Perennial Ryegrass Contains Gluten-Like Proteins That Could Contaminate Cereal Crops

Abstract

Background: To ensure safe consumption of gluten-free products, there is a need to understand all sources of unintentional contamination with gluten in the food chain. In this study, ryegrass (Lolium perenne), a common weed infesting cereal crop, is analysed as a potential source of gluten-like peptide contamination. Materials and Methods: Ten ryegrass cultivars were analysed using shotgun proteomics for the presence of proteins from the prolamin superfamily. A relative quantitative assay was developed to detect ryegrass gluten-like peptides in comparison with those found in 10 common wheat cultivars. Results: A total of 19 protein accessions were found across 10 cultivars of ryegrass for the protein families of PF00234-Tryp_alpha_amyl, PF13016-Gliadin, and PF03157-Glutenin_HMW. Protein and peptide homology searches revealed that gliadin-like peptides were similar to avenin and gamma-gliadin peptides. A total of 20 peptides, characteristic of prolamin superfamily proteins, were selected for liquid chromatography mass spectrometry (LC-MS) with multiple reaction monitoring (MRM). Only two of the monitored peptides were detected with high abundance in wheat, and all others were detected in ryegrass. Glutenin and alpha-amylase/trypsin inhibitor peptides were reported for the first time in ryegrass and were noted to be conserved across the Poaceae family. Conclusion: A suite of gluten-like peptides were identified using proteomics that showed consistent abundance across ryegrass cultivars but were not detected in wheat cultivars. These peptides will be useful for differentiating wheat gluten contamination from ryegrass gluten contamination.

Keywords: LC-MS/MS; cereal; gluten; proteomics; ryegrass; wheat; wild grass.

Figure 1

(A) Number of proteins (purple) and peptides (green) detected at 1% FDR (99% confidence) for 10 ryegrass cultivars. (B) Proteins identified with the protein family search (Pfam) that belong to three specific families: PF00234-Tryp_alpha_amyl, PF13016-Gliadin, and PF03157-Glutenin_HMW. The size of the circle represents the number of identification occurrences among 10 cultivars of ryegrass.

Figure 2

The Heatmap and hierarchical clustering show the relative abundance of the peptides representing two major groups detected across 10 ryegrass cultivars (n = 3) and 10 wheat cultivars (n = 4). The colour in each cell represents the log (peak area) of each peptide monitored (red = max value, blue = min value, grey = NA). The column to the right indicates the protein family membership for the peptides (orange = PF00234-Tryp_alpha_amyl family, blue = PF13016-Gliadin, green = PF03157-Glutenin_HMW).

Figure 3

Relative quantitation expressed as multiple reaction monitoring (MRM) peak area for peptides from proteins of the family PF13016 across 10 cultivars of Lolium perenne. Data are presented as the mean ± SD (n = 3) with one to three peptides from each protein. (A) Avenin protein (G8ZCU8); (B) Avenin (I4EP57); (C) Avenin (I4EP61); (D) Gamma-gliadin (F2X0K8); (E) Gamma-gliadin (F2X322); (F) Gamma-gliadin (B6DQD5); and (G) LMW-glutenin (J9QGY5).

Figure 4

Relative quantitation of glutenin peptides across the 10 varieties of L. perenne from proteins of the family PF03157. Data are presented as the mean ± SD (n = 3) with one to three peptides from each protein. (A) HMW-glutenin (X5CHT6); and (B) HMW-glutenin (H6UQP6).

Figure 5

Relative quantitation of peptides across the 10 varieties of L. perenne from proteins of the Pfam family PF00234. Graphs show the MRM peak area for each cultivar. Data are presented as the mean ± SD (n = 3) with one peptide from each protein. (A) Dimeric alpha-amylase inhibitor (T1WIP3); (B) Alpha-gliadin (A8VZG4); (C) Dimeric alpha-amylase inhibitor (T1WI30); and (D) Trypsin inhibitor (Q84VT9).