Fischer Matthew S, Rogers Holden T, Chapman Emily A, Jin Song, Ge Ying
Department of Chemistry, University of Wisconsin─Madison, 1101 University Ave., Madison, Wisconsin 53706, United States.
Department of Cell and Regenerative Biology, University of Wisconsin─Madison, 1111 Highland Ave., Madison, Wisconsin 53705, United States.
Anal Chem. 2025 Jul 1;97(25):13663-13671. doi: 10.1021/acs.analchem.5c02341. Epub 2025 Jun 20.
Protein complexes are essential for virtually all biological processes, yet their structural characterization remains a major challenge due to their heterogeneous, dynamic nature and the complexity of the proteome. Native top-down mass spectrometry (nTDMS) has emerged as a powerful tool for comprehensive structural characterization of purified protein complexes, but its application to endogenous protein complexes in the proteome is challenging and typically requires labor-intensive and time-consuming prefractionation. Here, for the first time, we develop a nondenaturing online two-dimensional liquid chromatography (2D-LC) method for native top-down proteomics (nTDP), enabling high-throughput structural analysis of endogenous protein complexes. The automated, online interfacing of size-exclusion and mixed-bed ion-exchange chromatography achieves high coverage of endogenous protein complexes. We further develop a multistage nTDMS approach that enables comprehensive structural characterization within the chromatographic time scale, capturing intact noncovalent complexes, released subunits/cofactors, and backbone fragments. Our analysis detected 133 native proteoforms and endogenous protein complexes (up to 350 kDa) from human heart tissue in less than 2 h. This work represents a significant technical advancement toward direct, high-throughput structural characterization of endogenous protein complexes from biological mixtures.
蛋白质复合物对于几乎所有生物过程都至关重要，然而由于其异质性、动态性质以及蛋白质组的复杂性，它们的结构表征仍然是一项重大挑战。原生自上而下质谱法（nTDMS）已成为纯化蛋白质复合物全面结构表征的强大工具，但其应用于蛋白质组中的内源性蛋白质复合物具有挑战性，通常需要耗费大量人力和时间的预分级分离。在此，我们首次开发了一种用于原生自上而下蛋白质组学（nTDP）的非变性在线二维液相色谱（2D-LC）方法，能够对内源性蛋白质复合物进行高通量结构分析。尺寸排阻色谱和混合床离子交换色谱的自动化在线联用实现了对内源性蛋白质复合物的高覆盖率。我们进一步开发了一种多级nTDMS方法，能够在色谱时间尺度内进行全面的结构表征，捕获完整的非共价复合物、释放的亚基/辅因子以及主链片段。我们的分析在不到2小时的时间内从人心脏组织中检测到了133种原生蛋白质形式和内源性蛋白质复合物（分子量高达350 kDa）。这项工作代表了从生物混合物中直接高通量表征内源性蛋白质复合物结构方面的一项重大技术进步。
