Absolute quantitation of glycosylation site occupancy using isotopically labeled standards and LC-MS.
Absolute quantitation of glycosylation site occupancy using isotopically labeled standards and LC-MS.
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DOI：
10.1007/s13361-014-0859-2
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发表时间：
2014-06
影响因子：
3.2
通讯作者：
Desaire, Heather
中科院分区：
文献类型：
--
作者：
Zhu, Zhikai;Go, Eden P.;Desaire, Heather
关键词：
N-连接聚糖是维持蛋白质适当生物功能所必需的。糖基化不足导致植物和动物中的许多疾病;因此，表征蛋白质上糖基化的程度是理解、诊断和治疗疾病的重要步骤。为了确定糖基化位点占有率，通常使用蛋白N-糖苷酶F（PNGase F）将聚糖与蛋白分离，在此期间，先前糖基化的天冬酰胺经历脱酰胺以变成天冬氨酸。通过比较所得含有天冬氨酸的肽与含有非糖基化天冬酰胺的肽的丰度，可以评估糖基化位点占有率。然而，当非糖基化天冬酰胺发生自发化学脱酰胺时，这种方法可能给出不准确的结果。为了克服这一局限性，我们开发了一种新的方法来测量糖基化位点占有率，该方法不依赖于将糖基化肽转化为其去糖基化形式。具体而言，通过使用重同位素标记的内标物结合LC-MS分析同时定量总蛋白浓度和蛋白质的非糖基化部分，并准确测定位点占用程度。通过定量牛胎球蛋白糖基化位点的占有率证明了该方法的有效性。开发的方法是第一个在不使用PNGase F的情况下测量糖基化位点占有率的工作，并且可以与糖肽分析平行进行，因为聚糖在整个工作流程中保持完整。
N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.
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影响因子：
7
作者：
Huelsmeier, Andreas J.;Paesold-Burda, Patricie;Hennet, Thierry
通讯作者：
Hennet, Thierry
影响因子：
4.3
作者：
Petrescu, AJ;Milac, AL;Wormald, MR
通讯作者：
Wormald, MR
糖pep探测器：一种根据其电子传递分离光谱分配N连接糖肽的质谱数据的工具。
影响因子：
7.4
作者：
Zhu, Zhikai;Hua, David;Clark, Daniel F.;Go, Eden P.;Desaire, Heather
通讯作者：
Desaire, Heather
影响因子：
7
作者：
Zhou, Hui;Froehlich, John W.;Lee, Richard S.
通讯作者：
Lee, Richard S.
影响因子：
4.4
作者：
Segu, Zaneer M.;Hussein, Ahmed;Mechref, Yehia
通讯作者：
Mechref, Yehia