Table 5.

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

Method	Advantages	Limitations
Isolation, fractionation and purification of mucins [128–130]	- 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, 133–135])	- 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.