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

Area of Science:

• Food Science and Technology
• Analytical Chemistry
• Immunology

Background:

• Wheat α-amylase/trypsin inhibitors (ATIs) are implicated in non-celiac wheat sensitivity (NCWS) and baker's asthma.
• Previous studies focused on major ATIs, leaving a gap in comprehensive analysis.
• A need exists for a method covering all known wheat ATIs.

Purpose of the Study:

• To develop a comprehensive workflow for characterizing and quantifying all wheat ATIs.
• To establish a robust analytical method encompassing all UniProt-listed wheat ATIs.
• To evaluate extraction and analytical techniques for improved ATI profiling.

Main Methods:

• Optimized extraction using chloroform/methanol (CM) and buffered denaturing conditions.
• Refined tryptic digestion, chromatographic separation, and targeted tandem mass spectrometry (HPLC-MS/MS).
• Utilized SDS-PAGE for purity assessment and demonstrated bifunctional inhibition activity.

Main Results:

• Successfully analyzed all 14 reviewed wheat ATI entries using the developed HPLC-MS/MS method.
• Identified 13 biomarkers for ATIs.
• Observed differential representation of major ATIs (7-34%) across 46 German and Turkish wheat samples.

Conclusions:

• The developed HPLC-MS/MS method provides the first comprehensive analysis of all known wheat ATIs.
• This advancement aids in understanding the role of ATIs in NCWS and allergic reactions.
• The method offers a powerful tool for wheat quality control and allergen detection.