Robust Capillary- and Micro-Flow Liquid Chromatography–Tandem Mass Spectrometry Methods for High-Throughput Proteome Profiling

Capillary- and micro-flow liquid chromatography-tandem mass spectrometry (capLC-MS/MS and μLC-MS/MS) is becoming a valuable alternative to nano-flow LC-MS/MS due to its high robustness and throughput. The systematic comparison of capLC-MS/MS and μLC-MS/MS systems for global proteome profiling has not been reported yet. Here, the capLC-MS/MS (150 μm i.d. column, 1 μL/min) and μLC-MS/MS (1 mm i.d. column, 50 μL/min) systems were both established based on UltiMate 3000 RSLCnano coupled to an Orbitrap Exploris 240 by integrating with different flowmeters. We evaluated both systems in terms of sensitivity, analysis throughput, separation efficiency, and robustness. capLC-MS/MS was about 10 times more sensitive than μLC-MS/MS at different gradient lengths. Compared with capLC-MS/MS, μLC-MS/MS was able to achieve higher analysis throughput and separation efficiency. During the 7 days' long-term performance test, both systems showed good reproducibility of chromatographic full width (RSD < 3%), retention time (RSD < 0.4%), and protein identification (RSD < 3%). These results demonstrate that capLC-MS/MS is more suitable for high-throughput analysis of clinical samples with a limited starting material. When enough samples are available, μLC-MS/MS is preferred. Together, capLC and μLC coupled to Orbitrap Exploris 240 with moderate sensitivity should well meet the needs of large-cohort clinical proteomic analysis.