A High-Throughput Chemical Proteomic Approach for Profiling Serum CA19-9 Protein Carriers and Their Characterization in Patients with Elevated CA19-9 Levels

Abstract Although the carbohydrate biomarker CA19-9 is widely used for cancer screening, its diagnostic specificity is severely compromised by false-positive elevations in benign inflammatory conditions. Characterizing specific CA19-9 protein carriers provides a promising alternative, yet high-throughput, micro-volume profiling directly from serum remains a challenge. Here, we present a 96-well chemical proteomic platform for the high-throughput profiling of CA19-9-modified proteins. Utilizing a tailored trifunctional chemical probe, photo-activated covalent crosslinking between anti-CA19-9 antibodies and target carriers enables high-stringency enrichment within streptavidin-coated microplates. The workflow identified 509 carriers in cancer cell lines and achieved highly sensitive quantification of established targets, including CD44, LGALS3BP, and mucin family, using as little as 10 μL serum. Notably, this represents the first mass spectrometry-based direct identification of mucin family in peripheral serum. In a clinical cohort of 91 hyper-CA19-9 individuals, CA19-9-modified CD44 was enriched in healthy controls, whereas CA19-9-modified MUC5B was significantly elevated in benign lesions over colorectal cancer (CRC), validated by ELISA across two independent cohorts with AUCs 0.715 and 0.756. Integrating the protein carrier dataset into a random forest machine learning classifier effectively discriminated CRC from benign lesions with an AUC of 0.864. Furthermore, comparative sub-analysis resolved a critical clinical dilemma by identifying 59 upregulated protein carriers in pancreatic ductal adenocarcinoma relative to benign gallstone-induced cholangitis. Overall, this 96-well chemical proteomic strategy enables parallelized isolation of CA19-9 modified proteins from low-volume samples, establishing a reproducible and scalable tool for the differential profiling of clinically relevant carbohydrate antigen carriers.