A rapid separation and characterization of mucins from mouse submandibular glands by supported molecular matrix...

Mucins are heavily glycosylated proteins with high molecular mass, and are involved in various diseases including infection, inflammation, and cancer. As easy separation method, such as gel electrophoresis, however, does not exist for mucins, due to their large molecular sizes and heterogeneities. In 2009, we published a supported molecular matrix electrophoresis (SMME) method that can be used to characterize mucins. For SMME analysis, mucins have been enriched by ultrafiltration of trypsin digests using a 100 KDa cutoff filter. However, this enrichment results in a loss of protein identification capability using proteomic approaches. In this study, we describe a simple enrichment of mucins without trypsinization for SMME analysis. The enrichment was developed using a porcine submandibular gland and then was applied to study and compare mouse submandibular glands between young and aged mice. From mouse submandibular glands, hyaluronic acid and some mucins were observed by SMME. One of the mucins was identified as MUC10 by proteomic analysis of the band on the SMME membrane and immunostaining using anti-MUC10 antibody. A major O-glycan of MUC10 was determined to be NeuAcα2-3Galβ1-3GalNAc. Furthermore, our experiments revealed that the concentrations of these molecules were lower in aged mice than in young mice, and that an unknown mucin-like molecule was detected only from the aged mouse submandibular gland.