Impaired wound healing is a common symptom resulting from diabetes. Keratinocytes, the main epidermal cell type, are deeply involved in the cell proliferation stage of wound healing. N-glycosylation increases the diversity of protein structures and functions. It has been reported that diabetes results in alterations of N-glycosylation and thus can serve as potential biomarkers for examining the molecular effects of diabetes. Differences in N-glycosylation may affect exosomes, which are extracellular vesicles generated by all types of cells that can transport bioactive cargos including lipids, genetic material, and proteins. The importance of exosomes for transport as well as intercellular communication indicates that they could be involved in the diabetes state. Our previous study found that exosomes play a critical role in the wound healing process with N-glycosylation facilitating exosome uptake by post-wounded macrophage tissue.<br/>We hereby report a study of post-wounded keratinocyte cells and keratinocyte-derived exosome glycopeptides using LC-MS/MS. Exosomes were isolated using an immunoprecipitation pull-down technique, yielding samples of single cell type origin. Glycoproteomics allows for multidimensional analysis at the glycan, peptide, and protein levels. By comparing diabetic cells to cell-derived exosomes and their counterparts from the healthy control group, we gain an increased understanding of glycosylation preferences upon the generation of exosomes and how glycosylation of both cells and exosomes are altered as an effect of diabetes.<br/>Our previously reported N-glycosylation 2D map was utilized to visualize our data. This format allows for straightforward visual comparisons of quantitative glycosylation patterns, which is often a challenging component of glycoproteomics experiments. To further compare these glycosylation patterns, we have developed a multi-dimensional correlation method to turn the degrees of differences into correlation coefficient values. This quantitative metric allows us to empirically quantify which glycosylation sites are most strongly regulated between healthy and diabetic animals.<br/>To summarize, we have quantified the N-glycoproteome of mouse keratinocyte cells and cell-derived exosomes under healthy and diabetic conditions. We have compared the glycosylation profiles from each glycosylation site and uncovered differences at the glycan, glycosylation site, and glycoprotein levels.

Quantitative N-glycoproteomic study of diabetic mouse keratinocyte cells and exosomes reveals site-specific changes

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