Presence of small resistant peptides from new in vitro digestion assays detected by liquid chromatography tandem mass spectrometry: An implication of allergenicity prediction of novel proteins?

Abstract

The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (β-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/μg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/ mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.

A Direct Assay for Measuring the Activity and Inhibition of Coactivator-Associated Arginine Methyltransferase 1

“…The addition of DTT was vital for avoiding disulfide bond formation, and the addition of BSA was found necessary to keep CARM1 in its active form by blocking aggregation and reducing unspecific binding of the CARM1 to the well plate. Sample workup consisted of quenching the enzyme reaction by the addition of 0.1% formic acid solution (known to be compatible with the MS conditions of the assay 28 ) and addition of the internal standard.…”

Mass spectrometric analysis of digesta does not improve the allergenicity assessment of GM crops

“…Increasingly, the evidence surrounding the allergenicity risk assessment for GM crops indicates that digestion assays are of little value in the context of protein allergenicity. Recent advancements in using MS to identify small peptides in digesta has not improved the value of digestion assays for the assessment of allergy risk (Mackie et al, 2019 ; Wang et al, 2020 , 2021 ). Therefore, the weight of the current evidence surrounding the allergenicity risk assessment for GM crops suggests that digestion assays should not be considered unless a validated assay with proven criteria is developed that can distinguish allergens from non-allergens with some reasonable level of reliability (Verhoeckx et al, 2019 ; Herman et al, 2020 ; Bøgh and Madsen, 2016 ).…”

“…Technical advancements in the application of mass spectrometry (MS) to identify small peptides in complex mixtures have been used to characterize the processing of known allergens and non-allergens exposed to digestive enzymes (Mackie et al, 2019 ; Korte et al, 2017 ; EFSA Panel on Genetically Modified Organisms et al, 2017; Wang et al, 2020 ; Di Stasio et al, 2020 ). No pattern of peptide fragmentation was found to be associated with the allergenic status of proteins (Torcello-Gómez et al, 2020 ; Wang et al, 2020 ). MS can be useful in the identification of epitopes contained in small peptides within the digestive residues of known allergens, which can then be further investigated using IgE antibodies from sensitized individuals, but such analyses are incapable of identifying unknown allergenic epitopes in proteins not known to cause allergy.…”

“…The effects of the food matrix and food processing, and the age, genetics, and environment (including microbiome composition) of individuals, further complicate predictions. With this backdrop, it is unclear how identifying small peptides in digesta will meaningfully inform the allergenicity risk assessment for newly expressed proteins in genetically engineered crops allergenic status of proteins (Torcello-Gómez et al, 2020;Wang et al, 2020). MS can be useful in the identification of epitopes contained in small peptides within the digestive residues of known allergens, which can then be further investigated using IgE antibodies from sensitized individuals, but such analyses are incapable of identifying unknown allergenic epitopes in proteins not known to cause allergy.…”

Scientific Opinion on development needs for the allergenicity and protein safety assessment of food and feed products derived from biotechnology

“…The pepsin resistance test is performed regularly, although several studies have demonstrated that there is a poor correlation between resistance to pepsin digestion and allergenicity (Kenna and Evans, 2000 ; Fu et al, 2002 ; Takagi et al, 2003 ; Thomas et al, 2004 ; Herman et al, 2007 ; Ofori‐Anti et al, 2008 ; Costa et al, 2020). In contrast, other studies show that the classical pepsin resistance assay and simple SDS–PAGE analysis, as developed by Astwood et al ( 1996 ), can distinguish between pepsin susceptible and resistant proteins and remains as the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a weight‐of‐evidence approach (Wang et al, 2017 , 2020 ). However, these studies only used small sets of proteins and a larger reference set is needed to make definite conclusions on the predictability of digestion tests.…”