A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics.
A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics.
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DOI：
10.1038/s41467-020-19052-w
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发表时间：
2020-10-19
影响因子：
16.6
通讯作者：
Urlaub H
中科院分区：
文献类型：
--
作者：
Fang P;Ji Y;Silbern I;Doebele C;Ninov M;Lenz C;Oellerich T;Pan KT;Urlaub H
蛋白质n -糖基化的调节在人体细胞中是必不可少的。然而，大规模、准确和位点特异性的糖基化定量在技术上仍然具有挑战性。我们在此介绍SugarQuant，这是一种基于质谱的集成管道，包括基于蛋白质聚集捕获（PAC）的样品制备，多缺口MS3采集（Glyco-SPS-MS3）和数据处理工具（GlycoBinder），可以对复杂生物样品中的完整糖肽进行自信的鉴定和定量。PAC大大减少了样品处理时间而不影响灵敏度。Glyco-SPS-MS3结合了高分辨率MS2和MS3扫描，增强了等压质量标签的报告信号，提高了n -糖肽片段的检测，降低了多路定量的干扰。glybinder能够简化处理Glyco-SPS-MS3数据，随后进行两步数据库搜索，与传统策略相比，糖肽的识别率提高了22%。我们使用SugarQuant来鉴定和量化伯基特淋巴瘤细胞中5000多种独特的糖型，并以高置信度确定在抑制聚焦化时发生的位点特异性糖基化变化。完整糖肽的全面定量分析在技术上仍然具有挑战性。为了解决这个问题，作者在这里开发了一个集成的定量糖蛋白组工作流程，包括优化的样品制备，多路量化和专用的数据处理工具。
Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence. Comprehensive quantitative profiling of intact glycopeptides remains technically challenging. To address this, the authors here develop an integrated quantitative glycoproteomic workflow, including optimized sample preparation, multiplexed quantification and a dedicated data processing tool.
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影响因子：
--
作者：
Holman, Jerry D;Tabb, David L;Mallick, Parag
通讯作者：
Mallick, Parag
影响因子：
7.4
作者：
Mysling, Simon;Palmisano, Giuseppe;Thaysen-Andersen, Morten
通讯作者：
Thaysen-Andersen, Morten
影响因子：
4.4
作者：
Lee, Hyoung-Joo;Cha, Hyun-Jeong;Paik, Young-Ki
通讯作者：
Paik, Young-Ki
通过固相提取N连接的聚糖和含糖材料的肽对蛋白质糖基化的全面分析。
影响因子：
46.9
作者：
Sun S;Shah P;Eshghi ST;Yang W;Trikannad N;Yang S;Chen L;Aiyetan P;Höti N;Zhang Z;Chan DW;Zhang H
通讯作者：
Zhang H
影响因子：
16.6
作者：
Hogrebe A;von Stechow L;Bekker-Jensen DB;Weinert BT;Kelstrup CD;Olsen JV
通讯作者：
Olsen JV