Disulfide Proteome Analysis of Buckwheat Seeds to Screen Putative Allergens

Abstract

Accumulating evidence suggests a correlation between disulfide bonding and the allergenicity of proteins. Also, a significant characteristic of most food allergens is that they are stable to proteases. We sought to identify putative allergens of buckwheat seed comprehensively by combining a disulfide proteome technique with an in vitro digestibility test. First, a dilute acetic acid fraction of buckwheat seed was found to be rich in disulfide proteins. Internal amino acid sequence analyses of these proteins showed that most of them were known allergens or putative allergens sharing high amino acid sequence similarities to known allergens. Next, the fraction was subjected to in vitro protease digestion, which revealed relatively large fragments that were resistant to prolonged enzymatic digestion. These protease‐resistant fragments contained disulfide bonds that should have protected the potential enzyme cleavage sites by forming compact structures. These results confirm and extend our knowledge of the correlations among the disulfide bonding of proteins, their protease stability, and their allergenicity. Also, these observations suggest a new strategy to identify putative allergens by proteomic approaches as well as to mitigate them.

Analysis of tartary buckwheat ( Fagopyrum tataricum ) seed proteome using offline two‐dimensional liquid chromatography and tandem mass spectrometry

“…Over the past several years, some studies have been made on the proteome of buckwheat seed, including the screening of buckwheat allergens (Yano, Kusada, Kuroda, & Kato‐Emori, ), the identification of endosperm and embryo proteins (Kamal et al, ), comparison of different buckwheat species (Capraro, Magni, Giorgi, Duranti, & Scarafoni, ), and the protein profile changes in stored buckwheat seeds (Hashiguchi et al, ). These results provide important information for the nutrition and storage research of buckwheat seed.…”

“…Over the past several years, some studies have been made on the proteome of buckwheat seed, including the screening of buckwheat allergens (Yano, Kusada, Kuroda, & Kato-Emori, 2006), the identification of endosperm and embryo proteins (Kamal et al, 2011), comparison of different buckwheat species (Capraro, Magni, Giorgi, Duranti, & Scarafoni, 2018), and the protein profile changes in stored buckwheat seeds (Hashiguchi et al, 2018). These results Here, a high-throughput proteomic workflow based on two-dimensional LC-MS/MS, which was connected by high-pH RPLC fractionation and LC-MS/MS, was used to deeply identify the proteome of TBS, and 3,363 proteins were identified, providing a comprehensive information at the protein molecular level for the future research of tartary buckwheat.…”

Quantitative plant proteomics

“…Both Trx and glutaredoxin are involved in the reduction of disulfide bonds in proteins 130, 131. For instance, the redox proteome has been studied in Trx‐linked reactions during seed germination 123, 126, 132–134. In addition, Alvarez et al 135 have investigated the changes in the proteins' redox status in response to methyl jasmonate using 2‐DE combined with monobromobimane labeling and Sypro Ruby staining, showing changes in the redox status and/or abundance for 35 proteins, 33 of which could be identified by MS analysis.…”

Introduction of the Disulfide Proteome: Application of a Technique for the Analysis of Plant Storage Proteins as Well as Allergens

“…Thus, we conducted a feasibility study to see whether specific labeling of disulfide proteins can be applied to allergen screening. After alkylation of free SH groups by iodoacetamide, disulfide proteins in crude extracts of mite, pollen 66 and buckwheat 67 were reduced to expose free sulfhydryl groups which were selectively labeled with mBBr. Fluorescent proteins detected on the subsequent 2D gel were then subjected to internal amino acid sequence analysis.…”