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Table 5 Advantages and limitations of methods and techniques used for measuring biochemical and biophysical mucus properties

From: Strategies for measuring airway mucus and mucins

MethodAdvantagesLimitations
Isolation, fractionation and purification of mucins [128,129,130]- Required steps for all initial molecular/biochemical characterizations.- Labor intensive and may be expensive.
- Loss of sample during the process, and therefore cannot be used solely for quantification purposes.
- Care must be taken that the mucins do not become degraded.
Glycosylation analysis [17] and mass spectrometry [127].- Provides valuable information on species- and organ-specific glycosylation and post-transcriptional modifications of mucins.
- Precise qualitative and quantitative information on different molecules in the sample, most often proteins and carbohydrates.
- Expensive system and materials.
- Identification of proteins/molecules require protein libraries for each animal species of interest and knowledge about glycosylation sites.
Viscoelasticity of mucus (laser/light scattering analysis [131], direct rheometry [132], and fluorescence recovery after photobleaching (FRAP) [13, 133,134,135])- Laser scattering or quasi-elastic (dynamic) scattering is used specifically for molecular size distribution and for mucin conformation and chain dimensions analysis.
- FRAP assay is easily applicable for in vitro/ex vivo studies.
- Viscosity/elasticity under shear stress conditions can be done directly in rheometer machines but requires higher amounts of sample.
- Use mathematical modeling equations to calculate the viscoelasticity, which can introduce errors if not performed or calculated correctly.
- They do not give information on the quantity or specificity of single mucin component.
- FRAP and other microrheology techniques depend on the diameter and non-adhesiveness of labelled particles used.
Chromatography separation and detection [128, 129, 136]- specific technique for separation and molecular analysis of biological substances.
- Provides information on molecular charge and size.
- Chromatography is expensive and labor-intensive.
- If radioactive detection use, handling and disposal, is expensive and environmentally unfriendly.
Metabolic labeling/ Radiolabel discharge measurement or autoradiography [129, 137].- Can be used to measure amount of secreted radioactive isotope-labelled substance (e.g. 3H-D-glucoseamine, or iodo[−14C]acetamide) incorporated easily in the newly produced mucins.
- Historically used for characterization of mucin size in chromatographically separated fractions and for quantitation of total mucus secretion after treatments (as ratio of radioactivity detected at baseline and after treatment).
- Requires radioactive substance handling and exposure.
- Materials are strictly regulated and expensive to dispose of.
- Not very sensitive to specific mucin secretion.
- Applicable only in vitro/ex vivo and on sputum samples from patients.