Su-Min Lee (Korea Basic Science Institute (KBSI))

Jin Young Kim (Korea Basic Science Institute (KBSI))

Su-Min Lee (Korea Basic Science Institute (KBSI))

Geul Bang (Korea Basic Science Institute (KBSI))

Sungwoo Choi (Korea Basic Science Institute (KBSI))

Jin Young Kim (Korea Basic Science Institute (KBSI))

Comprehensive profiling of the serum proteome is crucial for identifying clinically relevant biomarkers. However, this is hindered by the serum's complexity and broad dynamic range, dominated by highly abundant proteins such as albumin, which mask low-abundance proteins and limit proteome coverage. To address this, we developed a cost-effective, depletion-free, and scalable workflow that integrates polyphenolic biomolecule–conjugated magnetic bead–based EVP enrichment using an automated device with S-Trap digestion. This enables rapid and high-throughput isolation of serum-derived extracellular vesicles and particles (EVPs), while SDS-based S-Trap digestion facilitates efficient lysis of lipid membranes, improving protein extraction. To evaluate and optimize the EVP enrichment strategy, we compared proteome profiles from native serum, top 14-depleted serum, and EVP-enriched serum. DIA-MS identified 273 and 433 proteins from native and depleted samples, respectively, whereas EVP enrichment enabled identification of 735 proteins. This strategy significantly enhanced proteome coverage and sensitivity without requiring depletion or fractionation. The optimized workflow was applied to 50 serum samples from patients with pancreatic, bile duct, gallbladder, and liver cancers (n = 10 per group), along with controls. Enriched samples were labeled using TMT 32plex and analyzed by Orbitrap-based LC-MS/MS, enabling high-throughput, multiplexed quantification of cancer-associated proteomic alterations across two TMT sets. In total, 1,070 proteins were identified, with 824 consistently quantified after excluding missing values. The consistent detection of all 13 EVP-associated marker proteins across all samples highlights the robustness, reproducibility, and quantitative reliability of the workflow. This EVP enrichment–based serum proteomics strategy is expected to support high-throughput analysis and facilitate the discovery of clinically relevant biomarkers.