Advancing Blood Proteome Analysis Past the Plasma Age: Mass Spectrometry of Whole Blood and Plasma Collected with...

Mass spectrometry-based proteomics of blood faces significant challenges due to the high dynamic range of proteins, with abundant proteins masking detection of low-abundance biomarkers. This study evaluated a novel volumetric absorptive microsampling (VAMS) method for blood proteome analysis compared with conventional sample processing. Plasma and whole blood samples were processed using three different methods: liquid sample analysis, VAMS-direct processing, and our VAMS-optimized protocol involving selective washing steps. All samples were analyzed using shotgun LC-MS/MS. The VAMS-optimized method demonstrated superior performance, increasing protein identifications 4-fold for plasma (745 vs 3024 proteins) and 2.1-fold for whole blood compared to liquid samples, while improving quantitative reproducibility with mean coefficients of variation below 11%. Further, the VAMS-optimized approach produced more protein identifications and lower %CVs for single-spun plasma and whole blood when compared with a commercially available protein corona bead assay. Whole blood samples showed greater robustness than plasma, with reduced variability from preanalytical processing steps and improved storage stability at room temperature for up to 14 days. The VAMS-optimized approach addresses key limitations in current blood proteomics workflows, enabling patient-centric sample collection for longitudinal studies while maintaining analytical depth and reproducibility, essential for biomarker discovery applications.