Article pubs.acs.org/JAFC
Characterization of Grain-Specific Peptide Markers for the Detection of Gluten by Mass Spectrometry Katherine L. Fiedler, Sara C. McGrath, John H. Callahan, and Mark M. Ross* Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, College Park, Maryland 20740, United States S Supporting Information *
ABSTRACT: Global and targeted mass spectrometry-based proteomic approaches were developed to discover, evaluate, and apply gluten peptide markers to detect low parts per million (ppm) wheat contamination of oats. Prolamins were extracted from wheat, barley, rye, and oat flours and then reduced, alkylated, and digested with chymotrypsin. The resulting peptides were subjected to LC-MS/MS analysis and database matching. No peptide markers common to wheat, barley, and rye were identified that could be used for global gluten detection. However, many grain-specific peptide markers were identified, and a set of these markers was selected for gluten detection and grain differentiation. Wheat flour was spiked into gluten-free oat flour at concentrations of 1−100,000 ppm and analyzed to determine the lowest concentration at which the wheat “contaminant” could be confidently detected in the mixture. The same 2D ion trap instrument that was used for the global proteomics approach was used for the targeted proteomics approach, providing a seamless transition from target discovery to application. A powerful, targeted MS/MS method enabled detection of two wheat peptide markers at the 10 ppm wheat flour-in-oat flour concentration. Because gluten comprises approximately 10% of wheat flour protein, the reported wheat gluten-specific peptides can enable detection of approximately 1 ppm of wheat gluten in oats. KEYWORDS: wheat, gliadin, proteomics, LC-MS/MS, celiac disease, gluten-free
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INTRODUCTION Gluten is a complex network of proteins present in the endosperm of cereals that is responsible for giving dough its viscoelasticity.1 Gluten is composed of two fractions: (1) monomeric, alcohol-soluble prolamins; and (2) polymeric, alcohol-insoluble glutelins.2 Gluten proteins from wheat, barley, and rye can trigger an autoimmune disorder known as celiac disease (CD), and some wheat gluten and other wheat proteins can cause an IgE-mediated allergic response.3,4 There are some celiac/allergy common immunoreactive proteins, and the only successful treatment for patients with a wheat allergy or CD is a wheat- or gluten-free (GF) diet devoid of these grains.5 Although some oat cultivars have proven to be celiacogenic, pure oats can be an important source of nutrition in the GF diet because it is generally believed that the prolamins from oats (avenins) do not trigger an autoimmune response in most patients with CD, even though they are homologous to the prolamins from wheat (gliadins), barley (hordeins), and rye (secalins).4,6,7 However, oats are frequently contaminated with one or more of the three celiacogenic grains during farming, transport, storage, and processing.7,8 Analytical techniques for the detection of gluten in food are important to ensure accurate food labeling and thereby protect consumers with CD from exposure to gluten. The U.S. FDA recently established that food labels bearing a “gluten-free” claim must contain
Characterization of Grain-Specific Peptide Markers for the Detection of Gluten by Mass Spectrometry Katherine L. Fiedler, Sara C. McGrath, John H. Callahan, and Mark M. Ross* Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, College Park, Maryland 20740, United States S Supporting Information *
ABSTRACT: Global and targeted mass spectrometry-based proteomic approaches were developed to discover, evaluate, and apply gluten peptide markers to detect low parts per million (ppm) wheat contamination of oats. Prolamins were extracted from wheat, barley, rye, and oat flours and then reduced, alkylated, and digested with chymotrypsin. The resulting peptides were subjected to LC-MS/MS analysis and database matching. No peptide markers common to wheat, barley, and rye were identified that could be used for global gluten detection. However, many grain-specific peptide markers were identified, and a set of these markers was selected for gluten detection and grain differentiation. Wheat flour was spiked into gluten-free oat flour at concentrations of 1−100,000 ppm and analyzed to determine the lowest concentration at which the wheat “contaminant” could be confidently detected in the mixture. The same 2D ion trap instrument that was used for the global proteomics approach was used for the targeted proteomics approach, providing a seamless transition from target discovery to application. A powerful, targeted MS/MS method enabled detection of two wheat peptide markers at the 10 ppm wheat flour-in-oat flour concentration. Because gluten comprises approximately 10% of wheat flour protein, the reported wheat gluten-specific peptides can enable detection of approximately 1 ppm of wheat gluten in oats. KEYWORDS: wheat, gliadin, proteomics, LC-MS/MS, celiac disease, gluten-free
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INTRODUCTION Gluten is a complex network of proteins present in the endosperm of cereals that is responsible for giving dough its viscoelasticity.1 Gluten is composed of two fractions: (1) monomeric, alcohol-soluble prolamins; and (2) polymeric, alcohol-insoluble glutelins.2 Gluten proteins from wheat, barley, and rye can trigger an autoimmune disorder known as celiac disease (CD), and some wheat gluten and other wheat proteins can cause an IgE-mediated allergic response.3,4 There are some celiac/allergy common immunoreactive proteins, and the only successful treatment for patients with a wheat allergy or CD is a wheat- or gluten-free (GF) diet devoid of these grains.5 Although some oat cultivars have proven to be celiacogenic, pure oats can be an important source of nutrition in the GF diet because it is generally believed that the prolamins from oats (avenins) do not trigger an autoimmune response in most patients with CD, even though they are homologous to the prolamins from wheat (gliadins), barley (hordeins), and rye (secalins).4,6,7 However, oats are frequently contaminated with one or more of the three celiacogenic grains during farming, transport, storage, and processing.7,8 Analytical techniques for the detection of gluten in food are important to ensure accurate food labeling and thereby protect consumers with CD from exposure to gluten. The U.S. FDA recently established that food labels bearing a “gluten-free” claim must contain
