毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

Capillary electrophoresis-mass spectrometry and its application to proteomic analysis

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱(MS)鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳(CE)技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳(CZE)、毛细管等电聚焦(CIEF)、毛细管电色谱(CEC)等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的“自下而上(bottom-up)”策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段(尤其是翻译后修饰肽段)的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下“top-down”策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱(native MS)是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。

Proteomic analysis plays an important role in basic biological studies and precision medicine.However,real samples contain numerous proteins with a wide dynamic distribution range.Such high complexity of the samples has a drastic effect on the identification coverage of proteins.Consequently,with advancements in mass spectrometry(MS)technology,concomitant improvements in separation technologies for simplifying the sample should be critical.With the advantages of small sample loading volume,high separation efficiency,and high speed,capillary electrophoresis(CE)coupled to MS has been gained much attention in the field of proteomics research.A nanoflow sheath liquid interface and a sheathless interface have been developed and commercialized,boosting the development of the CE-MS technology.Capillary zone electrophoresis(CZE),capillary isoelectric focusing(CIEF),and capillary electrochromatography(CEC)have been successfully combined with MS,and CZE-MS has widespread application.In proteomic research,the“bottom-up”strategy,which is based on the separation and identification of enzymatic peptides,is widely applied.With the limit of detection as low as 1 zmol for peptides,CE-MS has been successfully applied to single-cell proteomic analysis.Besides,CE is complementary to reversed-phase liquid chromatography(RPLC),providing a new approach for the separation and identification of peptides with similar hydrophobic properties(especially,post-translational peptides).The“top-down”strategy,which is based on the separation and identification of intact proteins,can directly provide more accurate and complete information about proteins.For protein separation,CE is advantageous in terms of the high separation resolution and high protein recovery,thereby improving the sensitivity and coverage of protein identification.Native MS enables successful identification and characterization of protein complexes under nondenaturing conditions.Because of the good compatibility of CE with MS,attempts have been made to use CE coupled with native MS for the separation and identification of protein complexes.In this review,the development of the CE-MS technology is first reported,including a robust and sensitive CE-MS interface,and a separation mode coupled to MS.Then,the application of the CE-MS technology to“bottom-up”,“top-down”and native MS analysis is discussed.The superiority of CE-MS in proteomic analysis is also emphasized.Finally,the promising future prospects of CE-MS are discussed.