基于电喷雾电离质谱(ESI-MS)的丙酮稳定同位素标记对N-糖链的相对定量分析方法的研究

doi:10.6023/A13101106

化学学报 ›› 2014, Vol. 72 ›› Issue (2): 220-226.DOI: 10.6023/A13101106 上一篇下一篇

研究论文

基于电喷雾电离质谱(ESI-MS)的丙酮稳定同位素标记对N-糖链的相对定量分析方法的研究

薛向东^a, 孙玉姣^a, 刘洋^a, 张萍^a,b, 王仲孚^a, 黄琳娟^a

a 西北大学生命科学学院西部资源生物与现代生物技术教育部重点实验室西安 710069;
b 咸阳师范学院化学与化工学院咸阳 712000

投稿日期:2013-11-06 发布日期:2014-01-05
通讯作者: 黄琳娟，E-mail：huanglj@nwu.edu.cn；Tel.：029-88305853；Fax：029-88303572 E-mail:huanglj@nwu.edu.cn
基金资助:
项目受国家自然科学基金（Nos. 31170773，21375103，31370804）资助.

A Novel Method for Relative Quantitation of N-Glycans via Acetone Stable Isotopic Labeling and ESI-MS Analysis

Xue Xiangdong^a, Sun Yujiao^a, Liu Yang^a, Zhang Ping^a,b, Wang Zhongfu^a, Huang Linjuan^a

a Key Laboratory of Ministry of Education for Western China Resource Biology and Biotechnology, The College of Life Sciences, Northwest University, Xi'an 710069;
b Chemistry and Chemical Engineering School, Xianyang Normal University, Xianyang 712000

Received:2013-11-06 Published:2014-01-05
Supported by:
Project supported by the National Natural Science Foundation of China (Nos. 31170773, 21375103, 31370804).

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1. 基于电喷雾电离质谱(ESI-MS)的丙酮稳定同位素标记对N-糖链的相对定量分析方法的研究.PDF(241KB)

摘要/Abstract

建立了一种基于电喷雾电离质谱的丙酮稳定同位素标记对N-糖链进行相对定量的研究方法. 与传统的PNGase F酶水解N-糖链的方法不同，采用非特异性蛋白酶Pronase E对N-糖蛋白进行处理，使N-糖蛋白被酶解为带有一个氨基酸的糖氨酸（Glycan-Asn），为N-糖链引入了一个氨基活性基团，然后用丙酮对氨基进行标记. 用d₀/d₆丙酮对Ribo B标准糖蛋白的Pronase E酶解产物进行标记，考察了4对d₀/d₆丙酮标记的Glycan-Asn（Man₅～Man₈-Asn）在电喷雾电离质谱中的线性、动态范围以及重现性. 结果表明，在10倍动态范围内，相对定量方法有良好的线性关系（R=0.9981）和重现性（CV<8.7%）. 并将建立的方法应用于不同含量的鸡卵清白蛋白中，进一步验证了该方法的可行性. 研究结果表明，该方法能准确分析样品中N-糖链的含量，对不同样品中N-糖链进行相对定量. 该方法成本低廉，后处理方法简单方便，适于微量样品通量化分析，对差异糖组的研究有一定的意义.

关键词: 丙酮稳定同位素标记, 电喷雾电离质谱, 还原烷化, N-糖链的定量

A new method for relative quantitation of N-glycans based on acetone stable isotopic labeling and ESI-MS analysis is reported. Unlike the traditional PNGase F enzymolysis method, a non-specific protease, pronase E, is employed to digest N-glycoproteins into free N-glycans bearing a single asparagine residue (N-glycan-Asns) in this study. Consequently, the Asn moiety of the obtained N-glycan-Asns exposes an active amino group for further derivatization, enabling introduction of several relative quantitation methods of proteins/peptides into glycan analysis. On this basis, the N-glycan-Asns were derivatized with acetone (d₀-acetone) and its stable isotope form (d₆-acetone) by reductive alkylation, which has been extensively employed for derivatization of proteins/peptides. As a result, the isotopically labeled N-glycan-Asns have the same ionization efficiency as their isotopic forms, and each N-glycan-Asn exhibits a pair of ion peaks with a stable mass difference (Dm=6 Da) in the mass spectrum. Hence, comparing the intensities of the two peaks of each glycan, the relative contents of N-glycans obtained from different glycoprotein samples can be quantified. In this work, ribonuclease B from bovine pancreas (Ribo B) was digested by pronase E and applied to ESI-MS analysis, and four peaks corresponding to high-mannose N-glycans were observed in the mass spectra. The molecular weights of these glycans were 114 Da bigger than those released by PNGase F from Ribo B, due to the persistence of the Asn residue on the pronase-obtained glycans, indicating that pronase E could stably hydrolyze the N-glycoprotein to N-glycan-Asn. Next, two same-quantity aliquots of the obtained N-glycan-Asn sample were labeled with d₀-and d₆-acetone, respectively, and mixed together for ESI-MS analysis. As a result, some doublet ion peaks with a 6-Da mass difference were observed in mass spectra, and the light and heavy forms of each glycan appeared almost with the same peak intensity, indicating the acetone stable isotopic labeling procedure could be steadily employed for the relatively quantitative analysis of N-glycans. Furthermore, the linearity and dynamic range of this N-glycan relative quantitation method were evaluated using mixtures of the d₀-and d₆-acetone labeled Ribo B N-glycan-Asn samples (Man₅～Man₈-Asn) in different molar ratios. The MS profiling results demonstrated that the relative quantitative method could provide the relative quantitation data with a dynamic range of 10-fold, adequate linearity (R>0.995) and reliable reproducibility with a coefficient of variation that was less than 8.7%. Moreover, the reliable relative quantitative method was successfully employed to compare the quantitative difference between different chicken egg albumin samples with different purities by MS analysis. The results showed that the relative quantitative method could accurately perform relative quantitative analysis of N-glycans.

Key words: acetone stable isotopic labeling, ESI-MS, reductive alkylation, quantitation of N-linked glycans

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薛向东, 孙玉姣, 刘洋, 张萍, 王仲孚, 黄琳娟. 基于电喷雾电离质谱(ESI-MS)的丙酮稳定同位素标记对N-糖链的相对定量分析方法的研究[J]. 化学学报, 2014, 72(2): 220-226.

Xue Xiangdong, Sun Yujiao, Liu Yang, Zhang Ping, Wang Zhongfu, Huang Linjuan. A Novel Method for Relative Quantitation of N-Glycans via Acetone Stable Isotopic Labeling and ESI-MS Analysis[J]. Acta Chimica Sinica, 2014, 72(2): 220-226.

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基于电喷雾电离质谱(ESI-MS)的丙酮稳定同位素标记对N-糖链的相对定量分析方法的研究

A Novel Method for Relative Quantitation of N-Glycans via Acetone Stable Isotopic Labeling and ESI-MS Analysis

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