High-Throughput, Ultralow-Input Proteomics Enabled by Narrow-Bore Open Tubular Liquid Chromatography

Picogram-scale proteomics faces significant challenges in balancing sensitivity and throughput, driven largely by the performance limitations of liquid chromatography (LC) separations. We addressed this challenge by implementing a low-loss sample injection method for narrow bore open tubular LC ( n OTLC). This approach enabled the injection of ∼20% of the sample, marking a 295-fold improvement over the high-loss injection used in previous n OTLC publications. Our platform identified 3955 proteins on average from <40 pg of HeLa digest loaded on the column at a throughput of 261 samples per day (SPD). We further demonstrated the ability to increase throughput to a projected 720 SPD while still identifying an average of 2460 proteins from only ∼20 pg of HeLa digest. The narrow chromatographic peaks combined with ultralow flow rates provided excellent electrospray ionization efficiency resulting in enhanced sensitivity for limited samples. The optimized n OTLC-MS system effectively balances the throughput-sensitivity trade-off in picogram-scale proteomics, providing a transformative platform for large-scale exploration of cellular heterogeneity with unmatched speed and sensitivity.