Open-tubular trap columns: towards simple and robust liquid chromatography separations for single-cell proteomics

Abstract

Nanoflow liquid chromatography-mass spectrometry is key to enabling in-depth proteome profiling of trace samples, including single cells, but these separations can lack robustness due to the use of narrow-bore columns that are susceptible to clogging. In the case of single-cell proteomics, offline cleanup steps are generally omitted to avoid losses to additional surfaces, and online solid-phase extraction/trap columns frequently provide the only opportunity to remove salts and insoluble debris before the sample is introduced to the analytical column. Trap columns are traditionally short, packed columns used to load and concentrate analytes at flow rates greater than those employed in analytical columns, and since these first encounter the uncleaned sample mixture, trap columns are also susceptible to clogging. We hypothesized that clogging could be avoided by using large-bore porous layer open tubular trap columns (PLOTrap). The low back pressure ensured that the PLOTraps could also serve as the sample loop, thus allowing sample cleanup and injection with a single 6-port valve. We found that PLOTraps could effectively remove debris to avoid column clogging. We also evaluated multiple stationary phases and PLOTrap diameters to optimize performance in terms of peak widths and sample loading capacities. Optimized PLOTraps were compared to conventional packed trap columns operated in forward and backflush modes, and were found to have similar chromatographic performance of backflushed traps while providing improved debris removal for robust analysis of trace samples.