Top-Down Proteomics Analysis of pg-Level Cell Lysate Using Spray-Capillary CE-FAIMS-MS

Abstract Capillary electrophoresis-mass spectrometry (CE-MS) is a promising technique for ultrasensitive top-down proteomics due to its high resolution and sensitivity compared with traditional liquid chromatography (LC)-based methods. However, CE-MS-based top-down proteomics of mass-limited samples remains challenging because of the need to handle ultralow sample amounts. Recently, we developed a spray-capillary CE-MS platform for ultrasensitive, low-volume top-down proteomics analysis, allowing for the detection of hundreds of intact proteoforms from pg-level samples. Here, we integrated this platform with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to enhance proteoform coverage in mass-limited samples (i.e., samples with limited input mass). Our results suggested that FAIMS effectively enhanced the signal-to-noise ratio for pg-level samples by filtering background noise. At low injection masses (e.g., 1 pg), experiments performed with FAIMS yielded higher relative abundance than those conducted without FAIMS. Interestingly, in all cases, we did not observe the substantial loss in intensity commonly reported for bottom-up proteomics with FAIMS. Additionally, we implemented 2D CE-FAIMS-MS by applying five different FAIMS compensation voltages (CVs) for gas-phase intact proteoform fractionation. This platform detected >3000 yeast proteoforms using 100 pg per CV. Overall, our results suggested that the spray-capillary CE-FAIMS-MS platform enables deep proteoform characterization of pg-level samples and holds potential for analyzing intact proteoforms in mass-limited biological samples.