High-throughput, single-cell top-down proteomics using multisegmented spray-capillary CE-MS

Introduction: Ultrasensitive top-down proteomics techniques provide valuable insights into PTM-regulated cellular functions in mass-limited samples, including single cells. However, top-down proteomics analysis of mass-limited samples and single cells is challenging due to high sample complexity, wide dynamic range, and wide isotopic/charge state distributions of proteoforms. We recently developed "Spray-Capillary", an ESI-assisted device for quantitative ultralow-volume sampling and online CE-MS analysis. In this study, we optimized the spray-capillary capillary device for improved sensitivity for intact protein analysis via PEI coating and reducing capillary inner diameter. We further coupled this platform droplet-based sample preparation to analyze single mammalian cells. Additionally, to improve throughput, we developed a multisegmented spray-capillary injection method that allows simultaneous separation of 10+ samples in a single analysis.

Methods: Spray-capillary devices were fabricated as discussed previously (Huang et al. 2019). An ECE-001 CE autosampler (CMP scientific) was used for automation and a customized 3D stage was used for droplet sample injection. Data was collected on a Thermo Orbitrap Exploris 240 or Orbitrap Ascend Tribrid MS. Single cells were obtained from Hui Zhang at Johns Hopkins. Biopharma Finder and TopPIC Suite were utilized for mass feature deconvolution and identification, respectively.

Results: We have developed and successfully applied spray-capillary CE-MS for quantitative single-cell metabolomics analysis in single onion cells (Huang et al. 2021). Here, we adapted this platform for automated, high-throughput single-cell top-down proteomics. First, we introduced a polyethyleneimine (PEI) coating to reduce sample loss due to protein adhesion and modulate EOF. The intensity for the coated capillary was 1.81E8 (RSD=17%), which represents a >500-fold increase compared with the bare capillary. Additionally, 20 and 50 µm I.D. PEI-coated spray-capillary devices were evaluated, and the 20 µm capillary resulted in a ~32-fold higher BPE intensity compared with 50 μm. The optimized spray-capillary device was used to analyze 50 pg intact E. coli lysate and >200 proteoforms were detected, representing one of the most sensitive detection methods for top-down proteomics. Additionally, we coupled a nanodroplet-based sample preparation method with this optimized spray-capillary CE-MS platform. We detected 867 and 711 intact proteoforms in ~45 HeLa and OVCAR-8 cells, respectively. We further applied this nanodroplet-based platform to single PC3 cells, detecting 60-80 proteoforms in single cells with high cell-to-cell reproducibility. Furthermore, to improve throughput, a multisegment sample injection method was coupled to our platform to improve throughput. Up to 17 sample segments could be injected and achieve baseline separation in a single spray-capillary CE-MS top-down proteomics analysis.