Chemical proteomic approach for in-depth glycosylation profiling of plasma carcinoembryonic antigen in cancer patients

Abstract

Carcinoembryonic antigen (CEA) of human plasma is a biomarker of many cancer diseases, and its N-glycosylation accounts for 60% of molecular mass. It is highly desirable to characterize its glycoforms for providing additional dimension of features to increase its performance in prognosis and diagnosis of cancers. However, to systematically characterize its site-specific glycosylation is challenging due to its low abundance. Here, we developed a highly sensitive strategy for in-depth glycosylation profiling of plasma CEA through chemical proteomics combined with multi-enzymatic digestion. A trifunctional probe was utilized to generate covalent bond of plasma CEA and its antibody upon UV irradiation. As low as 1 ng/mL CEA in plasma could be captured and digested with trypsin and chymotrypsin for intact glycopeptide characterization. Twenty six out of 28 potential N-glycosylation sites were well identified, which were the most comprehensive N-glycosylation site characterization of CEA on intact glycopeptide level as far as we known. Importantly, this strategy was applied to the glycosylation analysis of plasma CEA in cancer patients. Differential site-specific glycoforms of plasma CEA were observed in patients with colorectal carcinomas (CRC) and lung cancer. The distributions of site-specific glycoforms were different as the progression of CRC, and most site-specific glycoforms were overexpressed in stage II of CRC. Overall, we established a highly sensitive chemical proteomic method to profile site-specific glycosylation of plasma CEA, which should generally applicable to other well-established cancer glycoprotein biomarkers for improving their cancer diagnosis and monitoring performance. In BriefA chemical proteomic approach for glycosylation profiling of proteins was established for glycosylation characterization of plasma CEA with low abundance. Although CEA has been widely used in diagnosis and prognosis of many cancers, it lacks specificity and sensitivity. We found that the glycosylation of CEA on intact glycopeptide level provided additional dimension of molecular features to improve the performance of CEA in cancer diagnosis and progression. HighlightsO_LIA chemical proteomic approach for glycosylation profiling of proteins with low abundance C_LIO_LIGlycosylation identification of plasma CEA on intact glycopeptide level with high sensitivity and reproducibility C_LIO_LIGlycosylation features of plasma CEA in cancer patients with CRC and lung cancer and in CRC patients at different progression stages C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/558933v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@73b539org.highwire.dtl.DTLVardef@7ed658org.highwire.dtl.DTLVardef@12a3e20org.highwire.dtl.DTLVardef@54a87f_HPS_FORMAT_FIGEXP M_FIG C_FIG