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?

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 (ß-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.

Variability of CP4 EPSPS expression in genetically engineered soybean (Glycine max L. Merrill).

The expression of the CP4 EPSPS protein in genetically engineered (GE) soybean confers tolerance to the Roundup® family of agricultural herbicides. This study evaluated the variability of CP4 EPSPS expression using an enzyme-linked immunosorbent assay in soybean tissues collected across diverse germplasm and 74 different environments in Argentina, Brazil and the USA. Evaluated material included single and combined (stacked) trait products with other GE traits in entries with cp4 epsps gene at one or two loci. The highest level of CP4 EPSPS was observed in leaf tissues, intermediate in forage and seed, and lowest in root tissues. Varieties with two loci had approximately twice the level of CP4 EPSPS expression compared to one locus entries. Variable and non-directional level of CP4 EPSPS was observed with other factors like genetic background, trait stacking, growing region or season. The maximum and average CP4 EPSPS expression levels in seed provided large margins of exposure (MOE of approximately 4000 and 11,000, respectively), mitigating concerns over exposure to this protein in food and feed from soybean varieties tolerant to Roundup® herbicides.

Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.