Tools for glycomics:: relative quantitation of glycans by isotopic permethylation using 13CH3I
Tools for glycomics:: relative quantitation of glycans by isotopic permethylation using 13CH3I
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DOI：
10.1093/glycob/cwm033
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发表时间：
2007-07-01
期刊：
影响因子：
4.3
通讯作者：
Orlando, Ron
中科院分区：
文献类型：
--
作者：
Alvarez-Manilla, Gerardo;Warren, Nicole Lynn;Orlando, Ron
通过质谱（MS）对寡糖进行分析，使得以高分辨率和高灵敏度研究生物体的聚糖库成为可能。然而，由于质谱用于定量目的存在重大局限性，很难将糖基转移酶的表达与特定细胞类型或组织中存在的聚糖结构相关联。出于这个原因，为了开发一种能够通过对两个样本进行质谱分析来对聚糖进行相对定量的技术，利用标准的全甲基化条件，开发了一种用碳 - 13标记的碘甲烷对寡糖进行同位素标记的方法。从人乳中分离出的寡糖的不同等份分别用碳 - 12或碳 - 13碘甲烷进行标记；将标记的和未标记的聚糖按已知比例混合，然后通过质谱对混合物进行分析。结果表明，此处描述的同位素标记能够提供动态范围至少为两个数量级的相对定量数据，具有足够的线性和重现性，变异系数平均为13%。该方法被用于分析从各种糖蛋白混合物（如α - 1酸性糖蛋白、人转铁蛋白和牛胎球蛋白）中释放的N - 连接聚糖，所使用的质谱技术包括基质辅助激光解吸电离 - 飞行时间质谱以及电喷雾电离 - 离子回旋共振 - 傅里叶变换质谱。从用（CH₃I） - C - 12或（CH₃I） - C - 13进行全甲基化的聚糖混合物中测得的C - 12∶C - 13比例与理论比例相符。该技术是一种通过质谱进行聚糖相对定量分析的有效方法。
Analysis of oligosaccharides by mass spectrometry (MS) has enabled the investigation of the glycan repertoire of organisms with high resolution and sensitivity. It is difficult, however, to correlate the expression of glycosyltransferases with the glycan structures present in a particular cell type or tissue because the use of MS for quantitative purposes has significant limitations. For this reason, in order to develop a technique that would allow relative glycan quanti. cation by MS analysis between two samples, a procedure was developed for the isotopic labeling of oligosaccharides with C-13-labeled methyl iodide using standard permethylation conditions. Separate aliquots of oligosaccharides from human milk were labeled with C-12 or C-13 methyl iodide; the labeled and non-labeled glycans were mixed in known proportions, and the mixtures analyzed by MS. Results indicated that the isotopic labeling described here was capable of providing relative quantitative data with a dynamic range of at least two orders of magnitude, adequate linearity, and reproducibility with a coefficient of variation that was 13% on average. This procedure was used to analyze N-linked glycans released from various mixtures of glycoproteins, such as a-1 acid glycoprotein, human transferrin, and bovine fetuin, using MS techniques that included matrix assisted laser desorption ionization-time of. ight MS and electrospray ionization with ion cyclotron resonance- Fourier transformation MS. The measured C-12: C-13 ratios from mixtures of glycans permethylated with either (CH3I)-C-12 or (CH3I)-C-13 were consistent with the theoretical proportions. This technique is an effective procedure for relative quantitative glycan analysis by MS.