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

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 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.

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

“…Other techniques can be used to study mucins including dynamic and multi-angle light scattering 1 , isopycnic and rate-zonal centrifugations 6 , electron microscopy, mass spectrometry 1,7,8 . However, with the investigation of novel pharmacological agents targeting mucin molecules, it is imperative to develop reliable laboratory techniques to study mucin biology.…”

“…In addition, rate-zonal centrifugation coupled with immunodetection and transmission electron microscopy are commonly used to determine the macromolecular conformation of mucins 6 . Mass spectrometry is also used to quantify mucins, detect proteolytic cleavage and analyze oligosaccharide composition 1,7,8 . Such techniques are costly, time consuming and often require large volumes and/or high concentrations of sample.…”

Identification and relative quantification of phosphopeptides by a combination of multi‐protease digestion and isobaric labeling

“…Furthermore, the basic principle presented is also not restricted to protein phosphorylation but has the potential to be extended to other PTMs, where the application of multi-protease digestion has been shown to be beneficial. [28]…”

Rapid global characterization of immunoglobulin G1 following oxidative stress

“…The use of multiple digestion enzymes to enhance sequence visibility and coverage has been reported. [38][39][40][41] Here, we digest each stressed sample separately in triplicate with either trypsin or chymotrypsin. 200 µg of protein was added to 100 µL of 20 mM DTT, 100 mM NH 4 HCO 3 pH 8.0, and incubated at 56°C for 45 min.…”