Optimization of bottom-up proteomic sample preparation by design of experiments for absolute quantification of 257 proteins in human plasma using UPLC-MRM-MS

INTRODUCTION: Targeted protein absolute quantification using mass spectrometry holds promise for identifying biomarkers for diagnosis, prognosis, and personalized medicine. However, complex and time-consuming workflows, particularly during sample preparation, present significant bottlenecks. Addressing these challenges is critical for the applicability of absolute quantification of proteins in clinical research settings. AREAS COVERED: We explore optimization strategies for protein digestion in bottom-up proteomics sample preparation. Design of experiments (DoE), a statistical approach for systematically evaluating multiple experimental factors, was used for simultaneous optimization of digestion time, temperature, enzyme-to-protein substrate ratio, and denaturing agent. Furthermore, the lower limit of quantification (LLOQ) for our platform was improved by using the Waters Xevo TQ-XS UPLC-MRM-MS. The integration of automated sample preparation into the workflow enabled reproducible absolute quantification of 257 proteins in human plasma. EXPERT OPINION: We successfully reduced protein digestion time from 18 hours (overnight) to 4 hours while maintaining relative digestion efficiency. We improved the sensitivity of the assay via the optimized workflow and were able to quantify proteins that previously fell below the LLOQ. These advancements, combined with automation, provide a practical, efficient, and reproducible workflow suitable for clinical research.