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首页> 美国卫生研究院文献>other >Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics
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Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics

机译：O-GlcNAc蛋白质组学中铜（I）催化炔叠氮化物环加成（CuAAC）和应变促进炔叠氮化物环加成（SPAAC）的比较分析

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O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein post-translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide-alkyne bioorthogonal reactions in click chemistry: copper-catalyzed azide-alkyne cycloaddition (CuAAC) with Biotin-Diazo-Alkyne and stain-promoted azide-alkyne cycloaddition (SPAAC) with Biotin-DIBO-Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O-GlcNAc modification were separated by SDS-PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O-GlcNAc modified proteins were identified with Biotin-Diazo-Alkyne conjugated sample and 188 proteins with Biotin-DIBO-Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin-Diazo-Alkyne conjugates and 46 verified proteins from Biotin-DIBO-Alkyne conjugates could be found in the O-GlcNAc modified proteins database dbOGAP (). These results suggested that CuAAC with Biotin-Diazo-Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity-based enrichment, SDS-PAGE separation, and mass spectrometry, would be adaptable for other post-translationally modified proteins in proteomics.
机译：O-连接的β-N-乙酰氨基葡萄糖（O-GlcNAc）在许多生物体中作为一种重要的蛋白质翻译后修饰而出现。它参与各种细胞过程，例如营养物感应，蛋白质降解，基因表达，并与许多人类疾病有关。尽管其重要性，鉴定O-GlcNAcylated蛋白仍是蛋白质组学的主要挑战。在这里，使用过乙酰化的N-叠氮基乙酰氨基葡萄糖（Ac4GlcNAz）作为生物正交化学处理，我们描述了一种基于凝胶的质谱方法，用于鉴定A549细胞中具有O-GlcNAc修饰的蛋白质。此外，我们对点击化学中两种叠氮化物-炔烃生物正交反应模式的标记效率进行了比较：铜与生物素-重氮-炔烃的叠氮化物-炔烃环加成（CuAAC）和污渍促进的叠氮化物-炔烃环化加成（SPAAC）生物素-DIBO-炔烃。在体外与点击化学结合并通过链霉亲和素树脂富集后，通过SDS-PAGE分离具有O-GlcNAc修饰的蛋白质，并通过质谱鉴定。蛋白质组学数据分析显示，与生物素-重氮-炔烃结合的样品鉴定出229个推定的O-GlcNAc修饰蛋白，与生物素-DIBO-炔烃结合的样品鉴定出188个蛋白质，其中114个蛋白质重叠。有趣的是，在O-GlcNAc修饰的蛋白质数据库dbOGAP（）中可以找到74种从生物素-重氮-炔烃结合物鉴定的蛋白质和46种从生物素-DIBO-炔烃结合物鉴定的蛋白质。这些结果表明，与SPAAC相比，具有生物素-重氮-炔烃的CuAAC代表了蛋白质组学中一种更强大的方法，具有更高的蛋白质鉴定和更好的准确性。蛋白质组学的可信度还通过分子功能和细胞成分基因本体论（GO）得以证实。总之，我们在这里报道的结合代谢标记，点击化学，基于亲和力的富集，SDS-PAGE分离和质谱的方法将适用于蛋白质组学中其他翻译后修饰的蛋白质。

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