A Simplified Workflow for Robust, High-Throughput and Quantitative Plasma Proteomics through Streamlined In-Tip Proteolysis

Abstract Blood plasma is a valuable matrix for biomarker discovery and disease monitoring, but LC-MS-based plasma proteomics remains limited by complex sample preparation workflows, analytical variability, and challenges in achieving reproducible quantitative measurements. To address these limitations, we developed a simplified “One-Tip” workflow that integrates protein extraction, reduction, alkylation, digestion, and peptide desalting within a single C18 disposable trap column (Evotip), minimizing handling, sample losses, and technical variability. Applied to undepleted human plasma, the One-Tip workflow achieved analytical performance comparable to established SP3 and S-Trap methods while requiring only 500 ng of starting material. More than 350 protein groups were reproducibly identified, with >54% overlap between workflows and median protein-level coefficients of variation below 20%. Plasma-loaded Evotips remained stable for up to 14 days at 4 °C without substantial loss of proteome coverage, and a shared core proteome of 234 proteins was consistently detected across five independent plasma lots. To improve quantitative consistency, we incorporated a stable isotopically labeled (SIL) mouse plasma internal standard directly into the workflow. Human/mouse peptide doublets enabled the quantification of approximately one hundred proteins (∼30% of the identified proteome), providing robust protein-level light/heavy ratio measurements. The SIL mouse plasma standard remained quantitatively stable during seven days of storage and across SIL mouse generations, supporting its use as a reliable biological reference material. Together, these results establish the One-Tip workflow as a low-handling, scalable, and reproducible platform for plasma proteomics and provide a foundation for standardized quantitative workflows based on biological isotope-labeled internal standards.