New applications of advanced instrumental techniques for the characterization of food allergenic proteins

Figures & data

Figure 1. Graphical representation of bioactive thermal proteome profiling strategy. Problem: uncharacterized bioactive compound in food matrix; Challenge: to obtain a proteome-wide food bioactive compound interaction map; Advanced method: bioactive thermal proteome profiling; Solution: unbiased identification of protein targets by MS; Opportunity: prediction of mechanisms of action of a novel compound by one single experiment and analytical technique.

Figure 2. Principle of surface plasmon resonance sensor. Surface plasmon resonance sensors detect a refractive index change within a detection area (<500 nm) as a modification of resonance angle (RA) (Yanase et al. 2014).

Figure 3. Microfluidic sensor. Schematic representation of the microfluidic ELISA sensor layout (not to scale) (A) and principle of the absorption detection with a miniaturized optical sensor device (B) (Weng, Gaur, and Neethirajan 2016).

Table 1. Novel advanced methods for application in the food allergy field.

Methods	Physical or chemical parameter	Readout	Novelty	Limitations	References
X-Ray free-electron lasers crystallography	Molecular structure	Three-dimensional structure	Structure information at room temperature	Extent of radiation damage in the nanocrystal target	Sugahara et al. (2015)
Solid-state nuclear magnetic resonance	Molecular structure	Epitope characterization, atomic level molecular structure information	Not limited by molecular size, solubility, and lack of long-range order. Proteins do not need to be crystallized	Overlapping resonances, poor to modarete sensitivity (μM), few software resources	Sun et al. (2012); Emwas et al. (2019)
Cryo-electron microscopy	Molecular structure	IgE epitope mapping	Proteins do not need to be crystallized	Limitations to obtain high-resolution structures	Bianchi et al. (2018)
Cryo-electron microscopy / mass spectrometry	Molecular structure	Three-dimensional reconstructions of macromolecular assemblies	Allow to identify protein regions that are non-covalently bound	Some secondary structures, such as beta sheets, impede cross-linking	Schmidt and Urlaub (2017)
Sequence-based methods	Amino acid sequence	Homology to known sequences. Identity percentage	Easy and fast to perform	Not explain the cross-reactivity between allergens with low sequence homology. Ignore the presence of discontinuous epitopes. Precision may be low to be of practical use	Stadler and Stadler (2003); Fiers et al. (2004)
Motif-based methods	Identification of characteristic motifs	Allergenicity potential	Increase the accuracy and specificity of the prediction. Take into account the 3 D structure and its characteristic motifs	Only assist in the prediction of the allergenicity. Experimental analysis are necessary to confirm the results	Ivanciuc, Schein, and Braun (2003); Maurer-Stroh et al. (2019)
Support Vector Machine-based methods	Amino acid composition or pair-wise sequence similarity	Allergenicity potential	Machine learning based-method. Increase the accuracy and specificity of the prediction. Allow the prediction of cross-reactivity in novel proteins that lack high sequence similarity		Cui et al. (2007); Muh, Tong, and Tammi (2009)
Thermal proteome profiling	Protein targets and mechanisms of action	Identification of protein targets by MS	Proteome-wide identification of targets from any cell type. It is not required structural or function characterization of compound. Applicability to novel compounds or uncharacterized mixtures	Limitation to proteins as drivers of cellular responses	Carrasco del Amor et al. (2019)
Surface plasmon resonance	Interactions between allergens and biomolecules	Molecular interactions	Real-time monitoring. Label-free detection. Increasing sample throughput	Immobilization effects	Willison et al. (2013); Michel, Xiao, and Alameh (2017)
Microfluidics			Small sample consumption, greater sensitivity	Complex chip fabrication processes	Cretich et al. 2009; Singh et al. (2010)
Electrochemical biosensors			High sensitivity, portability	Antibodies limitations	Čadková et al. (2015)
Cytometry by time-of-flight	Protein expression	Expression of cellular markers involved in the immune response to allergens	Detection of more than 40 targets at a single cell resolution	Development of dimensionality reduction methods for the analysis of generated data	Simoni et al. (2018)
RNA sequencing	Gene expression	Measurement of transcriptionally active genes expressed by immune cells	Detection of novel transcripts at single cell level		Croote et al. (2018)
Cellular indexing of transcriptomes and epitopes by sequencing	Protein and gene expression	Combined identification of cellular markers and active genes expressed by immune cells	Measurement of immune markers together with a genome-wide expression profiling		Stoeckius et al. (2017)

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