In-depth N-glycome profiling of paired colorectal cancer and non-tumorigenic tissues reveals cancer-, stage- and EGFR-specific protein N-glycosylation.

Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-liquid chromatography-tandem mass spectrometry-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-Glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6 ± 13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by western blotting and immunohistochemistry. Detailed N-glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N-glycans. CRC-specific N-glycosylation phenotypes were observed including an overrepresentation of high mannose, hybrid and paucimannosidic type N-glycans and an under-representation of complex N-glycans (P < 0.05). Sialylation, in particular α2,6-sialylation, was significantly higher in CRC tumors relative to non-tumorigenic tissues, whereas α2,3-sialylation was down-regulated (P < 0.05). CRC stage-specific N-glycosylation was detected by high α2,3-sialylation and low bisecting β1,4-GlcNAcylation and Lewis-type fucosylation in mid-late relative to early stage CRC. Interestingly, a novel link between the EGFR status and the N-glycosylation was identified using hierarchical clustering of the N-glycome profiles. EGFR-specific N-glycan signatures included high bisecting β1,4-GlcNAcylation and low α2,3-sialylation (both P < 0.05) relative to EGFR-negative CRC tissues. This is the first study to correlate CRC stage and EGFR status with specific N-glycan features, thus advancing our understanding of the mechanisms causing the biomolecular deregulation associated with CRC.

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