Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag.
Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag.
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DOI：
10.1039/c6mb00138f
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发表时间：
2016-05-24
影响因子：
--
通讯作者：
Hsieh-Wilson LC
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文献类型：
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作者：
Griffin ME;Jensen EH;Mason DE;Jenkins CL;Stone SE;Peters EC;Hsieh-Wilson LC
蛋白质的丝氨酸或苏氨酸残基与单个N-乙酰葡糖胺单糖的翻译后修饰（O-GlcNAc化）对于细胞存活和功能是必不可少的。然而，由于从复杂样品中富集和检测O-GlcNAc酰化肽的困难，相对较少的O-GlcNAc修饰位点已被定位。在这里，我们描述了一种改进的方法来定量标记和富集O-GlcNAcylated蛋白质的位点识别。化学酶标记后进行铜（I）催化的叠氮化物-炔环加成（CuAAC）安装了一种新的质谱（MS）兼容接头，旨在从细胞裂解物中轻松纯化O-GlcNAc酰化蛋白质。接头还允许定量释放O-GlcNAc酰化蛋白质用于下游MS分析。我们通过明确识别蛋白质α-晶状体蛋白和O-GlcNAc转移酶（OGT）上的几个已建立的O-GlcNAc位点以及发现OGT上的新的、先前未报道的位点来验证该方法。值得注意的是，OGT上的这些新位点位于蛋白质的关键功能域中，强调了这种位点识别方法如何揭示蛋白质活性和调控的重要生物学见解。蛋白质O-GlcNAc化位点的定位对于理解这种丰富的翻译后修饰的功能至关重要。本文中，采用了一种新的方法，利用化学可切割的Dde-基标签定量标记和释放O-GlcNAc酰化蛋白质，导致质谱鉴定以前未鉴定的糖基化位点。
The post-translational modification of serine or threonine residues of proteins with a single N-acetylglucosamine monosaccharide (O-GlcNAcylation) is essential for cell survival and function. However, relatively few O-GlcNAc modification sites have been mapped due to the difficulty of enriching and detecting O-GlcNAcylated peptides from complex samples. Here we describe an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labelling followed by copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) installs a new mass spectrometry (MS)-compatible linker designed for facile purification of O-GlcNAcylated proteins from cell lysates. The linker also allows quantitative release of O-GlcNAcylated proteins for downstream MS analysis. We validate the approach by unambiguously identifying several established O-GlcNAc sites on the proteins α-crystallin and O-GlcNAc transferase (OGT) as well as discovering new, previously unreported sites on OGT. Notably, these novel sites on OGT lie in key functional domains of the protein, underscoring how this site identification method may reveal important biological insights into protein activity and regulation. Mapping sites of protein O-GlcNAcylation is crucial for understanding the functions of this abundant post-translational modification. Herein, a novel approach utilizing a chemically cleavable Dde-based tag is employed to quantitatively label and release O-GlcNAcylated proteins, leading to the mass spectrometric identification of previously unidentified glycosylation sites.