In a groundbreaking advance in cereal crop research, an international team led by Prof. Dr. Katharina Scherf at the Leibniz Institute for Food Systems Biology at the Technical University of Munich has, for the first time, successfully quantified amylase/trypsin-inhibitors (ATIs) in barley with unprecedented precision. These proteins, long recognized for their role in triggering immune responses in humans, particularly in relation to wheat, have remained poorly characterized in barley until now. This breakthrough, enabled by the development of a cutting-edge targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique, sheds new light on the molecular composition of barley, promising new paths towards breeding barley varieties that are better tolerated by individuals suffering from food sensitivities.
The study meticulously analyzed 181 barley accessions sourced globally, encompassing both two-row and six-row genetic types, distinguished by their distinct grain morphology. Two-row barley, notable for producing a single, robust grain per spike node rich in starch, remains the preferred choice in brewing industries, while six-row barley typically yields smaller grains in clusters of three and is commonly relegated to animal feed. This diverse collection allowed the researchers to examine the natural variability of ATI content across a broad genetic spectrum, providing invaluable insight into the protein’s distribution and abundance within the species.
Utilizing their bespoke LC-MS/MS method combined with stable isotope dilution analysis, Scherf and her colleagues identified and quantified ten barley-specific ATI isoforms. This methodological innovation overcomes prior limitations that hindered accurate measurement and precise characterization of ATI proteins in barley matrices. The analytical sensitivity and specificity achieved not only enable reliable quantitation but also establish a robust framework for future investigations into ATI dynamics during barley development and processing.
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Quantitative results revealed a striking variation in total ATI concentrations among the accessions, ranging from 1.1 to 5.2 milligrams per gram of flour, corresponding to approximately 0.7% to 3.6% of the total protein content. Interestingly, statistical analyses demonstrated that grain row number, a fundamental morphological trait distinguishing two-row from six-row barley, did not significantly influence ATI levels. This suggests that ATI expression is governed more by genetic and environmental factors than by this gross morphological characteristic.
The relevance of these findings extends beyond agricultural science into the realm of human health. ATIs have emerged as potential contributors to adverse reactions in consumers who are sensitive to wheat and related cereals, with symptoms including gastrointestinal distress, neurological complaints such as “brain fog,” and systemic inflammation. In particular, ATIs are implicated as key triggers in non-celiac wheat sensitivity (NCWS), a condition affecting an estimated 0.6% to 6% of Western populations but notoriously difficult to diagnose due to its complex etiology. The identification of barley-specific ATI profiles holds promise for developing barley cultivars with reduced immunogenic potential, thereby expanding dietary options for sensitive individuals.
Co-author Dr. Sabrina Geisslitz highlights the clinical implications, noting that many sufferers of NCWS report symptom relief upon adoption of gluten-reduced or gluten-free diets. “Understanding the ATI landscape in barley is a vital step towards mitigating food-induced immune responses,” she explains. By pinpointing barley varieties exhibiting naturally low ATI concentrations, breeders can judiciously select materials that minimize immunological risks, facilitating the production of more tolerable food products.
Sarah Joestl, the study’s first author and a doctoral researcher under Prof. Scherf, underscores that the discovery of three six-row landraces from Eritrea, Greece, and Ethiopia with notably low ATI contents is particularly encouraging. These genetic resources represent invaluable candidates for introgression into breeding pipelines aimed at creating barley crops that maintain agronomic performance while enhancing consumer health safety.
Importantly, barley’s role in the global food system surpasses its traditional use in animal feed and beer manufacturing. As the fourth most significant cereal by production volume, with over 142 million tons harvested in the 2023/24 season, barley’s versatility is expanding into breakfast cereals, baked goods, and even plant-based meat analogs. This diversification increases the imperative to understand and regulate potentially immunogenic compounds such as ATIs, ensuring that emerging barley-based products are both nutritious and safe for a broad consumer base.
Future research directions highlighted by the team involve extending the ATI quantification methodology to processed barley goods. Considering that thermal treatment and food processing can alter protein structures and bioactivity, assessing ATI content in end products will be critical for developing effective strategies to reduce or eliminate immunostimulatory components, particularly for individuals coping with allergies or chronic inflammatory diseases.
The technical prowess of this study lies in the innovative combination of liquid chromatography with tandem mass spectrometry, augmented by stable isotope dilution analysis. This approach ensures the absolute quantification of specific ATI peptides, overcoming historical obstacles posed by the protein’s low abundance and structural complexity. Such precision analytical methodologies are defining a new era in cereal protein research, allowing for accurate molecular profiling that was previously unattainable.
The barley samples analyzed were meticulously curated by the Leibniz Institute of Plant Genetics and Crop Plant Research, ensuring a representative and genetically diverse collection that strengthens the validity and applicability of the findings. This collaboration underscores the importance of interdisciplinary and cross-institutional efforts in addressing complex challenges at the intersection of agriculture, food science, and human health.
Funded in part by the European Union’s ERC under the GLUTENOMICS project (grant number 101040437), this research exemplifies the cutting-edge initiatives driving the sustainable transformation of staple crop production. Through rigorous experimental design and analytical innovation, the team led by Prof. Scherf has paved the way for a new generation of cereal crops that harmonize agricultural productivity with rising consumer health demands.
As the world grapples with increasing incidences of food sensitivities and allergic diseases, the ability to precisely characterize and modulate biochemically active components like ATIs holds profound implications. The findings not only contribute to the fundamental scientific understanding of barley proteins but also inform practical applications ranging from breeding programs to food processing technologies. Ultimately, the work holds the promise of enhancing food tolerance and quality of life for millions globally.
Subject of Research: Not applicable
Article Title: Quantitation of amylase/trypsin-inhibitors in barley using targeted LC-MS/MS
News Publication Date: 23-Jun-2025
Web References: http://dx.doi.org/10.1016/j.foodres.2025.116910
References: Joestl, S., Alomari, D.Z., Alqudah, A.M., Börner, A., Geisslitz, S., and Scherf, K.A. (2025). Quantitation of amylase/trypsin inhibitors in barley using targeted LC-MS/MS. Food Res Int, 116910. 10.1016/j.foodres.2025.116910.
Image Credits: Prof. Dr. Katharina Scherf
Keywords: Amylase/trypsin-inhibitors, barley, LC-MS/MS, food intolerances, non-celiac wheat sensitivity, protein quantification, crop breeding, gluten sensitivity, food allergens, proteomics
Tags: allergy-triggering proteins in barleyamylase trypsin inhibitors quantificationbarley breeding for food sensitivitiesbarley protein composition analysiscereal crop research advancementsfood tolerance and sensitivitiesglobal barley accessions studyimmune responses to food proteinsimproving food safety through genetic diversityinnovative agricultural research techniquestargeted liquid chromatography mass spectrometrytwo-row vs six-row barley genetics
