ACCESSING SITE-SPECIFIC GLYCOSYLATION OF CARCINOEMBRYONIC ANTIGEN (CEA)

Carcinoembryonic antigen (CEA) quantitation is currently utilized in clinical diagnostics of colorectal cancer (CRC), tumor staging and recurrence prediction. However, the sensitivities for detecting CRC before clinical presentation are 13-25%, respectively, at 95% specificity. CEA was reported to have a sensitivity of 100% in detecting metastatic liver disease. Hence we hypothesize that circulating tumor-originating CEA, being involved in tumor progression, may exhibit site-specific glycosylation changes that can provide additional accuracy to the diagnostics and monitoring of CRC. Since CEA has 28 potential N-glycosylation sites, literature suggests that it's glycosylation pattern could be used as a more specific biomarker.In this work we developed an analytical platform to study the glycosylation of commercially available CEA from colon carcinoma. Enzymatic CEA digestion was followed by capillary electrophoresis coupled to mass spectrometry via electrospray ionization (CE-ESI-MS) with a sheathless interface and the addition of dopant enriched nitrogen gas. The glycopeptide pool obtained with trypsin, chymotrypsin, and their combination, was evaluated. A higher coverage (17 sites) was found compared to solely using trypsin (10 sites) or chymotrypsin (11 sites). Sialylation was not very common, but the overall level of fucosylation was high; certain sites (e.g. N-665, N-204) showed high levels of fucosylation, whereas others (e.g. N-197/N553) revealed no fucosylated glycans. Also increased branching of glycans was observed. Since we aim to characterize serum-derieved CEA VHH single-domain antibodies coupled to beads were evaluated as capturing method. First results for spiked PBS (3.3-67 µg/ml) revealed a CEA recovery of roughly 50-60%, which is a promising result to transfer the method on biofluids. Further optimization of recovery and method sensitivity is needed to obtain full coverage for even lower (10 ng/ml) concentrations in biological samples.