Background: Multidrug resistance (MDR), a major hurdle in the cancer treatment, has been closely associated with aberrant regulation of N-Glycosylation; identification of the related differentially expressed N-glycoproteins is essential for understanding the underlying molecular mechanisms and developing efficient drugs and therapies.
Methodologies: Differentially expressed N-glycosylation in MCF-7/ADR cells was characterized with our recently developed GPSeeker-enable quantitative structural N-glycoproteomics pipeline. With trypsin digestion, zicHILIC enrichment, isotopic diethyl labeling, 1:1 mixture of the labeled intact N-glycopeptides from MCF-7/ADR and MCF-7 cells was analyzed using C18-RPLC-nanoESI-MS/MS (HCD with stepped NCEs) on a Q Exactive Orbitrap MS; three technical replicates were acquired and the raw datasets were searched by intact N-glycopeptide search engine GPSeeker; intact N-glycopeptide IDs were further quantified with the abundance of the precursor ions in the MS spectra using GPSeekerQuan; final differentially expressed intact N-glycopeptides (DEGPs) were obtained with the criteria of observation of at least twice among the three technical replicates, ≥1.5 fold-change and p<0.05.
Findings: intact N-glycopeptides in MCF-7/ADR and MCF-7 cells each with the comprehensive structural information of both the peptide backbone (amino acid sequence, N-glycosite) and the N-glycan moiety (monosaccharide composition, sequence, linkages) were identified; unique N-glycan structures were characterized with structure-specific fragment ions including cross-ring A/X ions. Putative drug resistance N-glycosylation markers were found at DEGPs.
Conclusions: putative drug resistance N-glycosylation markers were discovered in the high-throughput omics fashion with GPSeeker-enable quantitative structural N-glycoproteomics pipeline at the intact N-glycopeptide level.
Figure 1. Intact N-glycopeptide SLSNSTAR-N2H8F0S0 on the N-glycosite N120 of N-glycoprotein Serpin H1 (SERPH_HUMAN, P50454) were identified from spectrum 3788 of TR1. From top to bottom, the isotopic envelopes of the paired precursor ions, the annotated MS/MS spectrum and the graphical fragmentation map with the matched fragment ions.