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 improve-ment 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.