| Protein glycosylation is one of the most important post-translational modifications (PTMs) and aberrant glycosylation on some important proteins is highly associated with various types of diseases such as cancer and neuromuscular disorders. Among which, N-glycosylation of proteins play a key role for disease prognosis, diagnosis, and therapeutic response to drugs. Consequently, the analysis of protein glycosylation is of great significance not only for biomedical research but also for biotechnology industries. Herein, a comprehensive urinary glycoproteome analysis of diabetic nephropahy has been performed and two kinds of materials have been proposed for glycopeptide enrichment.(1) The identification of biomarkers to noninvasively detect pre-diabetic nephropathy/diabetic nephropathy (DN) can greatly facilitate interventions designed to prevent or delay progression of end-stage renal disease. As identification of markers for prediction of the clinical course of DN still remains to be a great challenge, in this study, the human urinary glycoproteome in type-2DN was characterized to identify potential biomarkers. Glycoproteins from urine samples of19healthy individuals and56DN patients with different disease stages were enriched by hydrazide method and characterized by liquid chromatography/tandem mass spectrometry, coupled with label-free quantification based on spectral counts. Totally295glycoproteins were unambiguously identified based on745unique glycopeptides with776distinct glycosites, among which25glycoproteins were demonstrated expression differences in abundance correlated with DN progression, and5of them were first time determined differentially expressed in DN. The results were further validated by western blot. Bioinformatics analysis identified bio-functions of the potential biomarkers including cellular function and maintenance, small molecule biochemistry and cellular movement; and the major pathway was TNF-a signaling. The present work provides a comprehensive urinary glycoproteome analysis for DN, which is helpful in viewing mechanisms perturbed in DN patients, thus merit further investigations of these potential biomarkers in a larger cohort of subjects.(2) Hydrophilic magnetic colloidal nanocrystal clusters coated with chitasan (Fe3O4@CS MCNCs) have been successfully prepared by an easily accessible one-pot solvothermal strategy, and characterized with SEM, TG, IR and hysteresiscurve. The performance of as-prepared mateiral was evaluated using IgG tryptic digests. The results showed it had high selectivity, high sensitivity and high binding capacity to glycopeptides. Furthermore, it was used to enrich glycopeptides from45μg protein sample extracted from HeLa cells, and determined283unique N-glycosylation sites corresponding to175glycosylated proteins. Compared with conventional method, which were tedious and time-consuming with at least four steps, the procedure of prepared Fe3O4@CS MCNCs was just one step and cost-effective, which demonstrated the great potential in detection and identification of low abundant glycopeptides in glycoproteome analysis.(3) MIL-53, a member of metal oganic frameworks family (MOFs), is of superior hydrophilicity in the internal surface of the pores and hydrophobicity in the outside pores, as well as the large pore window. In this study, MIL-53(Al) was chosen from MIL-53serial materials for its high specificity, high sensitivity and high recovery for glycopeptides by using IgG tryptic digests. Following experimental resutls indicated that the possible enrichment mechanism was the superior hydrophilic interation of the materials, as well as the pore restricted access. It was the first time to introduce MOFs for glycopeptide enrichment, which provided a new mateiral for glycoproteome analysis. |
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