A simplified perchloric acid workflow with neutralization (PCA N) for democratizing deep plasma proteomics at population scale.

Large scale plasma proteomics studies offer tremendous potential for biomarker discovery but face significant challenges in balancing analytical depth, throughput and cost-effectiveness. We present an optimized perchloric acid-based workflow with neutralization - PCA-N - that addresses these limitations. By introducing a neutralization step following protein precipitation, PCA-N enables direct enzymatic digestion without additional purification steps, reducing sample volume requirements to only 5 μL of plasma while maintaining deep plasma proteome coverage. The streamlined protocol allows preparation of over 10,000 samples per day using 384-well formats at costs comparable to undepleted plasma analysis (NEAT). Rigorous validation according to the recently introduced CLSI C64 guideline demonstrated that despite somewhat higher technical variability compared to NEAT, PCA-N maintained excellent biological resolution and reproducibility. We confirmed the workflow's exceptional stability through analysis of over 1,700 quality control samples systematically interspersed among more than 40,000 plasma samples measured continuously over 353 days. Technical performance remained consistent across multiple instruments, sample preparation batches and nearly a year of measurements. Compared to NEAT plasma proteomics, PCA-N doubled the proteomic depth while maintaining comparable reagent costs and throughput. The minimal sample requirements, operational simplicity while using only common laboratory chemicals and exceptional scalability positions PCA-N as an attractive approach for population-level plasma proteomics, democratizing access to deep plasma proteomics analysis.