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

Abstract

Amylase/trypsin inhibitors (ATIs) are major wheat allergens and they are also implicated in causing non-celiac gluten sensitivity and worsening other inflammatory conditions. With only few studies on ATI contents in different Triticum species available so far, we developed a targeted liquid chromatography-tandem mass spectrometry (LC–MS/MS) method based on stable isotope dilution assays to quantitate the 13 most important ATIs in a well-defined sample set of eight cultivars of common wheat and durum wheat (modern species), as well as spelt, emmer and einkorn (ancient species) grown at three locations in Germany, respectively. Only few ATIs with low contents were detected in einkorn. In contrast, spelt had the highest total ATI contents. Emmer and common wheat had similar total ATI contents, with durum wheat having lower contents than common wheat. Due to the lack of correlation, it was not possible to estimate ATI contents based on crude protein contents. The wheat species had a higher influence on ATI contents than the growing location and the heritability of this trait was high. Despite comparatively low intra-species variability, some cultivars were identified that may be promising candidates for breeding for naturally low ATI contents.

Wheat ATIs: Characteristics and Role in Human Disease

“…Consistent with the low ATI concentrations determined by MS (44,60), einkorn showed no inhibition of Coleopteran insect or mammalian α-amylases (70-74) and the lowest TRL4activating potential compared to other wheat species (50). However, einkorn inhibited α-amylase from Lepidopteran insects (72) and contains a trypsin inhibitor similar to rye and barley (70) which may explain the high trypsin inhibitory activity (44).…”

“…hexaploid wheat whereas the proportion of tetrameric CM-type inhibitors is higher in tetraploid wheats (60).…”

“…A combination of discovery and targeted proteomics by MS is clearly the most attractive approach for identifying and quantifying ATIs. Comparative results on the contents of monomeric, dimeric and tetrameric ATIs have been obtained by absolute and relative quantification (25,59,60). Especially, targeted MS analysis is characterized by very low limits of detection (LOD) with 0.1-3.9 µg peptide per g flour ( 60), while discovery driven proteomics has at least 10 times higher LOD (61).…”

Comparative Characterization of Gluten and Hydrolyzed Wheat Proteins

“…This indicates the presence of proteins with M r around about 14 kDa or lower. This may result from, e.g., α-amylase/trypsin-inhibitors that have a M r of about 12-16 kDa, but further analyses are required to unambiguously identify these proteins [41].…”

Characterization of 150 Wheat Cultivars by LC-MS-Based Label-Free Quantitative Proteomics Unravels Possibilities to Design Wheat Better for Baking Quality and Human Health

“…Importantly, some wheat proteins can cause adverse inflammatory reactions in humans: (1) certain gluten peptide sequences trigger small intestinal and extraintestinal T cell activation and inflammation in patients with celiac disease [10,11]; (2) the family of wheat alpha-amylase/trypsin inhibitors (ATIs) stimulate intestinal innate immune cells via the activation of Toll-like receptor 4 and promote intestinal and extraintestinal inflammation [12][13][14][15][16][17]; (3) numerous wheat albumins and globulins, such as serpins, lipid transfer proteins, β-amylases and ATIs, as well as a few gluten proteins, can elicit respiratory and nutritional immediate allergic reactions [18][19][20][21]; and (4) a novel form of nutritional wheat allergy with an immediate intestinal, but a delayed clinical, reaction to wheat proteins is highly prevalent among patients with "irritable bowel syndrome" [22,23]. The first studies showed that wheat cultivars largely differ in the compositions of ATIs [24,25] or of the 33-mer α-gliadin peptide, a key immunogen for T cells in patients with celiac disease [26]. However, investigations of a large number of proteins across many wheat cultivars grown under different environmental conditions are lacking.…”