用于蛋白质足迹自上而下分析的MALDI源内衰变(ISD)方法。

IF 16.4 1区 化学 Q1 CHEMISTRY, MULTIDISCIPLINARY
Accounts of Chemical Research Pub Date : 2023-10-01 Epub Date: 2023-09-26 DOI:10.1177/14690667231202695
Ruidong Jiang, Don L Rempel, Michael L Gross
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引用次数: 0

摘要

不可逆蛋白质足迹是一种基于质谱的方法,其中蛋白质的溶剂可及位点被修饰以评估高阶蛋白质结构。可以通过确定修改的位置和范围来获得结构见解。获得“足迹”的通常方法是通过自下而上的LC-MS/MS分析蛋白质。在这种方法中,蛋白质被消化以产生肽的混合物,然后通过LC分离,然后通过MS/MS定位修饰位点。该过程消耗大量时间,并且对于需要快速和高通量分析的应用程序来说很难加速。在这里,我们描述了使用基质辅助激光解吸/电离(MALDI)源衰变(ISD)通过自上而下的方法分析足迹小测试蛋白(泛素)。基质辅助激光解吸/电离很容易适用于高通量分析,自上而下的策略可以避免长时间的蛋白水解和LC分离。我们用模型肽优化了该方法,然后证明了其在泛素上应用于两种足迹的可行性。我们发现MALDI ISD可以为小蛋白质产生一套全面的片段离子,以快速的方式提供足迹信息,并给出与既定方法一致的结果,并作为蛋白质溶剂可及性的粗略测量。为了帮助实现MALDI方法,我们开发了一种自上而下处理ISD数据的方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Toward a MALDI in-source decay (ISD) method for top-down analysis of protein footprinting.

Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the "footprint" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.

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来源期刊
Accounts of Chemical Research
Accounts of Chemical Research 化学-化学综合
CiteScore
31.40
自引率
1.10%
发文量
312
审稿时长
2 months
期刊介绍: Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance. Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.
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