Static in vitro digestion tests to assess allergenic risk of novel proteins - Use of a peptidomics workflow based on free softwares

IntroductionThe resistance of proteins to digestion may play a role in determining their allergenic potential. In order to evaluate the digestibility of proteins, in vitro protocols seem appropriate when ethical constraints hinder in vivo studies. In our study, the ‘pepsin resistance test’, currently used by the European Food Safety Authority to assess allergenicity of proteins is compared to more physiologically relevant in vitro digestion protocols. A peptidomics workflow based on free softwares is used to identify, quantify and perform statistical analysis on the peptides produced.MethodsComparison of an infant, early phase adult, and late phase adult model is being applied on a panel of allergen/non-allergen proteins from animal and vegetal origin. The proteolysis rate in gastric and intestinal compartment is determined in order to detect persistent intact protein (SDS-PAGE) and hydrolysis products larger 9 amino acids (LC-MS).All peptidomics data (NanoLC fitted to Quadrupole-Orbitrap MS) were processed with free softwares: from data conversion to peptide identification, quantification and statistical analysis.ResultsMore than two thousands NanoLC-MS/MS runs were acquired during the course of this project and the number of peptides identified ranged from one to five hundred per proteins depending on their resistance to enzymatic hydrolysis. All peptides identified (X!tandempipeline software [1]) were automatically quantified (MassChroQ [2]) in all samples, regardless the fact that it was identified at this particular time of digestion. Peptide quantification results processed in the R software environment allowed the clustering of peptides based on their behavior regarding the digestion protocol used and the digestion time (0,5 to 60min). Proteins that are not pepsin resistant show slower kinetics of proteolysis for the infant model of digestion, highlighting the fact that, for this population, they may still cause undesired immune responses via sensitization in the duodenum.ConclusionsDifferences in the kinetics of proteolysis have been found across models for proteins that are not pepsin resistant. This project highlights the importance of a multi-test protocol to assess protein digestibility.Novel AspectUsing a full pipeline of free softwares for peptidomics data treatment, we were able to point out peptide clusters which are only abundant when more physiologically relevant models of digestion were used