Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT.
Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT.
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DOI：
10.1021/acs.analchem.6b00465
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发表时间：
2016-08-02
影响因子：
7.4
通讯作者：
Mechref Y
中科院分区：
文献类型：
--
作者：
Zhou S;Hu Y;Veillon L;Snovida SI;Rogers JC;Saba J;Mechref Y
蛋白质糖基化在蛋白质功能修饰、蛋白质相互作用调控、蛋白质周转率控制等生物学过程中起着重要作用。此外，聚糖已被认为是许多哺乳动物疾病的潜在生物标志物，并且异常糖基化谱的发展是疾病或癌症病理学的重要指标。因此，定量是全面糖组学研究的一个重要方面。尽管在过去的几十年中已经开发了许多基于MS的定量策略，但仍然存在一些影响定量灵敏度和准确性的问题，并且仍然需要开发更有效的定量策略。氨氧基串联质量标签（AminoxyTMT™）试剂是最近商业化的同量异序标签，其能够同时相对定量多达六种不同的聚糖样品。在这项研究中，液相色谱和质谱条件已经优化，以实现可靠的LC-MS/MS定量糖分析使用aminoxyTMT™试剂。将样品重悬于0.2 M氯化钠溶液中以促进钠加合物前体离子的形成，这导致更高的MS/MS报告离子产率。首先使用来自模型糖蛋白和合并人血清样品的聚糖评价该方法。观察到的报告离子比率的变化相对于理论比率通常小于10%。即使对于高度复杂的次要N-聚糖，变化仍低于15%。该策略进一步应用于从患有不同食管疾病的患者的血清样品中释放的N-聚糖的糖组学分析。我们的研究结果表明，利用aminoxyTMT试剂的生物糖样品的可靠定量的好处。
Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (AminoxyTMT™) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT™ reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophagus diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.
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