Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications
Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications
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DOI：
10.1074/mcp.m200048-mcp200
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发表时间：
2002-10-01
影响因子：
7
通讯作者：
Hart, GW
中科院分区：
文献类型：
--
作者：
Wells, L;Vosseller, K;Hart, GW
蛋白质翻译后修饰位点的识别是蛋白质组学研究中的一个主要挑战。O-连接的β-N-乙酰葡糖胺（O-GlcNAc）是一种动态核质修饰，与经典的复杂O-糖基化相比，更类似于磷酸化。我们描述了一种基于质谱的方法，用于识别O-GlcNAc修饰的位点，该方法依赖于温和的β-消除，然后与二硫苏糖醇（BEMAD）进行Michael加成。使用合成肽，我们还表明，生物素戊胺可以取代二硫苏糖醇作为亲核试剂。修饰的肽可以通过亲和色谱法有效地富集，并且可以使用串联质谱法绘制位点。这种相同的方法可以应用到映射的丝氨酸和苏氨酸磷酸化位点，我们提供了一种策略，使用修饰特异性抗体和酶来区分这两个翻译后修饰。BEMAD的方法进行了验证，通过映射三个先前确定的O-GlcNAc网站，以及三个新的网站，从大鼠脑纯化的突触蛋白1。然后将BEMAD用于纯化的核孔复合物制剂，以绘制核纤层蛋白B受体和核孔蛋白Nup 155上O-GlcNAc修饰的新位点。该方法适用于进行定量质谱分析，也可用于定量半胱氨酸残基。此外，我们的研究强调了在使用β-消除/Michael加成方法绘制丝氨酸和苏氨酸翻译后修饰位点时区分O-磷酸与O-GlcNAc的重要性。
Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked, beta-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild beta-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin 1 purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using beta-elimination/Michael addition methods.