Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, the Proteomics Center of Excellence, and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University , Evanston, Illinois 60208, United States.
Anal Chem. 2015 Mar 3;87(5):3032-8. doi: 10.1021/ac504678d. Epub 2015 Feb 20.
The cadre of protein complexes in cells performs an array of functions necessary for life. Their varied structures are foundational to their ability to perform biological functions, lending great import to the elucidation of complex composition and dynamics. Native separation techniques that are operative on low sample amounts and provide high resolution are necessary to gain valuable data on endogenous complexes. Here, we detail and optimize the use of tube gel separations to produce samples proven compatible with native, multistage mass spectrometry (nMS/MS). We find that a continuous system (i.e., no stacking gel) with a gradient in its extent of cross-linking and use of the clear native buffer system performs well for both fractionation and native mass spectrometry of heart extracts and a fungal secretome. This integrated advance in separations and nMS/MS offers the prospect of untargeted proteomics at the next hierarchical level of protein organization in biology.
细胞中蛋白质复合物的组成执行着一系列生命所必需的功能。它们多样的结构是其执行生物功能的基础，这对于阐明复合物的组成和动态具有重要意义。对于低样本量和高分辨率的内源复合物，需要使用能够进行有效分离的天然分离技术来获取有价值的数据。在这里，我们详细介绍并优化了管凝胶分离的使用，以产生与天然多阶段质谱（nMS/MS）兼容的样品。我们发现，具有交联度梯度的连续系统（即没有堆积胶）和使用透明天然缓冲液系统，对于心脏提取物和真菌分泌液的分级分离和天然质谱分析都非常有效。这种分离和 nMS/MS 的综合进展，为生物学中蛋白质组织的下一个层次的靶向蛋白质组学提供了前景。
