用于结构蛋白质组学的可丰富蛋白质足迹。

IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Jamison D Wolfer, Benjamin B Minkoff, Heather L Burch, Michael R Sussman
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引用次数: 0

摘要

蛋白质足迹法是研究蛋白质高阶结构以及通过在蛋白质上添加共价修饰与各种配体相互作用而引起的构象变化的有效方法。与其他提供单氨基酸级结构分辨率的方法(如低温电子显微镜、X 射线衍射和核磁共振)相比,基于质谱(MS)的方法对蛋白质数量和纯度的要求较低,因此具有优势。与其他基于质谱的蛋白质组学方法(如翻译后修饰分析)一样,在分析高度复杂的蛋白质组时,为了获得最佳灵敏度和序列覆盖率,富集技术已被证明是必要的。目前使用的共价标记足迹试剂(如羟基自由基和基于碳二亚胺的方法)还没有合适的富集方法,这限制了它们在全蛋白质组分析中的适用性。在此,我们报告了一种可富集的共价标记方法,该方法建立在常用于共价标记天冬氨酸和谷氨酸残基的 GEE/EDC 系统之上。含有炔基官能团的新型标记试剂可以通过铜催化叠氮-炔基环加成(CuAAC)"点击 "到任何含叠氮的分子上,从而实现富集或进一步标记。我们测试了多种含叠氮和炔烃的 GEE 类分子,确定最有效的方法是通过 EDC 促进甘氨酸丙炔酰胺 (GPA) 与蛋白质的偶联。我们报告的管道包括通过 CuAAC 与含有光可裂解连接体的市售生物素-叠氮化物进行点击,然后使用链霉亲和素树脂进行富集,随后在紫外光下进行裂解。通过对可点击的胺、偶联试剂、富集支架和方法的筛选,纯模式蛋白质的富集过程得到了优化,而且还被应用于从模式植物拟南芥中分离出来的复杂蛋白质混合物,这表明我们的方法最终可用于蛋白质组规模的蛋白质构象测量。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Enrichable Protein Footprinting for Structural Proteomics.

Protein footprinting is a useful method for studying protein higher order structure and conformational changes induced by interactions with various ligands via addition of covalent modifications onto the protein. Compared to other methods that provide single amino acid-level structural resolution, such as cryo-EM, X-ray diffraction, and NMR, mass spectrometry (MS)-based methods can be advantageous as they require lower protein amounts and purity. As with other MS-based proteomic methods, such as post-translational modification analysis, enrichment techniques have proven necessary for both optimal sensitivity and sequence coverage when analyzing highly complex proteomes. Currently used reagents for footprinting via covalent labeling, such as hydroxyl radicals and carbodiimide-based methods, do not yet have a suitable enrichment method, limiting their applicability to whole proteome analysis. Here, we report a method for enrichable covalent labeling built upon the GEE/EDC system commonly used to covalently label aspartic acid and glutamic acid residues. Novel labeling reagents containing alkynyl functionality can be "clicked" to any azido-containing molecule with copper-catalyzed azide-alkyne cycloaddition (CuAAC), allowing for enrichment or further labeling. Multiple azide- and alkyne-containing GEE-like molecules were tested, and the most efficient method was determined to be the EDC-facilitated coupling of glycine propargyl amide (GPA) to proteins. The pipeline we report includes clicking via CuAAC to a commercially available biotin-azide containing a photocleavable linker, followed by enrichment using a streptavidin resin and subsequent cleavage under ultraviolet light. The enrichment process was optimized through the screening of clickable amines, coupling reagents, and enrichment scaffolds and methods with pure model proteins and has also been applied to complex mixtures of proteins isolated from the model plant, Arabidopsis thaliana, suggesting that our method may ultimately be used to measure protein conformation on a proteomic scale.

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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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