A chromatographic and immunoprofiling approach to optimising workflows for extraction of gluten proteins from flour

Ingestion of gluten proteins from wheat, and related prolamin proteins from barley, rye, and oats, can cause adverse reactions in individuals with coeliac disease and IgE-mediated allergies. As there is currently no cure for these conditions, patients must practice avoidance of gluten-containing foods. In order to support patients in making safe food choices, foods making a “gluten-free” claim must contain no more than 20 mg/Kg of gluten. Mass spectrometry methods have the potential to provide an alternative method for confirmatory analysis of gluten that is complementary to analysis currently undertaken by immunoassay. As part of the development of such methodology the effectiveness of two different extraction procedures was investigated using wholemeal wheat flour before and after defatting with water-saturated butan-1-ol. A single step extraction with 50 % (v/v) propan-2-ol containing 2 M urea and reducing agent (buffer 1) was compared with a two-step extraction using 60 % (v/v) aqueous ethanol (buffer 2) followed by re-extraction of the pellet using buffer 1, using either wheel mixing under ambient conditions (19 ◦C) or sonication at 60 ◦C. The procedures were compared based on total protein extraction efficiency and the composition of the extracts determined using a combination of HPLC, SDSPAGE and immunoblotting with a panel of four gluten-specific monoclonal antibodies. Defatting generally had a detrimental effect on extraction efficiency and sonication at 60 ◦C only improved extraction efficiency with buffer 2. Although the single-step and two-step procedures were equally effective at extracting protein from the samples, analysis of extracts showed that the two-step method gave a more complete extraction of gluten proteins. Future studies will compare the effectiveness of these procedures when applied in the sample workflows for mass spectrometry based methods for determination of gluten in food

Dynamic changes in the brain protein interaction network correlates with progression of Aβ42 pathology in Drosophila

Alzheimer’s disease (AD), the most prevalent form of dementia, is a progressive and devastating neurodegenerative condition for which there are no effective treatments. Understanding the molecular pathology of AD during disease progression may identify new ways to reduce neuronal damage. Here, we present a longitudinal study tracking dynamic proteomic alterations in the brains of an inducible Drosophila melanogaster model of AD expressing the Arctic mutant Aβ42 gene. We identified 3093 proteins from flies that were induced to express Aβ42 and age-matched healthy controls using label-free quantitative ion-mobility data independent analysis mass spectrometry. Of these, 228 proteins were significantly altered by Aβ42 accumulation and were enriched for AD-associated processes. Network analyses further revealed that these proteins have distinct hub and bottleneck properties in the brain protein interaction network, suggesting that several may have significant effects on brain function. Our unbiased analysis provides useful insights into the key processes governing the progression of amyloid toxicity and forms a basis for further functional analyses in model organisms and translation to mammalian systems

The pharmacometabodynamics of gefitinib after intravenous administration to mice: a preliminary UPLC–IM–MS study

The effects of intravenous gefitinib (10 mg/kg), an anilinoquinazoline thymidylate kinase inhibitor (TKI), selective for the epidermal growth factor receptor (EGFR), on the urinary metabotypes of mice were studied. We hypothesized that, in response to the administration of gefitinib, there might be significant changes in the excretion of many endogenous metabolites in the urine, which could be correlated with the plasma pharmacokinetics (PK) of the drug. In order to investigate this conjecture, urine from male C57 BL6 mice was collected before IV dosing (10 mg/kg) and at 0–3, 3–8, and 8–24 h post-dose. The samples were profiled by UPLC/IM/MS and compared with the profiles obtained from undosed control mice with the data analyzed using multivariate statistical analysis (MVA). This process identified changes in endogenous metabolites over time and these were compared with drug and drug metabolite PK and excretion. While the MVA of these UPLC/IM/MS data did indeed reveal time-related changes for endogenous metabolites that appeared to be linked to drug administration, this analysis did not highlight the presence of either the drug or its metabolites in urine. Endogenous metabolites affected by gefitinib administration were identified by comparison of mass spectral, retention time and ion mobility-derived collision cross section data (compared to authentic standards wherever possible). The changes in endogenous metabolites resulting from gefitinib administration showed both increases (e.g., tryptophan, taurocholic acid, and the dipeptide lysyl-arginine) and decreases (e.g., deoxyguanosine, 8-hydroxydeoxyguanosine, and asparaginyl-histidine) relative to the control animals. By 8–24 h, the post-dose concentrations of most metabolites had returned to near control values. From these studies, we conclude that changes in the amounts of endogenous metabolites excreted in the urine mirrored, to some extent, the plasma pharmacokinetics of the drug. This phenomenon is similar to pharmacodynamics, where the pharmacological effects are related to the drug concentrations, and by analogy, we have termed this effect “pharmacometabodynamics”

A multiple reaction monitoring method for determining peanut (Arachis hypogea) allergens in serum using quadrupole and time-of-flight mass spectrometry

Peanut is a major cause of severe IgE-mediated food allergic reactions, which can be exacerbated by factors, such as exercise, that may increase allergen uptake into the circulation. Enzyme-linked immunosorbent assays (ELISAs) have been used to determine allergen uptake into serum, but there are concerns over their specificity and a confirmatory method is required. Mass spectrometry (MS) methods have the potential to provide rigorous alternatives for allergen determination. A suite of peptide targets representing the major clinically relevant peanut allergens previously applied in food analysis were used to develop a targeted multiple reaction monitoring (MRM) method for determination of peanut in serum. Depletion of serum using affinity chromatography was found to be essential to allow detection of the peptide targets. A comparison of triple quadrupole and Q-TOF methods showed that one Ara h 2 peptide was only detected by the Q-TOF, the other peptide targets giving similar assay sensitivities with both MS platforms, although transitions for all the peptides were detected more consistently with the Q-TOF. The Q-TOF MRM assay detected peanut from spiked serum more effectively than the triple quadrupole assay, with Ara h 3 being detected down to 3 mg total peanut protein/L of serum, comparable with an Ara h 3-specific ELISA. The poor recoveries observed for both methods are likely due to loss of peanut immune complexes during the serum depletion process. Nevertheless, the Q-TOF MRM method has much promise to confirm the uptake of peanut proteins in serum samples providing immune complexes can be disrupted effectively prior to depletion

The Fate of IgE Epitopes and Coeliac Toxic Motifs during Simulated Gastrointestinal Digestion of Pizza Base

Understanding how food processing may modify allergen bioaccessibility and the evolution of immunologically active peptides in the gastrointestinal tract is essential if knowledge-based approaches to reducing the allergenicity of food are to be realised. A soy-enriched wheat-based pizza base was subjected to in vitro oral–gastro–duodenal digestion and resulting digests analysed using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The digestion profile of pizza base resembled that of bread crust where higher temperatures during baking reduced protein solubility but still resulted in the generation of a complex mixture of peptides. MS profiling showed numerous peptides carrying IgE epitopes, and coeliac toxic motifs were in excess of 20–30 residues long and were only released after either 120 min of gastric digestion or a combination of gastric and duodenal digestion. In silico prediction tools showed an overestimated number of cleavage sites identified experimentally, with low levels of atypical peptic and chymotryptic cleavage sites identified particularly at glutamine residues. These data suggest that such alternative pepsin cleavage sites may play a role in digestion of glutamine-rich cereal foods. They also contribute to efforts to provide benchmarks for mapping in vitro digestion products of novel proteins which form part of the allergenicity risk assessment

Quantitative Proteomic Profiling of Peanut Allergens in Food Ingredients Used for Oral Food Challenges

Profiling allergens in complex food ingredients used in oral food challenges and immunotherapy is crucial for regulatory acceptance. Mass spectrometry based analysis employing data independent acquisition coupled with ion mobility mass spec-trometry-mass spectrometry (DIA-IM-MS) was used to investigate the allergen composition of raw peanuts and roasted peanut flour ingredients used in challenge meals. This comprehensive qualitative and quantitative analysis using label-free approaches identified and quantified 123 unique protein accessions. Semi-quantitative analysis indicated that allergens Ara h 1 and Ara h 3 were the most abundant proteins and present in approximately equal amounts, and were extracted in reduced amounts from roasted peanut flours. The clinically significant allergens Ara h 2 and 6 were less abundant but relative quantification was unaffected by roasting. Ara h 5 was undetectable in any peanut sample, whilst the Bet v 1 homologue Ara h 8 and the lipid transfer protein aller-gen, Ara h 9, were detected in low abundance. The oleosin allergens, Ara h 10 and 11 were moderately abundant in the raw peanuts but were 100-fold less abundant in the defatted roasted peanut flour than the major allergens Ara h 1, 3, 2 and 6. Certain isoforms of the major allergens dominated the profile. The relative quantitation of the major peanut allergens showed little variation between different batches of roasted peanut flour. These data will support future development of targeted approaches for absolute quantifica-tion of peanut allergens which can be applied to both food ingredients used in clinical studies, extracts used for skin testing and to identify trace levels of allergens in foods.</p

Quantitative Proteomic Profiling of Peanut Allergens in Food Ingredients Used for Oral Food Challenges

Profiling allergens in complex food ingredients used in oral food challenges and immunotherapy is crucial for regulatory acceptance. Mass spectrometry based analysis employing data-independent acquisition coupled with ion mobility mass spectrometry–mass spectrometry (DIA–IM–MS) was used to investigate the allergen composition of raw peanuts and roasted peanut flour ingredients used in challenge meals. This comprehensive qualitative and quantitative analysis using label-free approaches identified and quantified 123 unique protein accessions. Semiquantitative analysis indicated that allergens Ara h 1 and Ara h 3 were the most abundant proteins and present in approximately equal amounts and were extracted in reduced amounts from roasted peanut flours. The clinically significant allergens Ara h 2 and 6 were less abundant, but relative quantification was unaffected by roasting. Ara h 5 was undetectable in any peanut sample, while the Bet v 1 homologue Ara h 8 and the lipid transfer protein allergen, Ara h 9, were detected in low abundance. The oleosin allergens, Ara h 10 and 11, were moderately abundant in the raw peanuts but were 100-fold less abundant in the defatted roasted peanut flour than the major allergens Ara h 1, 3, 2, and 6. Certain isoforms of the major allergens dominated the profile. The relative quantitation of the major peanut allergens showed little variation between different batches of roasted peanut flour. These data will support future development of targeted approaches for absolute quantification of peanut allergens which can be applied to both food ingredients used in clinical studies and extracts used for skin testing and to identify trace levels of allergens in foods

Microfluidic Separation Coupled to Mass Spectrometry for Quantification of Peanut Allergens in a Complex Food Matrix

Peanut is an important food allergen, but it cannot currently be reliably detected and quantified in processed foods at low levels. A level of 3 mg protein/kg is increasingly being used as a reference dose above which precautionary allergen labeling is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred with 0, 3, 10, or 50 mg/kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid-labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on the detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionization efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimized MRM method. Although other peptide targets were detected with three transitions at the 50 mg/kg peanut protein level in both matrices, only Arah2­(Q6PSU2)^147–155 could be quantified reliably and only in the chocolate dessert at 10 mg/kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30–50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using a simple extraction methodology