N -Glycoproteomics Study of Putative N -Glycoprotein Biomarkers of Drug Resistance in MCF-7/ADR Cells

Currently, drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis. Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improvement of prognosis. Here we present our N -glycoproteomics study of putative N -glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7 (MCF-7/ADR) relative to parental michigan cancer foundation-7 (MCF-7) cells. Intact N -glycopeptides (IDs) from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), labeled with stable isotopic diethylation (SIDE), and analyzed with C18-RPLC-MS/MS (HCD with stepped normalized collision energies); these IDs were identified with database search engine GPSeeker, and the differentially expressed intact N -glycopeptides (DEGPs) were quantified with GPSeekerQuan. With target-decoy searches and control of spectrum-level FDR ≤ 1%, 322 intact N -glycopeptides were identified; these intact N -glycopeptides come from the combination of 249 unique peptide backbones (corresponding to 234 intact N -glycoproteins) and 90 monosaccharide compositions (corresponding to 248 putative N -glycosites). The sequence structures of 165 IDs were confirmed with structure-diagnostic fragment ions. With the criteria of observation at least twice among the three technical replicates, ≥ 1.5-fold change and p value < 0.05, 20 DEGPs were quantified, where five of them were up-regulated and 15 of them were down-regulated; the corresponding intact N -glycoproteins as putative markers of drug resistance were discussed.