Background and Aims: Type 2 diabetes (T2D) is a chronic metabolic disorder with a growing number of patients. Glycosylation, a typical post-translational modification (PTMs) of proteins, plays an important role in protein folding, signal transduction, and immune response. Mass spectrometry (MS) has been a powerful tool for glycoproteomics research. However, the complexity, low abundance and poor ionization efficiency of glycopeptides make the enrichment of glycopeptides prior to MS analysis. Among various strategies, HILIC-based enrichment method has been widely developed because of unbiased enrichment, excellent compatibility, and good repeatability. Here, We aimed to modify glucose-6-phosphate (G-6-P) with outstanding biocompatibility and hydrophilicity onto the surface of metal organic framework (MOF) for efficient enrichment of glycopeptides, and further for the glycoproteomics research of T2D.
Methods: We synthesized UiO-66-NH₂ by solvothermal method, then modified G-6-P groups to increase the hydrophilicity. The nanomaterials (denoted as UiO-66-NH₂/G-6-P) were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Human serum immunoglobulin G (IgG) and horseradish peroxidase (HRP) tryptic digests were used to evaluate the enrichment performance of UiO-66-NH₂/G-6-P by MALDI-TOF MS. The serum N-glycoproteome of 3 patients with T2D and 3 healthy controls were analyzed by nano-LC-MS/MS.
Results: The characterization results showed the successful synthesis of UiO-66-NH₂/G-6-P. MALDI-TOF MS analysis revealed that UiO-66-NH₂/G-6-P exhibited high sensitivity, good selectivity and repeatability of enrichment. Proteomics and glycoproteomics analysis showed that 3690 peptides corresponding to 1067 proteins and 160 N-glycopeptides corresponding to 118 N-glycoproteins were identified, and there were significant differences in the identified proteins and N-glycoproteins between healthy controls and T2D patients.
Conclusion: We developed a hydrophilic and biocompatible glycopeptides enrichment nanomaterial and applied it to serum N-glycoproteome research of T2D. We found some proteins and N-glycoproteins changed significantly in T2D, which may provide a new insight for the glycoproteomics research of T2D.

Yanruyu Feng

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