A Streamlined High-Throughput Plasma Proteomics Platform for Clinical Proteomics with Improved Proteome Coverage, Reproducibility, and Robustness

[Image: see text] Mass spectrometry-based clinical proteomics requires high throughput, reproducibility, robustness, and comprehensive coverage to serve the needs of clinical diagnosis, prognosis, and personalized medicine. Oftentimes these requirements are contradictory to each other. We report the development of a streamlined High-Throughput Plasma Proteomics (sHTPP) platform for untargeted profiling of the blood plasma proteome, which includes 96-well plates and simplified procedures for sample preparation, disposable trap column for peptide loading, robust liquid chromatographic system for separation, data-independent acquisition in tandem mass spectrometry, and DIA-NN, FragPipe, and in-house peptide spectral library-based data analysis. Using the optimized platform at a throughput of 60 samples per day, over 600 protein groups including 57 FDA-approved biomarkers can be consistently identified from whole human plasma, and more than 85% of the detected proteins have 100% completeness in quantitative values across 300 samples. The balance achieved between proteome coverage, throughput, and reproducibility of this sHTPP platform makes it promising in clinical settings, where a large number of samples are to be measured quickly and reliably to support various needs of clinical medicine.