Zwitter-ionic monolith-based spintip column coupled with Evosep One liquid chromatography for high-throughput proteomic analysis

A high-throughput proteomic workflow with good sensitivity and reproducibility is highly demanding for proteomic studies of large clinical cohorts. We present a workflow that seamlessly integrates the zwitter-ionic monolith-based spintip (ZIM-Tip) with the Evosep One liquid chromatography system to address this challenge. Disposable ZIM-Tips were prepared with satisfying permeability based on photo-initiated free radical polymerization. Sample preparation steps, including ion-exchange-based protein concentration, reduction, alkylation, and enzymatic digestion, were processed on the ZIM-Tips in 2 h with about 10% sample loss. The peptides recovered from ZIM-Tips were directly loaded on Evotips for desalting and proteomic data acquisition. In one-hour high performance liquid chromatography-MS/MS run, more than 4000 proteins were consistently identified from 1 µg of cell lysate using timsTOF Pro-mass spectrometer in data-dependent acquisition mode (DDA). At least 20 samples with protein amount of 1 µg could be processed each day. Good intra- and inter-day precision in quantification were demonstrated with median coefficient of variation (CV) values of less than 20% and 30%, respectively. The average Pearson correlation coefficients of each two sets of samples are 0.934 and 0.901, respectively. Collectively, the ZIM-Tip technology offers an useful solution for clinical cohort studies with demand for large sample amounts and low sample input while maintaining in-depth proteome coverage.