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首页> 外文期刊>Analytical Biochemistry: An International Journal of Analytical and Preparative Methods >Relative quantitation of glycans using stable isotopic labels 1-(d0/d5) phenyl-3-methyl-5-pyrazolone by mass spectrometry
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Relative quantitation of glycans using stable isotopic labels 1-(d0/d5) phenyl-3-methyl-5-pyrazolone by mass spectrometry

机译：使用稳定同位素标记的1-（d0 / d5）苯基-3-甲基-5-吡唑啉酮相对定量的聚糖

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摘要

A deuterium reagent, 1-(d5) phenyl-3-methyl-5-pyrazolone (d5-PMP), has been synthesized and used for relative quantitative analysis of oligosaccharides by mass spectrometry (MS) using d0/d5-PMP stable isotopic labeling. Previously reported permethylation-based isotopic labels generate variable mass differences, and reductive amination-based isotopic labels cause a loss of some acid-labile groups in carbohydrates. In contrast, d0/d5-PMP stable isotopic labeling is performed at the reducing end of glycans under basic conditions without desialylation, and the mass difference (Δm = 10 Da) between the heavy form (d5-PMP derivative) and light form (d0-PMP derivative) of each glycan is invariable. When the two derivative forms of a glycan are mixed in equimolar amounts, a pair of peaks with a 10-Da mass differences is observed in the MS profile. The difference at relative intensity between the d0- and d5-PMP derivatives reflects the difference in quantity of glycans in two samples, making it possible to carry out both qualitative and relative quantitative analyses of glycans in glycomic studies. Application of this method on DP _2 to DP_6 maltodextrin oligosaccharides and N-linked glycans released from ribonuclease B and bovine fetuin demonstrates a 10-fold relative quantitative dynamic range, a satisfying reproducibility (coefficient of variation [CV] ≤ 8.34%), and good accuracy (relative error [RE] ≤ 5.1%) of the method. The suggested technique has been successfully applied for comparative quantitative analysis of free oligosaccharides in human and bovine milk.
机译：合成了氘试剂1-（d5）苯基-3-甲基-5-吡唑啉酮（d5-PMP），并将其用于使用d0 / d5-PMP稳定同位素标记的质谱（MS）对寡糖进行相对定量分析。先前报道的基于全甲基化的同位素标记物会产生可变的质量差异，而基于还原胺化的同位素标记物会导致碳水化合物中某些酸不稳定基团的损失。相反，在碱性条件下，在聚糖的还原端进行d0 / d5-PMP稳定同位素标记，而没有去唾液酸化作用，重形式（d5-PMP衍生物）和轻形式（d0）之间的质量差（Δm= 10 Da）每个聚糖的-PMP衍生物是不变的。当两种衍生物形式的聚糖以等摩尔量混合时，在质谱图中观察到一对质量差为10 Da的峰。 d0-和d5-PMP衍生物之间的相对强度差异反映了两个样品中聚糖的数量差异，因此可以进行糖类研究中糖的定性和相对定量分析。该方法在DP _2上的应用（从核糖核酸酶B和牛胎球​​蛋白释放的DP_6麦芽糖糊精寡糖和N-连接的聚糖）具有10倍的相对定量动态范围，令人满意的重现性（变异系数[CV]≤8.34％），并且质量良好方法的准确度（相对误差[RE]≤5.1％）。所建议的技术已成功用于人和牛乳中游离寡糖的比较定量分析。

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