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

Abstract

The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker’s asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60–80%)/trypsin (10–20%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7–34%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported.

Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars

“…In this context, two commercially available wheat samples, whole meal and extracted wheat flour type 405 were analyzed. The analytical method used for the ATI analysis of wheat samples was the one developed in our previous work [ 20 ]. Compared to the average ATI contents in the two wheat samples, the results revealed that the relative amounts of ATIs were 2–3 times lower in sorghum samples SOR 1099, B-Sc, SOR 1236, SOR 958, SOR 20 and SOR 557 and up to 60 times lower in nine of the analyzed sorghum cultivars (SOR 777, SOR 618, R-Sc, SOR 654, SOR 759, SOR 226, SOR 1055, SOR 844 and SOR 555); while considering the corresponding averages ( Figure 4 a).…”

“…The protein extraction and the in-solution digestion steps were based on the method established and optimized in our previous work [ 20 ] using ammonium bicarbonate (100 mM) buffer containing urea (4 M). Figure S7 (Supplementary Materials) provides the different extraction steps.…”

“…They are small molecules, with molecular weights often between 10 and 16 kDa, containing in their structures 8–10% of cysteine residues, which can constitute correspondingly 4–5 disulfide bonds [ 19 ]. ATIs in wheat have been found to associate themselves to dimer or tetramer structures [ 20 ]. To our knowledge, no such associations have been reported for sorghum ATIs.…”

“…ATIs have been mentioned as being responsible for a type of allergy related to wheat consumption, also called non-celiac wheat sensitivity (NCWS). It is known that the risk of developing an allergy depends on the level/concentration of these molecules in a cereal [ 20 ]. Although extensive studies referring to the characterization of wheat ATIs and their related toxicity have been recently addressed, nevertheless, relatively little information about characteristics of sorghum ATIs, their relative proportion and their potential to induce such disorders are known.…”

“…Recent studies and our own work indicate that selected peptides originating from different proteins can be identified, being specific to these proteins and may be used to assess the corresponding proteins on a quantification scale [ 20 , 30 , 31 , 32 ]. The strategy involves different steps: selection of marker proteins based on the thorough research of literature and databases, in silico digestion with different proteolytic enzymes, potential matches to test for the specificity of the peptides liberated and to encompass their response by a multiple reaction monitoring (MRM) approach for chromatography coupled with tandem mass spectrometry (LC–MS/MS).…”