In planta chemical cross-linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis
In planta chemical cross-linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis
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拟南芥蛋白质结构和相互作用的植物化学交联和质谱分析
DOI：
10.1002/pmic.201500310
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发表时间：
2016
期刊：
影响因子：
3.4
通讯作者：
Li Ning
中科院分区：
文献类型：
--
作者：
Zhu Xinliang;Yu Fengchao;Yang Zhu;Liu Shichang;Dai Chen;Lu Xiaoyun;Liu Chenyu;Yu Weichuan;Li Ning
位点特异性化学交联结合质谱分析已成为研究蛋白质复合物三维结构和绘制蛋白质-蛋白质相互作用（PPI）的强大蛋白质组学方法。基于体外交联蛋白质的MS分析的成功，其已被广泛用于研究诱饵蛋白及其靶标在各种生物体中的特异性相互作用，我们报告了多细胞真核生物中体内化学交联和MS分析的工作流程。该方法优化了拟南芥体内蛋白质交联条件，建立了MudPIT富集交联肽的方法，并开发了一个完整的软件程序，即穷举交联肽鉴定工具（ECL），用于鉴定植物化学交联肽的MS谱。总之，从两个独立的生物学重复中鉴定出两对高置信度的体内交联肽。这项工作标志着在体内蛋白质三级结构和PPI在多细胞真核生物的研究开始的替代蛋白质组学方法。
Site‐specific chemical cross‐linking in combination with mass spectrometry analysis has emerged as a powerful proteomic approach for studying the three‐dimensional structure of protein complexes and in mapping protein–protein interactions (PPIs). Building on the success of MS analysis of in vitro cross‐linked proteins, which has been widely used to investigate specific interactions of bait proteins and their targets in various organisms, we report a workflow for in vivo chemical cross‐linking and MS analysis in a multicellular eukaryote. This approach optimizes the in vivo protein cross‐linking conditions inArabidopsis thaliana, establishes a MudPIT procedure for the enrichment of cross‐linked peptides, and develops an integrated software program, exhaustive cross‐linked peptides identification tool (ECL), to identify the MS spectra ofin plantachemical cross‐linked peptides. In total, two pairs of in vivo cross‐linked peptides of high confidence have been identified from two independent biological replicates. This work demarks the beginning of an alternative proteomic approach in the study of in vivo protein tertiary structure and PPIs in multicellular eukaryotes.