Site-Specific N-Linked Glycosylation Analysis of Human Carcinoembryonic Antigen by Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry

Abstract

With 28 potential N -glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N -linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N -glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N -glycopeptides and 128 unique N -glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N -glycosylation sites. Moreover, notable differences were found on certain N -glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: ].

Cancer glycomics offers potential biomarkers and therapeutic targets in the framework of 3P medicine

“…Of them, three more N -glycosylation sites were identified by gluc digestion than trypsin digestion. This research provides a better understanding of the heterogeneity of CEA glycosylation pattern ( 88 ). Virtual multi-stage MS was utilized to simultaneously obtain glycan, peptide sequence, and glycosylation sites.…”

Mass Spectrometry-Based Techniques to Elucidate the Sugar Code

“…This led to the application of more specific chromatographic methods such as PGC and HILIC, which have unique properties to resolve both peptides and glycopeptides, on an isomeric level. Furthermore, capillary electrophoresis showed its potential as a promising separation technique as it allows the separation of isomeric glycopeptides in very short time frames. , Further details on sample preparation and separation strategies are summarized in recent review articles. ,, …”

“…Furthermore, capillary electrophoresis showed its potential as a promising separation technique as it allows the separation of isomeric glycopeptides in very short time frames. 555,556 Further details on sample preparation and separation strategies are summarized in recent review articles. 4,557,558 7.2.2.…”

Mining the Immunopeptidome for Antigenic Peptides in Cancer

“…Other glycosylated immunopeptides have also been investigated as vaccines, tumor-selective antibodies, CAR T cells, nanoparticles, and DC therapy [ 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 ]. Critical to this effect is knowledge of the structure of glycoproteins for their synthesis and antibody binding properties and their surface localization and occurrence in the immunopeptidome [ 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 ]. Interestingly, MHC-I also undergoes glycosylation, which must be taken into account for antigen presentation [ 101 ].…”