Protein-Specific Differential Glycosylation of Immunoglobulins in Serum of Ovarian Cancer Patients

Abstract

Previous studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer; however, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight into biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n = 84) and matched healthy controls (n = 84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA, and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed in serum of EOC patients compared with controls. The protein-specific glycosylation profiles showed their potential in the diagnosis of EOC. In particular, IgG-specific glycosylation profiles are the most powerful in discriminating between EOC case and controls. Additional studies of protein- and site-specific glycosylation profiles of immunoglobulins and other proteins will allow further elaboration on the characteristics of biological functionality and causality of the differential glycosylation in ovarian cancer and thus ultimately lead to increased sensitivity and specificity of diagnosis.

Keywords: N-glycosylation; biomarker; immunoglobulin; ovarian cancer; serum.

Figure 1

PLS-LDA analysis of the glycopeptides for the immunoglobulin IgG (A), IgA (B), and IgM (C) in the OC1 discovery set. Clear separation between the EOC cases (n = 40) and the controls (n = 40) is observed for IgG and IgA, while the separation between the disease groups is less for IgM.

Figure 2

Differential analysis of peptide and glycopeptide variables from immunoglobulins in EOC. Closed dots indicate significantly different abundance levels between EOC cases and healthy controls, while open dots indicate no significance was achieved at the false discovery rate (FDR) < 0.05. Red dots indicate increased levels in EOC cases compared with controls, while blue dots indicate decreased levels in EOC compared with controls.

Figure 3

ROC curves for the best performing individual glycopeptides of all three immunoglobulins in the OC1 discovery set and OC2 test set. Curves are shown for IgG1-H₅N₅F₁ (A), IgA N^144/131 H₄N₅ (B), and IgM N²⁰⁹ H₄N₅F₁S₁ (C). The ROC curve for the OC1 discovery set is shown in solid black, while the ROC curve for the OC2 test set is shown in dotted gray. Glycan symbol key: blue square is N-acetylglucosamine, green ball is mannose, yellow ball is galactose, red triangle is fucose, and purple diamond is N-acetylneuraminic acid. The glycan structures presented here are putative structures.

Figure 4

ROC curves for the four multiclassifier models developed. Curves are shown for IgG, IgA, IgM, and the combined immunoglobulins, respectively. The ROC curve for the OC1 discovery set is shown in solid black, while the ROC curve for the OC2 test set is shown in dotted gray.