Human plasma provides a minimally invasive source of circulating cancer biomarkers, yet its extreme protein dynamic range and interindividual heterogeneity hinder the detection of low-abundance, tumor-derived markers. In colorectal cancer (CRC), where early detection, risk stratification, and therapy monitoring remain major unmet needs, these analytical barriers limit clinical translation. Here, we present a single-particle enrichment workflow that combines P2 nanoparticles with iST sample preparation and Spectronaut® 20 DIA analysis to enable deeper proteome coverage, improved quantitative precision, and enhanced biological insights in human EDTA-plasma biomarker discovery. Human EDTA-plasma samples from a pilot study of CRC and healthy specimens (n = 6 per group) were processed using the P2-iST Plasma workflow (PreOmics), employing proprietary nanoparticles that selectively capture low-abundance proteins while depleting high-abundance species. Enriched samples were subsequently digested and purified using the optimized iST technology (PreOmics), ensuring standardized peptide generation. Peptides were analyzed on a timsTOF HT (Bruker) operated in dia-PASEF® mode and data processed in Spectronaut® 20 (Biognosys) using the directDIA+ framework for deep, quantitative profiling. Compared with non-enriched plasma samples, the integrated workflow yielded deeper proteome coverage and high quantitative precision (median protein-level CV <15% across replicates). Principal-component and hierarchical clustering analyses demonstrated clear discrimination between CRC and control samples. Functional enrichment revealed distinct biological pathways differentiating disease from control groups, highlighting the sensitivity and discriminative potential of the workflow. The P2-iST Plasma workflow integrates nanoparticle-based enrichment, standardized digestion, and advanced DIA analysis to generate deep, precise, and reproducible plasma proteomes that reflect disease-relevant biology in colorectal cancer. By unlocking new possibilities in circulating biomarker discovery, it supports research in early detection, molecular subtyping, prognosis, and treatment monitoring. Its scalable, automation-friendly design further enables large-cohort validation and longitudinal studies in colorectal and other cancers.