Mass spectrometry-based shotgun proteomic analysis of C. elegans protein complexes.
Mass spectrometry-based shotgun proteomic analysis of C. elegans protein complexes.
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DOI：
10.1895/wormbook.1.171.1
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发表时间：
2014-06-24
期刊：
影响因子：
--
通讯作者：
Yates JR 3rd
中科院分区：
文献类型：
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作者：
Fonslow BR;Moresco JJ;Tu PG;Aalto AP;Pasquinelli AE;Dillin AG;Yates JR 3rd
基于质谱 (MS) 的鸟枪蛋白质组学是研究秀丽隐杆线虫蛋白质的一项支持技术。当与免疫共沉淀 (CoIP) 结合时，可以发现蛋白质之间新的相互作用和功能。我们提供蛋白质复合物的一般背景及其分析方法，以及最终定义蛋白质复合物的可识别成分的蛋白质的生命周期和相互作用类型。我们重点介绍传统的生化方法来评估复合物是否足够纯净和丰富，适合鸟枪法蛋白质组学分析。我们提出了两个来自秀丽隐杆线虫蛋白质复合物的 CoIP-MS 案例研究，使用内源性和融合蛋白抗体来说明其分析的重要方面。我们讨论质量准确度和分辨率存在差异的质谱仪结果，以及可以从生成的数据中提取的相关信息，例如蛋白质相对丰度、翻译后修饰和识别置信度。最后，我们说明比较分析如何揭示候选结合伙伴以进行生物学后续和验证。本章应作为 Walhout 和 Boulton 2006 年分子生物学和生物化学章节的补充和扩展，该章节描述了用于质谱分析的蛋白质复合物的串联亲和纯化 (TAP)。
Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a compliment and extension to Walhout and Boulton’s 2006 Molecular Biology and Biochemistry Chapter which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry.