A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies.
A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies.
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用于亲和力分离的内源性蛋白质组件的天然质谱分析的强大工作流程。
DOI：
10.1021/acs.analchem.5b04477
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发表时间：
2016-03-01
影响因子：
7.4
通讯作者：
Chait BT
中科院分区：
文献类型：
--
作者：
Olinares PD;Dunn AD;Padovan JC;Fernandez-Martinez J;Rout MP;Chait BT
大多数细胞活动的中心参与者是大分子的集合体。这些组件的结构和功能表征需要了解它们的亚基化学计量学和亚基间连接性。获取这种信息的最直接手段之一是所谓的自然质谱学(MS)，其中准确地确定完整组件及其部件的质量。将天然MS应用于内源性蛋白质组装的研究尤其令人感兴趣--即那些组分蛋白质在其自然功能状态下以内源性水平表达，而不是经典结构研究中通常采用的过度表达(有时是部分)构建，其组装可能会引入化学计量学伪影和其他有害的影响。到目前为止，天然MS在阐明内源性蛋白质复合体方面的应用一直受到限制，因为难以在足够高的浓度下获得原始的细胞来源的组装，以便进行有效的分析。为了解决这一挑战，我们在这里提出了一个强大的工作流程，结合快速和有效的亲和分离内源蛋白质复合体与灵敏的天然MS读数。由此产生的工作流程有可能提供关于内源蛋白质组件的化学计量学和亚基间连接性的丰富数据--这些信息是成功地对生物系统进行综合结构阐明的关键。
The central players in most cellular events are assemblies of macromolecules. Structural and functional characterization of these assemblies requires knowledge of their subunit stoichiometry and intersubunit connectivity. One of the most direct means for acquiring such information is so-called native mass spectrometry (MS), wherein the masses of the intact assemblies and parts thereof are accurately determined. It is of particular interest to apply native MS to the study of endogenous protein assemblies—i.e., those wherein the component proteins are expressed at endogenous levels in their natural functional states rather than the overexpressed (sometimes partial) constructs commonly employed in classical structural studies, whose assembly can introduce stoichiometry artifacts and other unwanted effects. To date, the application of native MS to the elucidation of endogenous protein complexes has been limited by the difficulty in obtaining pristine cell-derived assemblies at sufficiently high concentrations for effective analysis. To address this challenge, we present here a robust workflow that couples rapid and efficient affinity isolation of endogenous protein complexes with a sensitive native MS readout. The resulting workflow has the potential to provide a wealth of data on the stoichiometry and intersubunit connectivity of endogenous protein assemblies—information that is key to successful integrative structural elucidation of biological systems.