Scalable Hydrogel Nanoparticle Enrichment for Enhanced Mass Spectrometry-based Proteomic Analysis of Plasma or...

Discovery-based proteomics for translational research requires workflows that reveal deep proteomes with a high degree of analytical precision, are scalable across large sample cohorts, and that can be adapted to different sample matrices. In our work, these sample matrices often extend beyond plasma to include volumetric dried blood samples (VDBS) that offer a minimally invasive alternative to standard venipuncture and enable remote, self-directed sampling. We describe the development and validation of a one-day, 96-well plate-based workflow built on the Ceres Nanotrap Protein Enrichment Affinity (PEAK) nanoparticle platform, optimized independently for both plasma and VDBS ahead of liquid chromatography-tandem mass spectrometry (LC-MS/MS). We benchmarked a tube-based Ceres workflow against both a Seer-encrichment and an unenriched SP3 workflow, assessed its suitability for VDBS, and translated this workflow to a plate-based protocol with a 2-hour digestion. The adapted workflow demonstrated robust reproducibility for both matrices: plasma yielded 4,937 unique proteins with a median inter-day CV of 11% whereas VDBS yielded 3,613 proteins with a median inter-day CV of 19%. Plasma and VDBS proteomes were highly concordant, with 75% of shared proteins quantified within one order of magnitude between matrices. We preliminarily evaluated the biological utility of this platform against a curated panel of 20 Alzheimer's Disease-associated proteins. Our results reveal the Ceres PEAK platform as a flexible, laboratory-adaptable workflow capable of deep and reproducible proteomic profiling of both plasma and VDBS.