Ivana Prodić1*, Teodora Đukić2, Vesna Jovanović3, Katarina Smiljanić3
1Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11042 Belgrade, Serbia
2University of Belgrade, Faculty of Medicine, Institute of Medical Chemistry, Višegradska 26, 11000 Belgrade, Serbia
3University of Belgrade – Faculty of Chemistry, Center of Excellence for Molecular Food Sciences & Department of Biochemistry, Serbia;
ivana.prodic [at] imgge.bg.ac.rs
Abstract
Porcine-derived trypsin generated proteomic data of the major peanut allergen Ara h 1 from the peanut was reassessed to search for possible facilitating/hindrance effects on trypsin digestion efficacy caused by post-translational and chemical modifications (PTMs) positioned on arginine or lysine (K/R) residues. If the potential effects caused by PTMs are observed with porcine trypsin, they can be just augmented and more pronounced within human intestinal digestion. The reasoning is in inferior performance of human trypsin compared to porcine-derived used in proteomic digestion protocols, also in the lower trypsin-to-sample ratio and much shorter digestion times, even though gastric digestion precedes and trypsin is not the sole digestive enzyme.
A novel method was developed to decipher cleavage or miscleavage outcomes at scissile bonds in each, modified and unmodified sequence counterparts, using PEAKS Studio-X+ (Bioinformatics Solutions Inc., Ontario, Canada) in the reassessment of high-resolution tandem mass spectrometry data, from 18-hour long trypsin digestion proteomic protocols.
In general, eight site-specific and modified K/R residues with methylation, dihydroxy and formylation showed significantly higher content of miscleaved bonds (at least >10%) compared to their unmodified counterpart peptides. Specifically, dihydroxylation and formylation hindered trypsin efficacy, while methylation on several K/R showed opposite effects.
It is essential to elucidate the specific impacts of modifications on trypsin digestion performance and if there are additional effects generated by food processing, which could influence digestion outcomes and allergenicity of food proteins/peptides.
Keywords: Trypsin, PTMs, PEAKS, K/R residues, mass spectrometry
Acknowledgement: Ministry of Science, Innovation and Technological Development of Republic of Serbia Grants No. 451-03-47/2023-01/200168 (UBFC) and 451-03-47/2023-01/200042 (IMGGE).
