Quantitative O-glycomics based on improvement of the one-pot method for nonreductive O-glycan release and simultaneous stable isotope labeling with 1-(d(0)/d(5))phenyl-3-methyl-5-pyrazolone followed by mass spectrometric analysis
Quantitative O-glycomics based on improvement of the one-pot method for nonreductive O-glycan release and simultaneous stable isotope labeling with 1-(d(0)/d(5))phenyl-3-methyl-5-pyrazolone followed by mass spectrometric analysis
复制标题
基于非还原性 O-聚糖释放一锅法的改进和同时使用 1-(d(0)/d(5))苯基-3-甲基-5-吡唑啉酮进行稳定同位素标记和质量分析的定量 O-糖组学
DOI：
10.1016/j.jprot.2016.08.012
复制
发表时间：
2017
影响因子：
3.3
通讯作者：
Wang Zhongfu
中科院分区：
文献类型：
--
作者：
Wang Chengjian;Zhang Ping;Jin Wanjun;Li Lingmei;Qiang Shan;Zhang Ying;Huang Linjuan;Wang Zhongfu
快速、简便、通用的糖蛋白O-聚糖定量分析方法是当前研究蛋白O-糖基化模式和寻找疾病O-聚糖生物标志物的迫切需要。利用稳定同位素标记结合质谱分析对O-聚糖进行相对定量是一种理想且有前途的技术。然而，由于缺乏可靠的非还原性O-聚糖释放方法，以及O-聚糖衍生物的轻重同位素形式之间的质量差异过大或过小，导致O-聚糖的质谱识别和定量分析困难。本文报道了一种简单易行的O-聚糖相对定量方法，该方法基于改进的一锅法，可以一步定量地实现完整粘蛋白型O-聚糖的非还原性释放和原位发色团标记。在本研究中，优化了一锅法，并应用于定量O-聚糖释放和标记非氘代（d 0-）或氘代（d5-）1-苯基-3-甲基-5-吡唑啉酮（PMP）。所获得的O-聚糖衍生物具有d 0-和d5-PMP形式之间永久性10-Da质量差异的特征，从而允许通过质谱技术完全区分和比较定量这些同位素标记的O-聚糖。此外，d 0-和d5-PMP O-聚糖衍生物还具有较高的疏水性和较强的紫外吸收性，特别适合于RP-HPLC-UV的高分辨率分离和高灵敏度检测。我们已经改进了一锅反应的条件以及相应的样品纯化方法。以牛胎球蛋白和猪胃粘蛋白为模型糖蛋白，验证了该方法的可行性、可靠性和线性。此外，我们还成功地将该方法应用于鲈鱼卵和鲑鱼卵中O-糖组学的定量比较，通过ESI-MS，MS/MS和在线RP-HPLC-UV-ESI-MS/MS，证明了该方法对各种复杂生物样品的良好适用性。生物学意义O-连接糖蛋白，通过广泛存在的新生蛋白的丝氨酸（Ser）或苏氨酸（Thr）残基的糖基化修饰过程产生，在一系列生物过程中发挥重要作用。糖蛋白中的O-聚糖作为一种信息分子，直接参与这些生物学机制。因此，O-聚糖表达水平的特征性差异或变化通常与许多疾病的病理学有关，并代表了揭示各种糖蛋白O-聚糖功能机制的重要机会。本文介绍的新策略为通过质谱法精确定量糖蛋白O-聚糖提供了一种简单而通用的分析方法，使得能够快速评估O-聚糖在表达水平上的差异或变化。这对于探索O-糖复杂的结构-功能关系、寻找一些重大疾病的O-糖生物标志物和药物的O-糖相关靶点等方面都具有重要意义。
Rapid, simple and versatile methods for quantitative analysis of glycoproteinO-glycans are urgently required for current studies on proteinO-glycosylation patterns and the search for diseaseO-glycan biomarkers. Relative quantitation ofO-glycans using stable isotope labeling followed by mass spectrometric analysis represents an ideal and promising technique. However, it is hindered by the shortage of reliable nonreductiveO-glycan release methods as well as the too large or too small inconstant mass difference between the light and heavy isotope form derivatives ofO-glycans, which results in difficulties during the recognition and quantitative analysis ofO-glycans by mass spectrometry. Herein we report a facile and versatileO-glycan relative quantification strategy, based on an improved one-pot method that can quantitatively achieve nonreductive release andin situchromophoric labeling of intact mucin-typeO-glycans in one step. In this study, the one-pot method is optimized and applied for quantitativeO-glycan release and tagging with either non-deuterated (d0-) or deuterated (d5-) 1-phenyl-3-methyl-5-pyrazolone (PMP). The obtainedO-glycan derivatives feature a permanent 10-Da mass difference between the d0- and d5-PMP forms, allowing complete discrimination and comparative quantification of these isotopically labeledO-glycans by mass spectrometric techniques. Moreover, the d0- and d5-PMP derivatives ofO-glycans also have a relatively high hydrophobicity as well as a strong UV adsorption, especially suitable for high-resolution separation and high-sensitivity detection by RP-HPLC-UV. We have refined the conditions for the one-pot reaction as well as the corresponding sample purification approach. The good quantitation feasibility, reliability and linearity of this strategy have been verified using bovine fetuin and porcine stomach mucin as modelO-glycoproteins. Additionally, we have also successfully applied this method to the quantitativeO-glycomic comparison between perch and salmon eggs by ESI-MS, MS/MS and online RP-HPLC-UV-ESI-MS/MS, demonstrating its excellent applicability to various complex biological samples.Biological significanceO-Linked glycoproteins, generated via a widely existing glycosylation modification process on serine (Ser) or threonine (Thr) residues of nascent proteins, play essential roles in a series of biological processes. As a type of informational molecule, theO-glycans of these glycoproteins participate directly in these biological mechanisms. Thus, the characteristic differences or changes ofO-glycans in expression level usually relate to pathologies of many diseases and represent an important opportunity to uncover the functional mechanisms of various glycoproteinO-glycans. The novel strategy introduced here provides a simple and versatile analytical method for the precise quantitation of glycoproteinO-glycans by mass spectrometry, enabling rapid evaluation of the differences or changes ofO-glycans in expression level. It is attractive for the field of quantitative/comparativeO-glycomics, which has great significance for exploring the complex structure-function relationship ofO-glycans, as well as for the search ofO-glycan biomarkers of some major diseases andO-glycan related targets of some drugs.