A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry.
A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry.
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DOI：
10.1002/elps.201200656
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发表时间：
2013-08
期刊：
影响因子：
2.9
通讯作者：
Zhang S
中科院分区：
文献类型：
--
作者：
Thannhauser TW;Shen M;Sherwood R;Howe K;Fish T;Yang Y;Chen W;Zhang S
糖基化是植物蛋白的常见翻译后修饰，影响大量重要的生物过程。然而，不同位点占用和特定糖型的性质的影响尚不清楚。从历史上看，由于肽基和聚糖部分不同的物理化学性质、糖基化过程的可变性和动态性、它们的异质性以及每种糖型的相对丰度较低，糖蛋白的表征一直很困难。在这项研究中，我们探索了一种新的管道，用于大规模实证鉴定番茄果实的 N 连接糖蛋白，作为我们持续表征番茄分泌组的努力的一部分。提出的工作流程涉及凝集素亲和力、胰蛋白酶消化、离子对 HILIC 和前体离子驱动的数据依赖性 MS/MS 分析与脚本的组合，以促进占用的 N 连接糖基化位点的识别和表征。本研究共鉴定了 212 种糖蛋白，其中仅在一种 HILIC 组分中就成功鉴定了来自 24 种糖蛋白的 26 种糖肽。进一步使用基于前体离子发现 (PID) 的 MS/MS 和去糖基化，然后进行高精度和分辨率 MS 分析来确认糖基化位点并确定位点占用率。报告的工作流程非常稳健，能够产生大量涉及 N 连接糖基化位点及其相关糖型的经验数据。
Glycosylation is a common post-translational modification of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has been difficult due to the distinct physicochemical properties of the peptidyl and glycan moieties, the variable and dynamic nature of the glycosylation process, their heterogeneous nature, and the low relative abundance of each glycoform. In this study, we explore a new pipeline developed for large-scale empirical identification of N-linked glycoproteins of tomato fruit as part of our ongoing efforts to characterize the tomato secretome. The workflow presented involves a combination of lectin affinity, tryptic digestion, ion-pairing HILIC and precursor ion-driven data dependent MS/MS analysis with a script to facilitate the identification and characterization of occupied N-linked glycosylation sites. A total of 212 glycoproteins were identified in this study, in which 26 glycopeptides from 24 glycoproteins were successfully characterized in just one HILIC fraction. Further precursor ion discovery (PID)-based MS/MS and deglycosylation followed by high accuracy and resolution MS analysis were used to confirm the glycosylation sites and determine site occupancy rates. The workflow reported is robust and capable of producing large amounts of empirical data involving N-linked glycosylation sites and their associated glycoforms.