MUC5B Is the predominant mucin glycoprotein in chronic otitis media fluid

Abstract

Chronic otitis media (COM), e.g. "glue" ear is characterized by middle ear effusion and conductive hearing loss. Although mucous glycoproteins (mucins), which contribute to increased effusion viscosity, have been analyzed in ear tissue specimens, no studies have been reported that characterize the molecular identity of secreted mucin proteins present in actual middle ear fluid. For this study, effusions from children with COM undergoing myringotomy at Children's National Medical Center, Washington, DC were collected. These were solubilized and gel fractionated, and the protein content was identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics approach. Western blot analyses with mucin specific antibodies and densitometry were performed to validate the mass spectrometry findings. LC-MS/MS results identified mucin MUC5B by >26 unique peptides in six of six middle ear effusion samples, whereas mucin MUC5AC was only identified in one of six middle ear effusions. These findings were validated by Western blot performed on the same six and on an additional 11 separate samples where densitometry revealed on average a 6.4-fold increased signal in MUC5B when compared with MUC5AC (p = 0.0009). In summary, although both MUC5AC and MUC5B mucins are detected in middle ear effusions, MUC5B seems to be predominant mucin present in COM secretions.

Figure 1. SDS-PAGE and Mucin Protein identification

A. SDS-PAGE of samples 1–6 stained with Coomassie. Each individual sample gel lane was cut into 32 gel segment bins where protein bands were noted with the staining. The gel cuts performed are shown for sample 1. The same cuts were performed for each sample prior to in gel digestion and LC-MS/MS analysis for peptide identification as described in the Methods section. B. SDS-PAGE of serum sample stained with Coomassie. Serum was run as a control because of the blood contamination of the effusion samples. It was important to ensure that identified mucins from the effusion samples would not be identified in serum as well. C. Localization of mucin peptides detected in each effusion sample by location of gel lane segment bin cut. The y-axis represents the number of mucin specific peptides identified by LC-MS/MS. The x-axis represented by the grayscale bars depicts the individual Coomassie blue stained gel band segment ‘bins’ (from 1 to 32 as shown in Figure 1A) that were cut out from the SDS page gels for protein identification from the middle ear effusion samples. Results highlight that the majority of peptides identied to correspond to mucins were found in the gel segment ‘bins’ located to the top of the gel lane (as expected for large molecular weight proteins such as mucins).

Figure 2. Representative MS/MS fragmentation spectrum of a MUC5B tryptic peptide

Parent tryptic peptides identified by MS are further fragmented along the amide backbone by collision induced dissociation in the mass spectrometer (MS/MS). These fragments, which differ in mass by the loss of amino acids, are displayed in the MS/MS spectrum and enable protein identification based on the amino acid sequence combined with the peptide mass. The figure shows LTQ-MS/MS spectrum of the parent MS tryptic peptide m/z 607.66 corresponding to a doubly charged ion. The series of y and b ion fragments are identified by their mass and resolved to the indicated peptide sequence. This peptide was unambiguously identified as MUC5B corresponding tryptic peptide [SEQLGGDVESYDK].

Figure 3. Western blots

A. Western blot results for 6 samples tested with proteomics. Strong signal compared to positive biological control (saliva) was noted in all samples blotted for MUC5B. Faint signal compared to positive biological control (Calu-3 cell extracts) was noted in samples blotted for MUC5AC. All wells were loaded with 40μg of protein. Lane numbers corresponds to sample numbers used in proteomics analysis. B. Western blot results revealed strong MUC5B (upper panel) signal intensity in 9/11 separate, additional effusion samples (not in lanes 4 and 9). MUC5AC (lower panel) demonstrated strong signal intensity in only 1/11 samples (lane 6). Faint MUC5AC signal was noted in another 4 samples (lanes 3,5,7,9). All wells were loaded with 40 μg of protein. Lane numbers correspond to 11 separate COM samples used as validation. Positive biologic control for MUC5B (upper panel) was saliva. Positive biologic control for MUC5AC (lower panel) was whole Calu-3 cell extracts.

Figure 4. Average western blot densitometry signal

A. Average densitometric signal intensity of all sample signal/positive biologic control signal combined. Y-axis represents dots per inch of average density signal of each sample normalized to the biological positive control signal on the same gel/blot. Error bars represent standard error of the mean (p=0.0009, two-tailed Student’s t-test).