Multiple Enzyme Approach for
the Characterization
of Glycan Modifications on the C‑Terminus of the Intestinal
MUC2Mucin
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Abstract
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 <i>N</i>-glycan consensus sites were identified as glycosylated. Fifty-six <i>N</i>-glycopeptides covering 10 <i>N</i>-glycan sites,
and 14 <i>O</i>-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