Novel aspects of quantitation of immunogenic wheat gluten peptides by liquid chromatography–mass spectrometry/mass spectrometry
Introduction
Gluten proteins comprise a large family of storage proteins found in cereal grain seeds. This large protein family consists of hundreds of proteins ranging in size from about 30 kDa into the millions of kDa [1]. Since the late1700s, proteins in this family have been studied extensively and several theories have been applied to further subdivide or classify the family into sub-groups [2], [3], [4], according to their characteristics, amino acid sequences, locations within the grain, solubility and relationship within other grains. Today's nomenclature generally refers to “gluten” as the water-insoluble seed storage proteins found in the Triticeae tribe of the grass (Gramineae) family that includes wheat, barley and rye.
Some gluten proteins associated with wheat, rye and barley grains are considered a trigger of a variety of immune diseases, including food allergies and intolerances [5]. One such disease, celiac sprue, is a complex autoimmune disorder of the small intestine that affects close to 1% of the world's population [6], [7]. Current knowledge about the pathogenesis of the disease involves environmental, genetic and immunological factors. In those who are genetically predisposed, exposure to gluten can trigger both innate and adaptive immune responses. Prolonged exposure to even modest quantities of dietary gluten can result in severe damage to the small intestine. In contrast, symptoms can vary from severe to limited or even absent [8], [9].
Two interesting solubility-based subgroups of the large water-insoluble gluten protein family are: (1) the aqueous alcohol-soluble prolamins and (2) the alcohol-insoluble glutelins (i.e. “gliadins” and “glutenins”, respectively, in wheat). In the wheat kernel, the monomeric gliadins and polymeric glutenins are found in the relative ratio 65:35 and form most of the storage protein. Similar groups of proteins, illustrated in Fig. 1, are found in barley and rye grains [10]. The prolamins (originally so-named for their high proline and glutamine content) and more recently, the glutelins of wheat, barley and rye grains, have all been identified as the environmental triggers for the immune reactions in celiac patients [11], [12], [13], [14]. Although all cereal grains contain prolamin and glutelin proteins, the amino acid sequences of the proteins in each grain are different. At present, only those of wheat, barley and rye have been shown to trigger the abnormal immune response that affects the intestinal lining of those genetically susceptible to celiac disease.
Currently, the only effective therapy available for those who suffer with celiac disease is a strict lifelong gluten-free diet [15], [16], [17]. Maintaining a true gluten-free existence is difficult, because gluten is used (under a variety of names) extensively, throughout the world in many manufacturing arenas [18]. In food manufacturing, it is used as a flavor enhancer, a thickener, fortification ingredient, filler, whitener etc. Gluten is also used in the manufacturing of personal care products, nutritional supplements and drug products. Fortunately, the presence of gluten in a product is usually noted by its inclusion in the ingredient list on product packaging (albeit referenced by a variety of names). Unfortunately, there are also many hidden sources of gluten; from unlabeled ingredients and from cross-contamination of manufacturing and transportation equipment. Therefore, people with celiac disease are destined to a life of label-reading to ensure that products are safe for personal use and consumption. Several groups of researchers are working to establish if there actually exists a “safe” quantity of gluten, tolerable for celiac patients to ingest on a daily basis [19], [20]. But in the meantime, a gluten content of “zero” is desired.
Therefore, from the perspectives of patient disease management, manufacturing quality control and worldwide consistency with respect to product labeling, there poses an obvious need to be able to accurately determine that consumer products are gluten-free and label them as such. However, the subject of product labeling is a controversial one and there is no concise definition of “gluten-free” that is accepted worldwide [21], [22], [23]. The United States Food and Drug Administration (FDA) has proposed a definition for the term “gluten-free”, which was posted in the Federal Register on January 3, 2007. The final form of the definition was expected to be published in August 2008; but it still remains in draft form [24].
Conversely, the Codex Alimentarius Commission (a joint committee with delegates from both the Food and Agriculture Organization of the United Nations [FAO-UN] and the World Health Organization [WHO]) revised its Standard for Foods for Special Dietary Use for Persons Intolerant to Gluten in July 2008 [25]. The Codex definition is now the standard for gluten content allowed in a food product to be considered gluten-free for international trade. In short, it states that for a product to be labeled “gluten-free”, it must not contain more than 20 ppm (parts per million) of wheat, barley or rye gluten. This correlates to about 1 mg of total gluten in 50 g of food.
There is also controversy surrounding the analytical method(s) considered acceptable to a variety of organizations to determine and quantify the amount of gluten in food and consumer products. Currently, both the FDA and the Codex endorse different methods, variations of an immunological antibody-based ELISA. Various ELISA methods are available. Each has proven to be efficient, yet each has limited application in trace gluten analysis [26], [27]. It is imperative to know which method to use in each analytical application, thus accurately ensuring food safety.
The work described here details the development of a sensitive and specific non-immunological analytical method, which can be considered complementary to the ELISA assay. This liquid chromatography–mass spectrometry (LC–MS) based method has been developed for the quantitative detection of trace levels of immunogenic gluten marker peptides from complex mixtures, such as food samples. Data shown will emphasize how this direct enzymatic digestion-LC–MS/MS method releases the physiologically relevant marker peptides, then detects and quantifies them, in a wide variety of native and processed foods in order to form a correlation between the quantities of the gluten marker peptides in the digested food to a known gluten adverse reaction.
Section snippets
Chemicals
Pepsin, trypsin, chymotrypsin, KHNaPO4 and NaOH were obtained from Sigma–Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile dithiothreitol (DTT) ethanol, isopropanol, tris-(hydroxymethyl) aminomethane and HCl were obtained from Fischer Scientific. Water was obtained from an in-house Milli-Q water purification system (Millipore, Billerica, MA, USA).
Samples of food and consumer products
Quinoa flour, stone ground whole grain corn flour, stone ground whole grain soy flour, vital wheat gluten flour, stone ground whole wheat flour
Discovery of physiologically relevant immunogenic peptides
Previous work with recombinant gliadin proteins has led to the discovery of two highly inflammatory, physiologically relevant, multivalent 33- and 28-residue gluten oligopeptides [29], [30]. In turn, those peptides have proven to be useful markers of gluten toxicity in several subsequent studies [31], [32], [33], [34]. Motivated by those findings, we wished to identify additional physiologically relevant gluten peptides that could be detected and quantified in trace amounts with high
Discussion
Increased interest, awareness and research surrounding gluten-related conditions, including celiac disease, over the last two decades has resulted in a wealth of new information, thus allowing proper diagnoses in both symptomatic and asymptomatic patients [43]. There is a growing understanding of the pathogenesis of celiac disease, new methods for screening patients and novel methodologies for analyzing gluten proteins and gluten-containing foods and products.
Nonetheless, the analysis,
Conclusion
High performance liquid chromatography–mass spectrometry is a powerful analytical technique for use in the analysis of food proteins. A rapid, sensitive and specific analytical LC–MS/MS method has been developed for the quantitative detection of immunogenic gluten marker peptides from complex mixtures. This versatile methodology allows both cooked and native food to be analyzed for the presence of wheat gluten.
LC–MS/MS was used to both detect and quantify the marker peptides in enzymaticaly
Acknowledgements
This research was conducted with support by NIH–NIDDK Grant 1R41DK68890-1. The authors also wish to thank Agilent Technologies and Sigma–Aldrich/Supelco for their support.
References (52)
J. Am. Diet. Assoc.
(1959)- et al.
Gastroenterology
(2003) - et al.
Gastroenterology
(2001) - et al.
J. Food Sci.
(2007) - et al.
Am. J. Clin. Nutr.
(2007) J. Am. Diet. Assoc.
(2008)- et al.
J. Am. Diet. Assoc.
(2008) - et al.
J. Cereal Sci.
(1999) - et al.
Chem. Biol.
(2006) - et al.
Gastroenterology
(2007)
Anal. Chim. Acta
J. Chromatogr. B
J. Chromatogr. A
Balliere's Clin. Gastroenterol.
Semin. Immunol.
Food Res. Int.
Adv. Food Nutr. Res.
J. Chromatogr. B
J. Cereal Sci.
The Vegetable Proteins
Immun. Endoc. Metab. Agents Med. Chem.
Arch. Intern. Med.
Gut
Immunol. Rev.
Cited by (80)
Characterization of physicochemical and immunogenic properties of allergenic proteins altered by food processing: a review
2024, Food Science and Human WellnessProteomic analysis of food allergens
2022, Food Proteomics: Technological Advances, Current Applications and Future PerspectivesData Treatment in Food Proteomics
2020, Comprehensive FoodomicsAre current analytical methods suitable to verify VITAL® 2.0/3.0 allergen reference doses for EU allergens in foods?
2020, Food and Chemical Toxicology