Comprehensive evaluation of plasma proteomics platforms toward practical applications using accessible mass spectrometry instrumentation

Abstract Background Blood plasma proteomics is a promising approach for biomarker discovery, but the broad dynamic range of plasma proteins limits the detection of low-abundance proteins. Depletion and enrichment platforms have been developed to address this challenge; however, most previous evaluations have relied on next-generation mass spectrometry instruments. Platform behavior therefore needs to be evaluated in more accessible liquid chromatography-mass spectrometry instrumentation. Methods Seven plasma preparation platforms were evaluated using accessible LC-MS instrumentation and compared with recent next-generation mass spectrometry benchmarks. A commercial pooled plasma sample was processed using neat plasma, Multiple Affinity Removal System Human 14, perchloric acid precipitation with neutralization, ENRICH-iST, Mag-Net, Proteonano™, and Proteograph XT. Samples were analyzed by data-independent acquisition mode. Results Depletion and enrichment platforms increased proteome coverage compared with neat plasma, which identified an average of 777 proteins. The depletion platforms identified an average of 1,278–1,468 proteins, whereas the enrichment platforms identified 1,891–6,060 proteins. Proteograph XT provided the greatest proteome coverage, with an average of 6,060 identified proteins, and showed the lowest missing value rate of 1.9%. However, greater proteome coverage did not always correlate with better quantitative precision. Platform choice significantly affected abundance profiles and clinically relevant protein coverage. These platform-specific differences reproduced recent next-generation mass spectrometry results. Conclusions Findings in this study demonstrate that more accessible liquid chromatography-mass spectrometry setups can effectively capture key differences among plasma preparation platforms. This study supports the broader approach of plasma proteomics in settings where next-generation instrumentation is not routinely available.