High throughput proteomics enabled by a fully automated dual-column liquid chromatography system coupled with mass spectrometry

This technical note presents a dual-column liquid chromatography system coupled with mass spectrometry (LC-MS), designed for high-throughput bottom-up proteomic analysis. Utilizing two 2-position 10-port valves, a binary nanoflow pump, and an integrated loading pump, the system enables the simultaneous operation of two parallel subsystems. Each subsystem comprises a trap column and an analytical column. A T-junction union is employed to divide the mobile phase from the loading pump into two parts, which permits one set of columns to undergo washing and equilibration while the subsequent sample is injected. Meanwhile, the previous sample elutes and undergoes analysis on the alternate set of columns, significantly increasing the analysis throughput. The approach demonstrates high reproducibility when analyzing HeLa tryptic digests, with average relative standard deviations (RSD) of 1.64% for protein identification across nine consecutive runs. This system exhibits comparable capacity for protein identification and proteome depth to that of traditional single-column systems. Owing to its straightforward equipment requirements and set up process, this method is highly accessible and easily adopted by other laboratories.