2DG-induced protein glycosylation alterations in colorectal cancer cells CX1 using differential glycosite profiling

A new strategy to compare cellular glycoproteomes, thereby identifying membrane proteins with altered glycan structures and the concerned glycosites. The workflow consists of membrane proteins digestion followed by lectin-based isolation of glycopeptides and their fractionation. Since alterations in the glycan part of a glycopeptide causes mass alterations, analytical size exclusion chromatography is applied to detect these mass shifts. A combination of N-glycosidase treatment with nanoUPLC coupled Exploris-Orbitrap mass spectrometry identifies the altered glycoproteins and the respective glycosites. The methodology was established using the human colon cancer cell line CX1 which was treated with 2-deoxy-glucose - a modulator of N-glycosylation.