Table 1.
Comparison of four epitope mapping techniques that consider the three-dimensional structure of the allergen: X-ray crystallography, NMR, cryo-EM and mass spectrometry.
| X-ray crystallography | Nuclear magnetic resonance |
|---|---|
| • Crystalline state, however, the crystals contain ~30–70% of disordered solvent | • Solution conditions (requires weeks of stability for data collection). |
| • Theoretically no structure size limit • Proteins purified from natural sources can be used |
• High resolution structures up to ~30 kDa. |
| • Expression with isotope is typically not required for proteins or DNA. Sometimes selenomethionine is incorporated instead of Met. | • Protein/DNA samples usually require 13C and 15N labeling (stable isotopes). Cost of expression is prohibitive except in prokaryotes. |
| • X-rays diffraction data are recorded, and the diffraction patterns are used to calculate initial electron density maps. The maps are used to trace a model of the macromolecule, that is later refined and validated | • Data is nuclear resonance frequencies of primarily 1H, 13C, and 15N. Distances between 1H atoms are used to build ensembles of possible structures. |
| • Highly flexible/disordered regions of proteins cannot be modeled and are absent in the final models | • Motion and disorder can be directly measured on many time scales. |
| Mass spectrometry | Cryo-electron microscopy |
| • Typically used in protection assays for epitope mapping. • High sensitivity/low sample requirements. • Atomic resolution identifies specific residues for protection from modification. • Residues that are convenient to modify in protection assays are not always useful for epitope mapping. • Chemistry of modification procedures can have off target effects. |
• Can determine atomic resolution structures frozen from solution in vitreous ice. • Low sample requirements. • Resolution occasionally as good as X-ray crystallography. • Performs better on very large samples with high symmetry, typically 100's of kDa, so it is currently not easily or generally applicable to allergen epitope mapping. |