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.

Global Insight into N-Glycome and N-Glycoproteome of Three Most Abundant Snake Venoms in Asia

Snake venom is a complex cocktail including a variety of biological active proteins and proteinaceous components, which have considerable medical and pharmacological importance. N-Glycosylation is widely impli- cated as a common modification in numerous venom proteins and impacts the in vivo venomic functions. However, systematic survey of N-glycome and N-glycoproteome on snake venoms has not been undertaken. In this study, em- ploying combination of N-glycomics and N-glycoproteomics strategies, we explored the N-glycosylation including both N-glycoproteins and N-glyco-chains in three venoms from Agkistrodon blomhoffii, Naja naja atra Cantor and Vipera russelii siamensis Smith, respectively, which are amongst the most abundant venomous snakes in Asia. As a result, numbers of N-glycoproteins and N-glycans were identified. However, the overlaps of N-glycoproteins and N-glycans of the three venoms were small. Thus, the exploration results of N-glycome and N-glycoproteome indicate that N-glycosylation increases the complexity and variety of the three venoms. Our research provided some new horizons for the comprehensive understanding of venoms variation, which is helpful for the basic venom re- search as well as the management of snake envenomation.