Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting.
Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting.
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DOI：
10.1021/acs.biochem.8b00648
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发表时间：
2018-10-09
期刊：
影响因子：
2.9
通讯作者：
Pratt MR
中科院分区：
文献类型：
--
作者：
Darabedian N;Thompson JW;Chuh KN;Hsieh-Wilson LC;Pratt MR
O-linked β- n -乙酰氨基葡萄糖（o - glcnac酰化）对细胞内蛋白的动态修饰在许多细胞过程中起着至关重要的作用。虽然已经开发了多种检测O-GlcNAc的方法，但监测糖基化化学计量学和状态（即单-、二-等，OGlcNAcylated）的技术很少。测量给定底物蛋白上的o - glcn酰化水平对于理解这种关键修饰的生物学和优先考虑底物的功能研究非常重要。解决这一限制的一个有效方法是将具有一定分子质量的聚乙二醇聚合物化学酶安装到o - glcn酰化蛋白上。这些“质量标签”在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）过程中产生蛋白质迁移的变化，可以通过Western blotting检测。然而，由于缺乏市售试剂和定义明确的蛋白质标准，科学界对这种方法的广泛采用受到了限制。在这里，我们开发了一种“点击化学”方法来使用完全商业化的试剂，并使用半合成的o - glcn酰化蛋白来证实该方法的准确性。我们的研究建立了一个新的，快速的实验工作流程和标准化的方法，可以很容易地利用非专业人员来量化任何细胞或组织裂解液中内源性蛋白质的O-GlcNAc化学计量和状态。
The dynamic modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAcylation) plays critical roles in many cellular processes. Although various methods have been developed for O-GlcNAc detection, there are few techniques for monitoring glycosylation stoichiometry and state (i.e., mono-, di-, etc., OGlcNAcylated). Measuring the levels of O-GlcNAcylation on a given substrate protein is important for understanding the biology of this critical modification and for prioritizingsubstrates for functional studies. One powerful solution to this limitation involves the chemoenzymatic installation of polyethylene glycol polymers of defined molecular mass onto O-GlcNAcylated proteins. These “mass tags” produce shifts in protein migration during sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) that can be detected by Western blotting. Broad adoption of this method by the scientific community has been limited, however, by a lack of commercially available reagents and well-defined protein standards. Here, we develop a “click chemistry” approach to this method using entirely commercial reagents and confirm the accuracy of the approach using a semisynthetic O-GlcNAcylated protein. Our studies establish a new, expedited experimental workflow and standardized methods that can be readily utilized by non-experts to quantify the O-GlcNAc stoichiometry and state on endogenous proteins in any cell or tissue lysate.
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DOI：
10.1002/path.4929
发表时间：
2017-09
期刊：
The Journal of pathology
影响因子：
--
作者：
Wang S;Yang F;Petyuk VA;Shukla AK;Monroe ME;Gritsenko MA;Rodland KD;Smith RD;Qian WJ;Gong CX;Liu T
通讯作者：
Liu T
DOI：
10.1083/jcb.201501101
发表时间：
2015-03-30
期刊：
The Journal of cell biology
影响因子：
--
作者：
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通讯作者：
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影响因子：
5.3
作者：
O'Donnell, N;Zachara, NE;Marth, JD
通讯作者：
Marth, JD
影响因子：
7.8
作者：
Chuh, Kelly N.;Pratt, Matthew R.
通讯作者：
Pratt, Matthew R.
影响因子：
7
作者：
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通讯作者：
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