Shaoshuai Xie, Leila Saba, Hua Jiang, Omar R Bringas, Mehrnoosh Oghbaie, Luciano Di Stefano, Vadim Sherman, John LaCava
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引用次数: 0
摘要
当使用全基因组的亲和标记细胞集时,免疫沉淀(IP)与质谱联用可有效绘制蛋白质-蛋白质相互作用图。此类研究记录了生理蛋白质复合物的大部分组成,提供了 "相互作用组 "草案;然而,蛋白质复合物的许多组成成分仍然未知。存在这一空白的部分原因是高通量方法无法优化每个 IP。IP优化的一个关键挑战是在从细胞转移到试管的过程中稳定体内的相互作用;如果做不到这一点,就会在IP过程中失去真正的相互作用,进而导致检测失败。我们的高内涵筛选方法探索了体外化学条件与 IP 结果之间的关系,从而实现了捕获目标大分子组装的条件的快速经验优化。
Immunoprecipitation (IP) coupled with mass spectrometry effectively maps protein-protein interactions when genome-wide, affinity-tagged cell collections are used. Such studies have recorded significant portions of the compositions of physiological protein complexes, providing draft 'interactomes'; yet many constituents of protein complexes still remain uncharted. This gap exists partly because high-throughput approaches cannot optimize each IP. A key challenge for IP optimization is stabilizing in vivo interactions during the transfer from cells to test tubes; failure to do so leads to the loss of genuine interactions during the IP and subsequent failure to detect. Our high-content screening method explores the relationship between in vitro chemical conditions and IP outcomes, enabling rapid empirical optimization of conditions for capturing target macromolecular assemblies.
期刊介绍:
BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies.
Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers:
Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions
Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices
Surveys of technical approaches related to broad fields of research
Reviews discussing advancements in techniques and methods related to broad fields of research
Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.