Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry
Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry
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DOI：
10.1016/j.ab.2005.10.036
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发表时间：
2006-01-15
影响因子：
2.9
通讯作者：
Hayakawa, T
中科院分区：
文献类型：
--
作者：
Harazono, A;Kawasaki, N;Hayakawa, T
铜蓝蛋白具有铁氧化酶活性，在铁代谢中起重要作用。本研究采用反相高效液相色谱-电喷雾电离串联质谱（LC-ESI-MS/MS）对人铜蓝蛋白（ceruloplasmin， CP）进行了位点特异性糖基化分析。采用LC-ESI-MS/MS对羧甲基化CP进行胰蛋白酶消化。利用数据依赖的产物离子光谱分别对m/z 204 （HexNAc）和m/z 366 （HexHexNAc）等碳水化合物b离子进行糖肽和多肽的区分。并根据衍生自该肽的b系列和y系列离子的存在来鉴定该糖肽的肽段。寡糖的组成由糖肽的观察质量和理论质量计算得到的分子量推断出来。在7个潜在的n -糖基化位点中，4个（Asn119、Asn339、Asn378和Asn743）被具有聚焦残基（0、1或2）的涎化双天线或三天线低聚糖占据。检测到少量唾液化的四蕨寡糖。外糖苷酶切表明，聚焦残基与双天线低聚糖的末端GlcNAc还原和三天线低聚糖的末端和/或α 1-3还原到外臂GlcNAc有关，并且三天线低聚糖中大约有一个天线被α 2-3唾液化，偶尔α 1-3在GlcNAc上聚焦。(C) 2005爱思唯尔公司版权所有。
Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc),. and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantermary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha 1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha 2-3 sialylated and occasionally alpha 1-3 fucosylated at GlcNAc. (C) 2005 Elsevier Inc. All rights reserved.