Structural classification of carbohydrates in glycoproteins by mass spectrometry and high-performance anion-exchange chromatography.
Structural classification of carbohydrates in glycoproteins by mass spectrometry and high-performance anion-exchange chromatography.
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通过质谱和高效阴离子交换色谱对糖蛋白中的碳水化合物进行结构分类。
DOI：
10.1016/0003-2697(91)90204-7
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发表时间：
1991
影响因子：
2.9
通讯作者：
Carr,SA
中科院分区：
文献类型：
--
作者：
Barr,JR;Anumula,KR;Vettese,MB;Taylor,PB;Carr,SA
研究人员已经开发出一种通用策略，用于确定n -连接低聚糖在糖蛋白特定附着位点上的结构类别（寡甘露糖、杂化糖、复合糖）、分支类型（双天线型、三天线型等）和分子微观异质性。该方法将质谱法、高性能阴离子交换色谱法与脉冲安培检测相结合，利用其高灵敏度和分析复杂低聚糖混合物的能力。糖肽的鉴定和分离是通过比较高效液相色谱图谱，在酶促碳水化合物释放之前和之后的蛋白质水解消化。每个分离的糖肽通过酶促释放寡糖，并通过快速原子轰击质谱（FAB-MS）对混合物的附着位点肽进行鉴定。然后将每个反应混合物的一部分进行过甲基化，并通过FAB-MS分析以确定碳水化合物部分的组成和分子异质性。FAB质谱中的片段离子可用于检测特定的结构特征，如聚乳糖胺单元和分割n -乙酰己糖胺残基，以及定位内核脱氧己糖残基。这些组分的甲基化分析提供了单体的连接。根据FAB-MS和甲基化分析数据，可以提出每个附着位点上碳水化合物的结构类别。从特定附着位点释放的碳水化合物的剩余部分通过高性能阴离子交换色谱进行分离，过甲基化，并通过FAB-MS进行分析。这些分析产生了色谱图中每个峰的电荷状态和组成，并提供了关于每个分子种类的相对数量的半定量信息。只需10 pmol的衍生化碳水化合物，就可以获得分析上有用的数据，并且达到了fmol的灵敏度。结合碳水化合物作图和结构指纹程序说明了重组形式的CD4受体糖蛋白。
A general strategy has been developed for determining the structural class (oligomannose, hybrid, complex), branching types (biantennary, triantennary, etc.), and molecular microheterogeneity of N-linked oligosaccharides at specific attachment sites in glycoproteins. This methodology combines mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection to take advantage of their high sensitivity and the capability for analysis of complex mixtures of oligosaccharides. Glycopeptides are identified and isolated by comparative HPLC mapping of proteolytic digests of the protein prior to, and after, enzymatic release of carbohydrates. Oligosaccharides are enzymatically released from each isolated glycopeptide, and the attachment site peptide is identified by fast atom bombardment mass spectrometry (FAB-MS) of the mixture. Part of each reaction mixture is then permethylated and analyzed by FAB-MS to identify the composition and molecular heterogeneity of the carbohydrate moiety. Fragment ions in the FAB mass spectra are useful for detecting specific structural features such as polylactosamine units and bisecting N-acetylhexosamine residues, and for locating inner-core deoxyhexose residues. Methylation analysis of these fractions provides the linkages of monomers. Based on the FAB-MS and methylation analysis data, the structural classes of carbohydrates at each attachment site can be proposed. The remaining portions of released carbohydrates from specific attachment sites are preparatively fractionated by high-performance anion-exchange chromatography, permethylated, and analyzed by FAB-MS. These analyses yield the charge state and composition of each peak in the chromatographic map, and provide semiquantitative information regarding the relative amounts of each molecular species. Analytically useful data may be obtained with as little as 10 pmol of derivatized carbohydrate, and fmol sensitivity has been achieved. The combined carbohydrate mapping and structural fingerprinting procedures are illustrated for a recombinant form of the CD4 receptor glycoprotein.