High-Throughput Top-Down Proteomic Analysis of Picogram-Level Complex Samples Using Multisegment Spray-Capillary...

Ultrasensitive top-down proteomics techniques provide valuable insights into PTM-regulated cellular functions in mass-limited samples. Capillary electrophoresis (CE) is a promising separation technique for top-down proteomics due to its high resolution, high sensitivity, and short cycle times compared to traditional liquid chromatography (LC)-based methods. We recently developed the "Spray-Capillary," an ESI-assisted device for quantitative ultralow-volume sampling and online CE-MS analysis, which successfully characterized hundreds of intact proteoforms from picogram-level cell lysate samples and showed promise for quantitative analysis of mass-limited complex biological samples. In this study, we further improved throughput for mass-limited top-down proteomics by integrating multisegment sample injection with our spray-capillary CE-MS analysis platform. By optimizing the spacer between sample plugs, we enabled the injection of multiple samples into the spray-capillary prior to a single CE-MS analysis, achieving baseline separation of identical proteins from different segments. Under optimized conditions, we quantifiedE. colilysate (10-250 pg) using a six-point calibration curve in a single analysis (e.g., ∼60 min run time), yielding a strong linear correlation (R² > 0.98). Our method supports up to 17 sample segments per run (e.g., ∼90 min run time) while maintaining baseline separation. This optimized multisegment injection platform has the potential to analyze hundreds of mass-limited samples (e.g., single cells) per day, significantly enhancing throughput in top-down proteomics.