Departments of Chemistry and Molecular Biosciences, The Chemistry of Life Processes Institute, The Proteomics Center of Excellence, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL 60208, United States.
Departments of Chemistry and Molecular Biosciences, The Chemistry of Life Processes Institute, The Proteomics Center of Excellence, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL 60208, United States.
Biochem Biophys Res Commun. 2014 Mar 21;445(4):683-93. doi: 10.1016/j.bbrc.2014.02.041. Epub 2014 Feb 17.
The rise of the "Top Down" method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious "inference" problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.
基于质谱的蛋白质组学领域中“自上而下”方法的兴起，为蛋白质的特征描述和鉴定带来了一个充满希望和挑战的新时代。将完整的蛋白质注入质谱仪可以更好地描述翻译后修饰，并避免与基于肽的蛋白质组学相关的一些严重的“推断”问题。然而，要成功地将自上而下的方法应用于内源性或其他具有生物学意义的样品，通常需要在质谱分析之前使用一种或多种分离形式，而这些形式对于完整蛋白质 MS 来说才刚刚开始成熟。仪器的最新进展与使用光子和电子进行新的离子碎裂相结合，使得即使在几年前还难以处理的情况下，也能够更好地（通常是完全地）对蛋白质进行特征描述。最后，使用天然电喷雾质谱法已显示出在 100 kDa 至 1 MDa 范围内鉴定和表征完整蛋白质复合物的巨大潜力，有望在未来几年内实现对内源性蛋白质组装的完整组成分析。
