Identification and Quantitation of Amylase Trypsin Inhibitors Across Cultivars Representing the Diversity of Bread Wheat

Abstract

α-Amylase/trypsin inhibitors (ATIs) may have a role in nonceliac wheat sensitivity (NCWS) and celiac disease (CD), but the ATI content and diversity across a range of wheat cultivars are not well characterized. 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 chloroform−methanol (CM) type (30%). Subsequently, the level of 18 ATI isoforms (63 peptides) grouped into four subtypes was monitored across 15 commercial wheat cultivars and the eight parental lines from a multiparent advancedgeneration intercross (MAGIC) population using liquid chromatography−multiple reaction monitoring-mass spectrometry (LC−MRM-MS). The ATI content of wheat cultivars Janz, Sunvale, Diamond Bird, and Longreach Scout was significantly lower than that of other wheat cultivars. The MAGIC parental cultivars Baxter and Xiaoyan 54 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 an 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 levels of ATIs.

Comparative quantitative LC–MS/MS analysis of 13 amylase/trypsin inhibitors in ancient and modern Triticum species

“…Recently, 18 ATIs, including isoforms of 0.19, 0.28, CM1, CM2, CM3, CM16 and CMX1/3, were monitored by SRM in several common wheat samples 35 . With this approach, it was possible to relatively compare different samples, but no absolute content can be calculated.…”

Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS

“…From these results, it is clear that option 2 of the Ambi/urea method is so far the best extraction method to achieve the relative quantification of wheat ATIs. Urea is, in principle, capable of denaturing proteins [ 39 ] and was also used in the study by Bose et al [ 26 ] to extract ATIs from wheat. The results of our own work provide significantly higher ATI contents compared to the C/M method for all wheat samples extracted with option 2.…”

“…They contribute 7–96% (S1, S11) to the total ATI content in the C/M extracts and 5–85% (S10, S11) in the option 2 extracts ( Figure S11, supplementary data ). In the available literature, the proteinogenic inhibitors of the CMX1/CMX3 type could only be relatively quantified by Bose et al [ 26 ]. For this reason, it would be desirable to extend the investigations in this respect.…”

“…In this work, tryptic digestion was performed in order to produce peptides before targeted LC−MS/MS analysis and untargeted based absolute quantification algorithm (iBAQ). More recently, 15 commercial wheat cultivars were used to analyze the ATI content [ 26 ]. The authors subsequently tried to maximize extraction of ATIs using isopropyl alcohol/dithiothreitol buffer (50 °C for 30 min) and then 8 M urea extraction buffer (at room temperature for 45 min).…”

“…Supernatants from both extracts were pooled before performing the tryptic digestion. The analysis was finally carried out using a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) [ 26 ].…”