A high-sensitivity low-nanoflow LC-MS configuration for high-throughput sample-limited proteomics

This study demonstrates how optimized liquid chromatography-mass spectrometry (LC-MS) conditions combined with a 50 {micro}m internal diameter (I.D.) column operated at 100 nL/min enables high-sensitivity and high-throughput sample-limited proteomics analysis, including single-cell proteomics (SCP). Through systematic parameter evaluation, an optimized method was developed, capable of identifying [~]3,000 protein groups from 250 pg of HeLa protein digest using a 10-min gradient in the direct injection workflow using data-independent acquisition (DIA) from a library-free search method. Further improvements reduced the cycle time from 20 to 14.4 min by employing a trap-and-elute workflow, permitting 100 runs/day with 70% MS utilization. A proof of principle study indicated that ca. 1,700 protein groups were identified from single-cell samples without a library for label-free quantification (LFQ). In conclusion, we demonstrate a high-sensitivity LC-MS/MS configuration that serves the needs for limited sample analysis, permitting 100 runs/day throughout.