In-depth structural proteomics integrating mass spectrometry and polyacrylamide gel electrophoresis
In-depth structural proteomics integrating mass spectrometry and polyacrylamide gel electrophoresis
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DOI：
10.3389/frans.2022.1107183
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发表时间：
2023-01
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影响因子：
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通讯作者：
Nobuaki Takemori;Ayako Takemori
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文献类型：
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作者：
Nobuaki Takemori;Ayako Takemori
建立一种高灵敏的方法来获取体内蛋白质和蛋白质复合体的结构信息一直是结构生物学中的一个技术挑战。近年来，利用自上而下的质谱学、自然质谱学和交联质谱学等方法进行蛋白质结构分析的方法已经发展起来，这些技术已经成为获得细胞蛋白质组全面结构信息的最有前途的方法。然而，仅通过MS获得的信息主要来自体内丰富的蛋白质组分，对低丰度组分的数据不足。对于那些低丰度组分的检测，先进行样品分馏再进行质谱分析是非常有效的，因为它可以降低样品的复杂性。聚丙烯酰胺凝胶电泳法(PAGE)是一种简单易行的蛋白质分离技术，被广泛应用于生化实验，也是一种很有前途的结构蛋白质组学分离工具。在凝胶中回收蛋白质的困难一直是一个障碍，到目前为止限制了其在结构质谱学中的应用。随着2020年出现的一种非常有效的蛋白质被动提取方法--Peppi-MS的突破，各种基于PAGE的蛋白质组分离工作流程被开发出来，导致了结构质谱学和PAGE的快速整合。在本文中，我们描述了一种简单而廉价的基于PAGE的样品制备策略，该策略加速了结构质谱学在生命科学研究中的广泛应用，并讨论了利用PAGE实现深度结构蛋白质组学的未来前景。
The establishment of a highly sensitive method for obtaining structural information on proteins and protein complexes in vivo has long been a technological challenge in structural biology. In recent years, protein structure analysis approaches using top-down mass spectrometry, native mass spectrometry, and cross-linking mass spectrometry, among others, have been developed, and these techniques have emerged as the most promising methods for obtaining comprehensive structural information on the cellular proteome. However, information obtained by MS alone is derived mainly from protein components that are abundant in vivo, with insufficient data on low abundance components. For the detection of those low abundance components, sample fractionation prior to mass spectrometry is highly effective because it can reduce the complexity of the sample. Polyacrylamide gel electrophoresis (PAGE), which is widely used in biochemical experiments, is an excellent technique for protein separation in a simple straightforward procedure and is also a promising fractionation tool for structural proteomics. The difficulty of recovering proteins in gels has been an obstacle, thus far limiting its application to structural mass spectrometry. With the breakthrough of PEPPI-MS, an exceptionally efficient passive extraction method for proteins in gels that appeared in 2020, various PAGE-based proteome fractionation workflows have been developed, resulting in the rapid integration of structural mass spectrometry and PAGE. In this paper, we describe a simple and inexpensive PAGE-based sample preparation strategy that accelerates the broad use of structural mass spectrometry in life science research, and discuss future prospects for achieving in-depth structural proteomics using PAGE.