{"title":"基于质谱的蛋白质组学研究线粒体转运蛋白的突变体和相互作用组。","authors":"Silke Oeljeklaus, Lakshita Sharma, Julian Bender, Bettina Warscheid","doi":"10.1016/bs.mie.2024.07.059","DOIUrl":null,"url":null,"abstract":"<p><p>The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are \"born\" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. In the last unifying part, we describe methods for LC-MS, data processing, statistical analysis and visualization of quantitative MS data, and provide a Python code for effective, customizable analysis.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins.\",\"authors\":\"Silke Oeljeklaus, Lakshita Sharma, Julian Bender, Bettina Warscheid\",\"doi\":\"10.1016/bs.mie.2024.07.059\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are \\\"born\\\" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. 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引用次数: 0
摘要
线粒体的多种功能受其蛋白质组支配,根据生物体的不同,蛋白质组由 1000-1500 种蛋白质组成。然而,只有少数蛋白质是在线粒体内合成的,而大多数蛋白质则是在线粒体外 "诞生 "的。为了到达目的地,这些线粒体蛋白质要遵循由线粒体转运酶网络建立的特定导入路线。要了解线粒体生物学以及线粒体如何融入细胞网络,就必须详细了解不同转运酶的作用和相互作用。事实证明,质谱法(MS)可以有效研究转运酶网络的组成、功能、相互作用以及功能障碍引起的细胞反应。在本章中,我们将为线粒体转运蛋白的突变体和相互作用组的质谱功能分析提供量身定制的方案。在第一部分中,我们举例说明了基于质谱的转位突变体蛋白质组学分析,用于描述中心转位蛋白 TOMM40 缺失后的人类线粒体导入组。该方案包括代谢稳定同位素标记、TOMM40基因敲除、线粒体组分制备和液相色谱-质谱样品制备。为了进行深度质谱分析,我们介绍了通过高pH反相液相色谱法对肽混合物进行预分馏的方法。在第二部分中,我们概述了一种亲和纯化 MS 方法,以揭示孤儿蛋白与转运酶 TIM23 的关联。该方案包括从线粒体组分中亲和性纯化蛋白复合物的 FLAG 标记,以及用于相互作用组分析的下游样品制备。在最后的统一部分中,我们介绍了定量 MS 数据的 LC-MS、数据处理、统计分析和可视化方法,并提供了有效、可定制分析的 Python 代码。
Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins.
The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are "born" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. In the last unifying part, we describe methods for LC-MS, data processing, statistical analysis and visualization of quantitative MS data, and provide a Python code for effective, customizable analysis.
期刊介绍:
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.