基因组水平和转录组水平的信息不能准确反映蛋白质组水平的信息,原因在于转录后的调控也会调节基因的表达。蛋白质翻译后修饰(PTMs)也会影响蛋白质的功能,并且细胞中的大多数蛋白质会与蛋白质、RNAs、金属离子或其他小分子等形成复合物而起作用,故研究蛋白质组的特征将有利于揭示蛋白质、蛋白质PTMs和蛋白质复合物在生物体发育和疾病过程中的作用机制。
自上而下的蛋白质组学(Top-down proteomics)是在完整蛋白质水平上对蛋白质组进行大规模表征的一种常用方法。此方法对于从同一基因衍生的各种蛋白质分子和蛋白质形式因基因水平发生变化、RNA水平的选择性剪接、以及蛋白质水平的PTMs进行高分辨率表征是非常有用的。人类蛋白质组中的蛋白质体(proteoform)数目估计超过100万个。在质谱分析(MS)之前对蛋白质进行高效分离对于深入的、高分辨率分析自上而下的蛋白质组学是必不可少的。毛细管区带电泳(CZE)被认为是自上而下蛋白质中对蛋白质进行高效分离的有力工具。
在此次演讲中,将介绍应用新型超低流速鞘流液接口和离子源(深圳市永道致远科学技术有限公司CMP Scientific品牌EMASS-II ion source)的CZE-MS技术而开发的自上而下蛋白质组学,并对自上而下蛋白质组学的历史及其主要挑战和机遇,对如何提高CZE-MS对自上而下蛋白质组学的灵敏度和峰容量的方法进行讨论,和对基于CZE-MS的自上而下蛋白质组学的未来发展方向进行一些思考。
Genome-level and transcriptome-level information cannot accurately reflect proteome level information because post-transcriptional regulations modulate gene expression, because protein post-translational modifications (PTMs) influence protein function, and because most proteins in cells function as complexes with proteins, RNAs, metals or other small molecules. Characterization of the proteome is imperative to understand the roles played by proteins, protein PTMs andprotein complexes in development and diseases.
Top-down proteomics is a well-known strategy for large-scale characterization of proteome at the intact protein level and is very useful for high-resolution characterization of proteoforms that represent all kinds of protein molecules derived from the same gene due to gene-level variations, RNA-level alternative splicing, and protein-level PTMs.
The number of proteoforms in the human proteome has been estimated to be over 1 million.High-capacity separation of proteoforms before mass spectrometry (MS)is essential for deep and high-resolution top-down proteomics. Capillary zone electrophoresis (CZE)-MS has been recognized as a powerful tool for top-down proteomics.
In my talk, I will introduce the history of CZE-MS-based top-down proteomics, talk about the major challenges and opportunities of CZE-MS for top-down proteomics, discuss our recent progress in improving CZE-MS regarding sensitivity and peak capacity for top-down proteomics, and summarize the talk with some thoughts about the future directions of CZE MS-based top-down proteomics.