Relative quantitation of glycans using stable isotopic labels 1-(d0/d5) phenyl-3-methyl-5-pyrazolone by mass spectrometry.
Relative quantitation of glycans using stable isotopic labels 1-(d0/d5) phenyl-3-methyl-5-pyrazolone by mass spectrometry.
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DOI：
10.1016/j.ab.2011.07.006
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发表时间：
2011-11
影响因子：
2.9
通讯作者：
Ping Zhang;Ying Zhang;Xiangdong Xue;Chenjian Wang;Zhongfu Wang;Linjuan Huang
中科院分区：
文献类型：
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作者：
Ping Zhang;Ying Zhang;Xiangdong Xue;Chenjian Wang;Zhongfu Wang;Linjuan Huang
合成了一种氘试剂1-（d5）苯基-3-甲基-5-吡唑啉酮（d5-PMP），并将其用于d 0/d5-PMP稳定同位素标记的寡糖质谱相对定量分析。先前报道的基于全甲基化的同位素标记产生可变的质量差异，并且基于还原胺化的同位素标记导致碳水化合物中一些酸不稳定基团的损失。相比之下，d 0/d5-PMP稳定同位素标记是在碱性条件下在聚糖的还原端进行的，没有去唾液酸化，并且每种聚糖的重链形式（d5-PMP衍生物）和轻链形式（d 0-PMP衍生物）之间的质量差（Δm= 10 Da）是不变的。当聚糖的两种衍生物形式以等摩尔量混合时，在MS图谱中观察到一对质量差为10 Da的峰。d 0-和d5-PMP衍生物之间相对强度的差异反映了两个样品中聚糖数量的差异，使得在糖组学研究中进行聚糖的定性和相对定量分析成为可能。将该方法应用于DP 2 ~ DP 6麦芽糊精低聚糖和核糖核酸酶B及牛胎球蛋白中N-连接聚糖的测定，结果表明该方法的相对定量范围为10倍，重复性好（变异系数[CV] ≥ 8.34%），准确度好（相对误差[RE] ≥ 5.1%）。该技术已成功地应用于人乳和牛乳中游离低聚糖的比较定量分析。
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=10Da) 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 DP2to DP6maltodextrin 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.