Comparative glycoproteomics study on the surface of SKOV3 versus IOSE80 cell lines

Abstract

Objective: Site- and structure-specific quantitative N-glycoproteomics study of differential cell-surface N-glycosylation of ovarian cancer SKOV3 cells with the non-cancerous ovarian epithelial IOSE80 cells as the control. Methods: C18-RPLC-MS/MS (HCD with stepped normalized collision energies) was used to analyze the 1: 1 mixture of labeled intact N-glycopeptides from SKOV3 and IOSE80 cells, and the site- and structure-specific intact N-glycopeptide search engine GPSeeker was used to conduct qualitative and quantitative search on the obtained raw datasets. Results: With the control of the spectrum-level false discovery rate ≤1%, 13,822 glycopeptide spectral matches coming from 2,918 N-glycoproteins with comprehensive N-glycosite and N-glycan structure information were identified; 3,733 N-glycosites and 3,754 N-glycan sequence structures were confirmed by site-determining and structure-diagnostic fragment ions, respectively. With the control of no less than two observations among the three technical replicates, fold change ≥1.5, and p-value ≤ 0.05, 746 DEPGs in SKOV3 cells relative to IOSE80 cells were quantified, where 421 were upregulated and 325 downregulated. Conclusion: Differential cell-surface N-glycosylation of ovarian cancer SKOV3 cells were quantitatively analyzed by isotopic labeling and site- and structure-specific N-glycoproteomics. This discovery study provides putative N-glycoprotein biomarker candidates for future validation study using multiple reaction monitoring and biochemical methods.

Keywords: GPSeeker; differential N-glycosylation; quantitative N-glycoproteomics; site-specific; structure-specific.

FIGURE 1

Qualitative and quantitative results and differentially expressed intact N-glycopeptides (DEPGs) in SKOV3 and IOSE80 cells. (A) Qualitative information on intact N-glycopeptides IDs including glycosites, peptides, and N-glycoprotein identified by GPSeeker with FDR ≤1% from C18-RPLC-MS/MS (HCD) analysis in 1:1 mixed SKOV3 and IOSE80 cells. (B) Quantitative results of differentially expressed N-glycopeptides searched by GPSeekerQuan.

FIGURE 2

Volcano plot of DEPGs (at least two technical duplications three times) in SKOV3 vs. IOSE80 cells. Red: upregulation. Blue: downregulation. p < 0.05.

FIGURE 3

Identification of the intact N-glycopeptide GNRTENFTK_N2H8. (A–C) Annotated MS/MS spectra from the three technical replicate RPLC-MS/MS runs; (D) fingerprinting map of the experimental (bar) and theoretical (dot) isotopic envelopes of the precursor ion; (E) graphical fragmentation map of the N-glycan moiety; and (F) extracted ion chromatograms of the precursor ion from the three technical replicate RPLC-MS/MS runs.= N-acetylglucosamine (Y),= mannose (M); au = arbitrary unit; IPMD = isotopic peak m/z deviation; IPAD = isotopic peak abundance deviation.

FIGURE 4

Identification of the intact N-glycopeptide LLSTAGTPENGSEPESR_N5H6F2S2. (A–C) Annotated MS/MS spectra from the three technical replicate RPLC-MS/MS runs; (D) fingerprinting map of the experimental (bar) and theoretical (dot) isotopic envelopes of the precursor ion; (E) graphical fragmentation map of the N-glycan moiety; and (F) extracted ion chromatograms of the precursor ion from the three technical replicate RPLC-MS/MS runs.=N-acetylglucosamine (Y);=mannose (M);= galactose (L); =sialic acid (S);= fucose (F); au = arbitrary unit; IPMD = isotopic peak m/z deviation; IPAD = isotopic peak abundance deviation.

FIGURE 5

Gene Ontology (GO) analysis of the identified N-glycoproteins from 736 DEPGs. (A) GO enrichment of top 10 biological process (BP), cellular component (CC), and molecular function (MF) terms for 421 upregulations. (B) GO enrichment of top 10 B P, CC, and MF terms for 325 downregulations.

FIGURE 6

Kyoto Encyclopedia of Genes and Genomes (KEEG) pathway enrichment analysis of the N-glycoproteins corresponding to the DEPGs in SKOV3 relative to IOSE80 cells. (A) Enrichment of 421 upregulated genes. (B) Enrichment of 325 downregulated genes.