Proteomics analysis of mass-limited samples has become increasingly important for understanding biological systems in physiologically relevant contexts such as patient samples, multicellular organoids, spheroids, and single cells. However, relatively low sensitivity in top-down proteomics methods makes their application to mass-limited samples challenging. Capillary electrophoresis (CE) has emerged as an ideal separation method for mass-limited samples due to its high separation resolution, ultralow detection limit, and minimal sample volume requirements. Recently, we developed “spray-capillary”, an electrospray ionization (ESI)-assisted device, that is capable of quantitative ultralow-volume sampling (e.g., pL–nL level). Here, we developed a spray-capillary-CE–MS platform for ultrasensitive top-down proteomics analysis of intact proteins in mass-limited complex biological samples. Specifically, to improve the sensitivity of the spray-capillary platform, we incorporated a polyethylenimine (PEI)-coated capillary and optimized the spray-capillary inner diameter. Under optimized conditions, we successfully detected over 200 proteoforms from 50 pg of E. coli lysate. To our knowledge, the spray-capillary CE–MS platform developed here represents one of the most sensitive detection methods for top-down proteomics. Furthermore, in a proof-of-principle experiment, we detected 261 ± 65 and 174 ± 45 intact proteoforms from fewer than 50 HeLa and OVCAR-8 cells, respectively, by coupling nanodroplet-based sample preparation with our optimized CE–MS platform. Overall, our results demonstrate the capability of the modified spray-capillary CE–MS platform to perform top-down proteomics analysis on picogram amounts of samples. This advancement presents the possibility of meaningful top-down proteomics analysis of mass-limited samples down to the level of single mammalian cells.

中文翻译：

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使用基于喷雾毛细管的毛细管电泳-质谱法对皮克级复杂样品进行自上而下的蛋白质组学分析


质量限制样品的蛋白质组学分析对于理解生理相关环境中的生物系统（例如患者样品、多细胞类器官、球体和单细胞）变得越来越重要。然而，自上而下的蛋白质组学方法的灵敏度相对较低，这使得它们在质量有限的样品中的应用具有挑战性。毛细管电泳 (CE) 因其分离分辨率高、检测限超低和样品体积要求极低而成为质量限制样品的理想分离方法。最近，我们开发了“喷雾毛细管”，一种电喷雾电离（ESI）辅助装置，能够定量超低体积采样（例如pL-nL水平）。在这里，我们开发了一个喷雾毛细管-CE-MS 平台，用于对质量有限的复杂生物样品中的完整蛋白质进行超灵敏的自上而下的蛋白质组学分析。具体来说，为了提高喷雾毛细管平台的灵敏度，我们采用了聚乙烯亚胺（PEI）涂层毛细管并优化了喷雾毛细管内径。在优化条件下，我们成功从 50 pg 大肠杆菌裂解液中检测到 200 多种蛋白质。据我们所知，这里开发的喷雾毛细管CE-MS平台代表了自上而下蛋白质组学最灵敏的检测方法之一。此外，在原理验证实验中，通过将基于纳米液滴的样品制备与我们优化的 CE-MS 平台相结合，我们分别从不到 50 个 HeLa 和 OVCAR-8 细胞中检测到了 261 ± 65 和 174 ± 45 个完整的蛋白质形式。总体而言，我们的结果证明了改进的喷雾毛细管 CE-MS 平台能够对皮克量的样品进行自上而下的蛋白质组学分析。 这一进步使得对质量有限的样品进行有意义的自上而下的蛋白质组学分析直至单个哺乳动物细胞的水平成为可能。