17，2012.
Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS2, Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e. g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS3, of selected fragment-ions from the CID-MS2 analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan-and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.013649, 1-17, 2012.