LC-MS/MS Quantitation of HILIC-Enriched N-glycopeptides Derived from Low-Abundance Serum Glycoproteins in Patients with Narcolepsy Type 1

Abstract

Glycoproteomic analysis is always challenging because of low abundance and complex site-specific heterogeneity. Glycoproteins are involved in various biological processes such as cell signaling, adhesion, and cell-cell communication and may serve as potential biomarkers when analyzing different diseases. Here, we investigate glycoproteins in narcolepsy type 1 (NT1) disease, a form of narcolepsy characterized by cataplexy-the sudden onset of muscle paralysis that is typically triggered by intense emotions. In this study, 27 human blood serum samples were analyzed, 16 from NT1 patients and 11 from healthy individuals serving as controls. We quantified hydrophilic interaction liquid chromatography (HILIC)-enriched glycopeptides from low-abundance serum samples of controls and NT1 patients via LC-MS/MS. Twenty-eight unique N-glycopeptides showed significant changes between the two studied groups. The sialylated N-glycopeptide structures LPTQNITFQTESSVAEQEAEFQSPK HexNAc₆, Hex₃, Neu5Ac₂ (derived from the ITIH4 protein) and the structure IVLDPSGSMNIYLVLDGSDSIGASNFTGAK HexNAc₅, Hex₄, Fuc₁ (derived from the CFB protein), with p values of 0.008 and 0.01, respectively, were elevated in NT1 samples compared with controls. In addition, the N-glycopeptide protein sources Ceruloplasmin, Complement factor B, and ITH4 were observed to play an important role in the complement activation and acute-phase response signaling pathways. This may explain the possible association between the biomarkers and pathophysiological effects.

Keywords: HILIC-enriched N-glycopeptides; NT1; biomarker glycoproteins.

Figure 1

Workflow for LC-MS/MS analysis. Symbols: blue squares, N-acetylglucosamine (GlcNAc); yellow circles, galactose (Gal); red triangles, fucose (Fuc); green circles, mannose (Man); blue circles, glucose (Glc); purple diamonds, N-acetylneuraminic acid (NeuAc/sialic acid).

Figure 2

HILIC enrichment and no enrichment comparison of N-glycopeptides: (A) Number of identified glycopeptides without and with enrichment, blue and pink color, respectively, (the error bar represents the SD calculated from the average number of glycopeptides in a biological replicate). (B) MS spectra of non-enriched N-glycopeptides from NT1 samples. (C) MS spectra of enriched N-glycopeptides from NT1 samples. Symbols are described in Figure 1.

Figure 3

Changes in N-glycan distribution including sialylated, sialylated–fucosylated, fucosylated, and high mannose in percentages based on abundance of glycopeptides for (A) control and (B) narcolepsy type 1 samples.

Figure 4

Unsupervised principal component analysis (PCA) at 95% confidence level for total identified glycopeptides.

Figure 5

Heatmap of N-glycopeptides with statistically significant expression structures between control and NT1 samples.

Figure 6

ROC curves of the N-glycopeptides with statistically significant differences between control and NT1 samples: (A) up-regulated glycopeptides; (B) down-regulated glycopeptides.