前景:蛋白质-代谢物相互作用的全局分析

Q1 Agricultural and Biological Sciences
Ewelina M. Sokolowska, Dennis Schlossarek, Marcin Luzarowski, Aleksandra Skirycz
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引用次数: 13

摘要

小分子不仅是代谢的中间体,而且在信号传导和调控细胞代谢、生长发育等方面发挥着重要作用。尽管已经进行了一些系统的研究,但生物系统中蛋白质-小分子相互作用的真实程度仍然未知。利用大小分离技术(PROMIS)研究蛋白质-代谢物相互作用是一种非靶向、蛋白质组和代谢组范围内研究蛋白质-小分子相互作用的方法。该方法采用尺寸排除色谱法,然后使用蛋白质组学和代谢组学液相色谱-质谱法对收集的部分进行分析。假设与蛋白质结合的小分子会一起共分离,我们发现许多小分子与蛋白质共洗脱,这强烈表明形成了稳定的复合物。利用PROMIS,我们确定了已知的小分子-蛋白质复合物,例如酶和辅因子之间的复合物,并发现了新的相互作用。©2019作者。基本方案1:从植物材料中制备天然细胞裂解液支持方案:Bradford测定蛋白质浓度基本方案2:使用尺寸排除色谱分离分子复合物基本方案3:使用单步提取同时提取蛋白质和代谢物基本方案4:代谢组学分析基本方案5:蛋白质组学分析
本文章由计算机程序翻译,如有差异,请以英文原文为准。

PROMIS: Global Analysis of PROtein-Metabolite Interactions

PROMIS: Global Analysis of PROtein-Metabolite Interactions

Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein–small molecule interactions in biological systems remains unknown. PROtein–metabolite interactions using size separation (PROMIS) is a method for studying protein–small molecule interactions in a non-targeted, proteome- and metabolome-wide manner. This approach uses size-exclusion chromatography followed by proteomics and metabolomics liquid chromatography–mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co-fractionate together, we found numerous small molecules co-eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule–protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors.

Basic Protocol 1: Preparation of native cell lysate from plant material

Support Protocol: Bradford assay to determine protein concentration

Basic Protocol 2: Separation of molecular complexes using size-exclusion chromatography

Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single-step extraction protocol

Basic Protocol 4: Metabolomics analysis

Basic Protocol 5: Proteomics analysis

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来源期刊
Current protocols in plant biology
Current protocols in plant biology Agricultural and Biological Sciences-Plant Science
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期刊介绍: Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.
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