Bridged Hybrid Monolithic Column Coupled to High-Resolution Mass Spectrometry for Top-Down Proteomics

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for comprehensive characterization of intact proteins. However, because of the high complexity of the proteome, highly effective separation of intact proteins from complex mixtures prior to MS analysis remains challenging. Monolithic columns have shown great promise for intact protein separation due to their high permeability, low backpressure, and fast mass transfer. Herein, for the first time, we developed bridged hybrid bis(triethoxysilyl)ethylene (BTSEY) monolith with C8 functional groups (C8@BTSEY) for highly effective protein separation and coupled it to high-resolution MS for identification of intact proteins from complex protein mixtures. We have optimized mobile phase conditions of our monolith-based reverse-phase chromatography (RPC) for online liquid chromatography (LC)-MS analysis and evaluated separation reproducibility of the C8@BTSEY column. We further assessed the chromatographic performance of this column by separating a complex protein mixture extracted from swine heart tissue. Using our monolithic column (i.d. 100 μm × 35 cm), we separated over 300 proteoforms (up to 104 kDa) from 360 ng of protein mixture in an 80 min one-dimensional (1D) LC run. The highly effective separation and recovery of intact proteins from this monolithic column allowed unambiguous identification of ∼100 proteoforms including a large protein, αactinin2 (103.77 kDa), by online 1D LC-MS/MS analysis for the first time. As demonstrated, this C8@BTSEY column is reproducible and effective in separation of intact proteins, which shows high promise for top-down proteomics.