Simple and robust high-throughput serum proteomics workflow with low-microflow LC-MS/MS.

Clinical proteomics has substantially advanced in identifying and quantifying proteins from biofluids, such as blood, contributing to the discovery of biomarkers. The throughput and reproducibility of serum proteomics for large-scale clinical sample analyses require improvements. High-throughput analysis typically relies on automated equipment, which can be costly and has limited accessibility. In this study, we present a rapid, high-throughput workflow low-microflow LC-MS/MS method without automation. This workflow was optimized to minimize the preparation time and costs by omitting the depletion and desalting steps. The developed method was applied to data-independent acquisition (DIA) analysis of 235 samples, and it consistently yielded approximately 6000 peptides and 600 protein groups, including 33 FDA-approved biomarkers. Our results demonstrate that an 18-min DIA high-throughput workflow, assessed through intermittently collected quality control samples, ensures reproducibility and stability even with 2 µL of serum. It was successfully used to analyze serum samples from patients with diabetes having chronic kidney disease (CKD), and could identify five dysregulated proteins across various CKD stages.