Identification and quantitation of amylase trypsin inhibitors across cultivars representing the diversity of bread wheat.

Alpha-amylase/trypsin inhibitors (ATIs) may have a role in non-coeliac wheat sensitivity (NCWS); but the ATI content and diversity across a range of wheat cultivars is not well characterised. Discovery proteomics was used to detect ATIs across two wheat cultivars: Chara and Magenta. Comprehensive mapping of detected ATIs with the ATIs from the recently published wheat genome RefSeq v1.0 shows the presence of three major subclasses: monomeric (9%), dimeric (61%) and CM-type (30%). Subsequently, the level of 18 ATI isoforms (63 peptides) grouped into four subtypes were monitored across 15 commercial wheat cultivars and the 8 parental lines from a multiparent advanced generation intercross (MAGIC) population using LC-MRM-MS. The ATI content of wheat cultivars Janz, Sunvale, Diamond Bird and Longreach Scout was significantly lower than other wheat cultivars. The MAGIC parental cultivars Baxter and Xiaoyan contain higher levels (~115% relative to the average wheat ATI content), whereas cultivar Pastor contained the lowest levels (~87%). Comprehensive sequence analysis, annotation, chromosomal locations and epitope mapping enabled us to build a LC-MRM-MS method to monitor and quantify the immunostimulatory ATI proteins potentially related to NCWS, autoimmune diseases and metabolic disorders. This provides an opportunity to select wheat cultivars with significantly lower level of ATIs.