Top-down single-cell proteomics (SCP) analysis allows for detecting cell-to-cell variations in proteoform expression to provide important insights into PTM-regulated cellular functions. Currently, bottom-up SCP studies use nRPLC-MS/MS for sample analysis which provides high resolution and sensitivity but typically requires sample dilution to the µL level prior to injection onto the LC column. This dilution leads to increased sample adsorption to surfaces resulting in sample loss which is negligible for traditional bulk cell proteomics but may be catastrophic at the single-cell level. Our group has developed a spray-capillary CE-MS platform capable of ultralow-volume microsampling (pL-level) and online CE-MS analysis to facilitate the study of mass-limited single-cell samples. Here, we integrated a nanodroplet-based sample preparation platform and spray-capillary CE-MS analysis for top-down single-cell analysis of cancer cells such as Hela and ovarian cancer cells.
Our spray-capillary device uses the pressure differential created during electrospray ionization to quantitatively inject ultralow-volume (pL-level) samples. After sampling, the spray-capillary can be used directly for online CE-MS analysis resulting in minimal sample loss for sensitive bottom-up or top-down proteomic analysis. To further improve the sensitivity of spray-capillary CE-MS analysis, we coated the spray-capillary with PEI, significantly reducing the sample adsorption loss on the inner wall during the CE separation. We applied this platform to analyze mass-limited samples of E. coli lysate as a proof of concept for single-cell analysis. We detected >200 mass features from 0.05 ng E. coli lysate (one-tenth of the quantity of protein in a single mammalian cell) with an injection volume of ~300 pL.
We further applied the top-down spray-capillary CE-MS platform to analyze a small number of HeLa cells prepared using a droplet-based method. Approximately 100 HeLa cells were lysed and prepared in droplets as discussed above. Whole droplets were sampled and analyzed using the spray-capillary CE-MS platform (N=3). We reproducibly detected 700-800 intact proteoforms from HeLa and OVCAR cell lines with high run-to-run reproducibility. Furthermore, our results demonstrated clear proteomic profile differences between the cell lines using PCA analysis.

Si Wu

The University of Oklahoma

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