A meta-analysis of affinity purification-mass spectrometry experimental systems used to identify eukaryotic and chlamydial proteins at the Chlamydia trachomatis inclusion membrane.
A meta-analysis of affinity purification-mass spectrometry experimental systems used to identify eukaryotic and chlamydial proteins at the Chlamydia trachomatis inclusion membrane.
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DOI：
10.1016/j.jprot.2019.103595
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发表时间：
2019-11
影响因子：
3.3
通讯作者：
M. Olson;S. Ouellette;E. A. Rucks
中科院分区：
文献类型：
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作者：
M. Olson;S. Ouellette;E. A. Rucks
专性细胞内细菌病原体，沙眼衣原体，在称为包涵体的膜结合空泡内发育。亲和纯化-质谱（AP-MS）实验研究衣原体包涵体膜上发生的相互作用，最近，结合C。沙眼遗传学然而，四个AP-MS发表的报告中的每一个都使用了不同的实验方法或统计工具来鉴定定位在包涵体处的蛋白质。我们批判性地分析了每种实验方法，并对每项研究报告的统计学显著性蛋白质进行了荟萃分析，发现只有少数真核生物蛋白质在所有四种实验方法中被共同鉴定。使用相同的统计分析工具，INTeractome的显著性分析（SAINT），比较了两个类似进行的体内标记研究，这揭示了原始分析鉴定的显著蛋白质数量的差异。我们进一步研究了方法，以确定潜在的背景污染物蛋白质，统计分析后仍然存在。总的来说，这项荟萃分析强调了仔细控制和分析AP-MS数据的重要性，以便从这些不同的AP-MS实验方法中获得相关信息。本研究提供了重要的指导方针和考虑使用这种方法来研究细胞内病原体居住在一个membrane-bound compartment.SignificanceChlamydia沙眼衣原体，一个专性细胞内病原体，生长在一个膜结合的空泡称为inclusion。内含物中布满了细菌膜蛋白，这些蛋白可能与宿主细胞发生了许多相互作用。虽然维持细胞内生态位是至关重要的，理解宿主-病原体的相互作用，发生在包涵体膜是有限的，在纯化膜蛋白组分从受感染的宿主细胞的困难。溶解疏水性蛋白质所必需的实验程序不能维持瞬时的蛋白质-蛋白质相互作用。C.进展沙眼遗传学使我们和其他人能够使用各种实验方法与亲和纯化质谱法（AP-MS）相结合来研究在衣原体空泡或包涵体膜上发生的相互作用。这是第一次，两个小组发表了AP-MS研究使用相同的工具，抗坏血酸过氧化物酶邻近标记系统（APEX 2），克服了过去的实验限制，因为膜蛋白相互作用在感染的情况下在体内标记。该系统的实用性突出了其研究衣原体III型分泌的包涵体膜蛋白（公司）的相互作用的能力。Inc在C.沙眼感染当仔细控制和分析时，获得的数据可以产生大量有用的信息。在这里，我们批判性地分析了四项先前发表的研究，包括与衣原体-宿主相互作用相关的AP-MS数据集的统计分析，以将数据置于背景中，并确定解释这些类型的复杂输出的最佳实践。
The obligate intracellular bacterial pathogen, Chlamydia trachomatis, develops within a membrane-bound vacuole termed the inclusion. Affinity purification-mass spectrometry (AP-MS) experiments to study the interactions that occur at the chlamydial inclusion membrane have been performed and, more recently, combined with advances inC. trachomatisgenetics. However, each of the four AP-MS published reports used either different experimental approaches or statistical tools to identify proteins that localize at the inclusion. We critically analyzed each experimental approach and performed a meta-analysis of the reported statistically significant proteins for each study, finding that only a few eukaryotic proteins were commonly identified between all four experimental approaches. The two similarly conducted in vivo labeling studies were compared using the same statistical analysis tool, Significance Analysis of INTeractome (SAINT), which revealed a disparity in the number of significant proteins identified by the original analysis. We further examined methods to identify potential background contaminant proteins that remain after statistical analysis. Overall, this meta-analysis highlights the importance of carefully controlling and analyzing the AP-MS data so that pertinent information can be obtained from these various AP-MS experimental approaches. This study provides important guidelines and considerations for using this methodology to study intracellular pathogens residing within a membrane-bound compartment.SignificanceChlamydia trachomatis, an obligate intracellular pathogen, grows within a membrane-bound vacuole termed the inclusion. The inclusion is studded with bacterial membrane proteins that likely orchestrate numerous interactions with the host cell. Although maintenance of the intracellular niche is vital, an understanding of the host-pathogen interactions that occur at the inclusion membrane is limited by the difficulty in purifying membrane protein fractions from infected host cells. The experimental procedures necessary to solubilize hydrophobic proteins fail to maintain transient protein-protein interactions. Advances inC. trachomatisgenetics has allowed us and others to use various experimental approaches in combination with affinity purification mass spectrometry (AP-MS) to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. For the first time, two groups have published AP-MS studies using the same tool, the ascorbate peroxidase proximity labeling system (APEX2), which overcomes past experimental limitations because membrane protein interactions are labeled in vivo in the context of infection. The utility of this system is highlighted by its ability to study chlamydial type III secreted inclusion membrane protein (Inc) interactions. Incs act as the mediators of host-pathogen interactions at the inclusion duringC. trachomatisinfection. When carefully controlled and analyzed, the data obtained can yield copious amounts of useful information. Here, we critically analyzed four previously published studies, including statistical analysis of AP-MS datasets related toChlamydia-host interactions, to contextualize the data and to identify the best practices in interpreting these types of complex outputs.