Top-Down Proteomics Analysis of Picogram-Level Complex Samples Using Spray-Capillary-Based Capillary Electrophoresis–Mass Spectrometry
Zhitao Zhao, Yanting Guo, Trishika Chowdhury, Samin Anjum, Jiaxue Li, Lushuang Huang, Kellye A. Cupp‐Sutton, Anthony W. G. Burgett, Dingjing Shi, Si Wu
Abstract
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.