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Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C-Terminus of the Intestinal MUC2Mucin.

Artikel i vetenskaplig tidskrift
Författare Sjoerd van der Post
Kristina A Thomsson
Gunnar C. Hansson
Publicerad i Journal of proteome research
Volym 13
Nummer/häfte 12
Sidor 6013-23
ISSN 1535-3907
Publiceringsår 2014
Publicerad vid Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi
Sidor 6013-23
Språk en
Länkar dx.doi.org/10.1021/pr500874f
Ämneskategorier Cell- och molekylärbiologi

Sammanfattning

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein's central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 N-glycan consensus sites were identified as glycosylated. Fifty-six N-glycopeptides covering 10 N-glycan sites, and 14 O-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins.

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