Department of Chemistry, Michigan State University, 578 S Shaw Lane, East Lansing, Michigan, 48824, United States.
Plant Resilience Institute, Michigan State University, East Lansing, Michigan, 48824, USA.
Angew Chem Int Ed Engl. 2024 Nov 25;63(48):e202408370. doi: 10.1002/anie.202408370. Epub 2024 Oct 24.
Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.
天然蛋白质组学使用天然质谱（nMS）与液相分离相结合，在近生理条件下测量内源性蛋白质形式和蛋白质复合物。天然蛋白质组学应该为理解几乎所有生物过程提供细胞内蛋白质组动力学的最准确的鸟瞰图。nMS 已广泛用于表征纯化良好的蛋白质复合物。然而，只有极少数尝试利用 nMS 测量复杂样品（即整个细胞裂解物）中的蛋白质形式和蛋白质复合物。在这里，我们通过将天然毛细管区带电泳（nCZE）与超高质量范围（UHMR）Orbitrap 质谱仪在线偶联，开创了从复杂蛋白质组中测量大蛋白质形式或蛋白质复合物（高达 400 kDa）的天然蛋白质组学测量方法。nCZE-MS 技术能够测量 115 kDa 的标准蛋白质复合物，而仅消耗约 0.1 ng 的蛋白质材料。nCZE-MS 分析大肠杆菌细胞裂解物，在单次运行中检测到 30-400 kDa 质量范围内的 72 种蛋白质形式或蛋白质复合物，而仅消耗 50-ng 蛋白质材料。检测到的蛋白质形式或蛋白质复合物的质量分布与质量光度测量的结果非常吻合。这项工作代表了天然蛋白质组学在测量复杂蛋白质组方面的技术突破。
