Top-down Proteomics Analysis of Picogram-level Complex Samples using Spray-Capillary-Based Capillary Electrophoresis Mass Spectrometry

Proteomics analysis, including post-translational modifications (PTMs) of mass-limited samples has become an important method for understanding biological systems in physiologically relevant contexts, such as patient samples and in multicellular organoids and spheroids. There is a growing need to develop ultrasensitive top-down proteomics techniques to provide valuable insights into PTM-regulated cellular functions in mass-limited samples, including single cells. Capillary electrophoresis–mass spectrometry (CE-MS) is a promising technique due to its high resolution and sensitivity compared to liquid chromatography (LC)-based separation techniques. We recently developed “Spray-Capillary”, an electrospray ionization (ESI)-assisted device for quantitative ultralow-volume sampling and online CE-MS analysis. In this study, we present an enhanced spray-capillary-based CE-MS platform using the polyethyleneimine (PEI) coated capillary for ultrasensitive top-down proteomics analysis. Under optimized conditions, we detected >200 proteoforms from 50 pg E. coli lysate, which is approximately one-tenth of the protein mass in a single mammalian cell. Using a nanodroplet-based sample preparation method and our optimized CE-MS platform, we reproducibly detected 867 intact proteoforms in HeLa cells and 711 intact proteoforms in OVCAR-8, a type of ovarian cancer cell (~45 cells per analysis). Overall, our results demonstrate the capability of the Spray-Capillary CE-MS system to perform top-down proteomic analysis on picogram amounts of sample, and this advancement presents the possibility of meaningful top-down proteomic analysis of mass-limited samples down to the level of single mammalian cells.