Abstract：Bisecting N-acetylglucosamine (GlcNAc) act an essential role in a variety of biological processes, such as signal transduction, cell adhesion and cell migration. In this study, glycoproteins with bisecting GlcNAc structures were identified in normal mouse (Mus musculus) mammary gland epithelial (NMuMG) cells and mouse breast cancer cells (4T1) with lectin enrichment. Briefly, proteins from cell lines were reduced and alkylated, and digested with Lys-C/trypsin, and the peptides were subjected to PHA-E capture, PNGase F digestion and LC-MS analysis. 378 of bisecting GlcNAc N-glycosylation sites, 248 of glycoproteins were identified in two cell lines. The bisecting GlcNAc N-glycosylation consensus sequence was determined with WebLogo tool. Results showed that N-glycosylation consensus sequence containing NXS accounted for 38.9% of total N-glycosylation sites, the amino acid next to asparagine was most likely to be glycine and valine; consensus sequence containing NXT accounted for 61.1% of total N-glycosylation sites, the amino acid next to asparagine was most likely to be alanine and glycine. Gene Ontology classification of 248 glycoproteins with bisecting GlcNAc structures was performed as cellular component, molecular function and biological process with DAVID website using the whole Mus musculus proteome as background. The main cellular component categories were intrinsic to membrane, integral to membrane and plasma membrane; the major biological process categories were cell adhesion, biological adhesion and proteolysis; the top three most common molecular functions were ion binding, cation binding and nucleotide binding. For determination of the differentially expressed glycoproteins with bisecting GlcNAc structures in two cell lines, quantification of each glycosites was accomplished by comparative analysis between the intensity of MS signals derived from NMuMG and 4T1. Eighty glycoproteins were differentially expressed between NMuMG and 4T1. Fifty glycoproteins (including LAMP1 and LAMP2) with bisecting GlcNAc structures were up-regulated in 4T1 comparing to NMuMG, and 30 glycoproteins (including EGFR, ITA6 and ITA3) with bisecting GlcNAc structures were down-regulated. Functional enrichment analysis was performed to identify enrichment terms associated with the differentially expressed glycoproteins. The most highly ranked molecular function in the significant enriched GO terms of up- and down-regulated glycoproteins were respectively pigment qranule and integrin complex; the enrichment of GO terms of molecular function in up- and down-regulated glycoproteins were mainly hexosaminidase activity and calcium ion binding, respectively; the enrichment of GO terms of biological process in up- and down-regulated glycoproteins were mainly heterophilic cell adhesion and integrin-mediated signaling pathway, respectively. Functional network analysis of differentially expressed glycoproteins were performed by uploading protein lists to the String website. And we found that cell adhesion, lysosome and PI3K-AKT signaling pathway was involved. Glycoproteomics analysis based on lectin enrichment could profile differentially expressed glycoproteins during the disease process, and provide essential information for disease diagnosis, treatment and prognosis.
