Top-Down Proteomic Analysis of Limited Samples Using Porous Layer Open Tubular Columns and High-Field Asymmetric Ion Mobility Spectrometry Coupled to Mass Spectrometry.

Despite significant recent progress in the field of mass spectrometry (MS)-based top-down proteomics (TDP), the analysis of limited samples is still a major challenge. Here, we explored the potential of ultralow flow (ULF) liquid chromatography (LC) porous layer open tubular (PLOT) columns interfaced with MS via high-field asymmetric waveform ion mobility spectrometry (FAIMS) to enable high-sensitivity TDP analysis of small populations of mammalian cells. The developed robust and easy-to-use platform delivered high reproducibility of retention times (RSD < 0.4%) and high separation performance for intact proteins (∼14-s peak full width at half-maximum and peak capacity of >125 for a 60 min effective gradient). The FAIMS-based experiments resulted in a ∼2-fold increase in identifications compared to the control experiments for ∼200 HeLa cell aliquots, i.e., 819 vs 454 proteins and 2645 vs 1305 proteoforms, respectively. The pilot ULF LC-MS analysis of six HeLa cells yielded 29 ± 3 proteins and 38 ± 2 proteoforms, on average, and a total of 44 proteins and 68 proteoforms. Data revealed a high degree of acetylation, methylation, phosphorylation, glycosylation, lactylation, and other relevant post-translational modifications. Notably, the presented protein identification results for limited samples are comparable to those of recent large-scale TDP studies of bulk samples, demonstrating the potential to enable informative single-cell TDP profiling.