Systematic Evaluation of Depletion and Enrichment Technologies for Platelet-Free Plasma Proteomics

High Resolution Image Download MS PowerPoint Slide Plasma proteomics is a rapid, noninvasive, and highly informative approach for identifying disease biomarkers. However, the wide dynamic range of protein concentration limits the depth of liquid chromatography–tandem mass spectrometry proteomics. To address this challenge, we evaluated, with an Orbitrap Astral instrument using DIA, the performance of several protein depleting and enriching technologies on platelet-free plasma. Specifically, we assessed: perchloric acid depletion, immunodepletion, ProteoMiner, MagNet-SAX, ENRICHplus, Proteonano and the combination of ProteoMiner and immunodepletion. All methods were assessed in terms of proteomic depths, protein quantification precision, and functional analysis. Proteonano exhibited the most effective enrichment for the lowest abundance proteins, confidently identifying 299 proteins with mapped blood concentrations below 10 6 pg/L. Immunodepletion yielded the highest proteome coverage in the moderate abundance range (660 confident proteins). Also, ENRICHplus quantitative profile closely matched that of the neat plasma (93% correlation). Additionally, high repeatability (median coefficient of variation) was demonstrated by MagNet-SAX (13%), ProteoMiner (15%), and immunodepletion (16%). Combining ProteoMiner and immunodepletion reduced the plasma protein dynamic range, enabling deeper low abundance protein analysis but decreased repeatability. These results obtained on platelet-free plasma deviate from previously reported results on platelet-rich plasma, highlighting the crucial sample preparation stage for plasma proteomics.