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

05 October 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


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.


: Single cell proteomics • Top-down proteomics • CE-MS • Spray-capillary• Quantitative proteomics


Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.