During the COVID-19 pandemic, development of diagnostic tests was vital to chart the course and to reduce the impact of the infection. Continued testing and surveillance of vaccine escape will continue for years to come which presents an opportunity to integrate such testing into clinical biochemistry laboratories that form part of integrated healthcare testing. Here we describe a protocol for a targeted mass spectrometry based proteomic assay (COVIDCAP) developed to detect SARS-CoV-2 peptides from oro-nasopharyngeal swabs (ONP) and saliva. This uses novel SISCAPA antibodies bound to magnetic beads and subsequent analysis of captured and purified SARS-CoV-2 nucleocapsid (NCAP) peptides. The method involves immediate deactivation of the sample using an ethanolic solution. This simultaneously inactivates the virus and denatures viral proteins at sampling in contrast to the approach for RT-PCR testing, with benefits for the assay as well as for downstream processing. A plate-based preparation of the samples involving acetone precipitation followed by a short tryptic digestion and subsequent immunocapture allows LC-MS detection and quantification of peptides from the NCAP protein, in a 3-minute inject-to-inject assay with an LOD of 20 attomoles from starting sample. For 576 ONP swab samples taken as exemplars here, the sensitivity and specificity of this analysis is shown to be 97.0% and 96.6% respectively.

A FASTA file containing WHO/IUIS wheat, barley and rye allergens and homologues identified through BLAST searching and 80mer sliding window FASTA searching in wheat, barley, rye and oats.

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 degrees C) or sonication at 60 degrees C. The procedures were compared based on total protein extraction efficiency and the composition of the extracts determined using a combination of HPLC, SDS-PAGE and immunoblotting with a panel of four gluten-specific monoclonal antibodies. Defatting generally had a detrimental effect on extraction efficiency and sonication at 60 degrees 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 pro-teins. 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.

Sensitisation to the lipid transfer protein Pru p 3 is associated with severe allergic reactions to peach, the proteins stability being thought to play a role in its allergenicity. Lipid binding increases susceptibility of Pru p 3 to digestion and so the impact of bile salts on the in vitro gastrointestinal digestibility of Pru p 3 was investigated and digestion products mapped by SDS-PAGE and mass spectrometry. Bile salts enhanced the digestibility of Pru p 3 resulting in an ensemble of around 100 peptides spanning the protein’s sequence which were linked by disulphide bonds into structures of ~ 5–6 kDa. IgE binding studies with a serum panel from peach allergic subjects showed digestion reduced, but did not abolish, the IgE reactivity of Pru p 3. These data show the importance of including bile salts in vitro digestion systems and emphasise the need to profile of digestion in a manner that allows identification of immunologically relevant disulphide-linked peptide aggregates.

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.

Suppl Information S1: Summary data on different Pru p 3 isoforms identified by Progenesis in the intestinal digests including protein accession, peptide count, number of unique peptides, confidence score, the theoretical mass, and normalized abundance; Sequence coverage on different Pru p 3 isoforms identified by Progenesis in the intestinal digests (reduced and non-reduced). Suppl Information S2: Summary data on peptides identified with either a phosphorylated serine or deamidated glutamine or asparagine residue, or both, by Progenesis in the non-reduced intestinal digests corresponding to Pru p 3 including protein accession, protein description, peptide sequence, charge, neutral mass, score, peptide modification and the normalised abundance. Suppl Information S3: Summary data on peptides identified by Progenesis in the reduced intestinal digests corresponding to Pru p 3 including protein accession, protein description, peptide sequence, charge, neutral mass, score, peptide modification and the normalised abundance.

Pure oats are generally accepted to be safe for most celiac patients, and consumption of oats provides advantageous dietary fibers. However, oats can be contaminated by gluten proteins from wheat, barley, and/or rye. The analytical challenge lies in the reliability of the quantification method and how to maintain the contamination level under a gluten-free food threshold of 20 mg/kg. In this study, we investigated barley-spiked oat flour samples at four levels using four gluten ELISA kits. The largest recovery variance was with the R5 kit that gave 5-6 times overestimation; the G12 kit cross-reacted with oat proteins and gave 4-5 times overestimation at all spiked levels. The Total Gluten and Morinaga kits gave satisfactory recoveries. Total barley hordeins were isolated and characterized to be used as a common calibrator in all four kits aiming at harmonizing the results and to test the kits' performance. Immunoblotting of total hordein isolate revealed that Total Gluten and Morinaga antibodies provided an overall detection, while R5 and G12 antibodies recognized specific hordein groups leading to a larger difference when wheat and barley were used as the calibrant. Calibration with total hordein isolate corrected the overestimation problem and decreased the variability between the four gluten kits.

Amylase/trypsin-inhibitors (ATIs) comprise about 2–4% of the total wheat grain proteins and may contribute to natural defense against pests and pathogens. However, they are currently among the most widely studied wheat components because of their proposed role in adverse reactions to wheat consumption in humans. ATIs have long been known to contribute to IgE-mediated allergy (notably Bakers' asthma), but interest has increased since 2012 when they were shown to be able to trigger the innate immune system, with attention focused on their role in coeliac disease which affects about 1% of the population and, more recently, in non-coeliac wheat sensitivity which may affect up to 10% of the population. This has led to studies of their structure, inhibitory properties, genetics, control of expression, behavior during processing, effects on human adverse reactions to wheat and, most recently, strategies to modify their expression in the plant using gene editing. We therefore present an integrated account of this range of research, identifying inconsistencies, and gaps in our knowledge and identifying future research needs. Note This paper is the outcome of an invited international ATI expert meeting held in Amsterdam, February 3-5 2020

Food allergies have risen in prominence over the last 20–30 years and currently, as there is no accepted cure, individuals usually have to practice life-long avoidance of their problem food(s). There are many different types of food allergy and intolerance, but those involving the immune system are amongst the most important. This chapter focuses on the food labelling of allergens that seeks to protect those with immune-mediated allergies.

Wheat gluten, and related prolamin proteins in rye, barley and oats cause the immune-mediated gluten intolerance syndrome, coeliac disease. Foods labelled as gluten-free which can be safely consumed by coeliac patients, must not contain gluten above a level of 20 mg/Kg. Current immunoassay methods for detection of gluten can give conflicting results and may underestimate levels of gluten in foods. Mass spectrometry methods have great potential as an orthogonal method, but require curated protein sequence databases to support method development. The GluPro database has been updated to include avenin-like sequences from bread wheat (n = 685; GluPro v1.1) and genes from the sequenced wheat genome (n = 699; GluPro v 1.2) and Triticum turgidum ssp durum (n = 210; GluPro v 2.1). Companion databases have been developed for prolamin sequences from barley (n = 64; GluPro v 3.0), rye (n = 41; GluPro v 4.0), and oats (n = 27; GluPro v 5.0) and combined to provide a complete cereal prolamin database, GluPro v 6.1 comprising 1,041 sequences. Analysis of the coeliac toxic motifs in the curated sequences showed that they were absent from the minor avenin-like proteins in bread and durum wheat and barley, unlike the related avenin proteins from oats. A comparison of prolamin proteins from the different cereal species also showed α- and γ-gliadins in bread and durum wheat, and the sulphur poor prolamins in all cereals had the highest density of coeliac toxic motifs. Analysis of ion-mobility mass spectrometry data for bread wheat (cvs Chinese Spring and Hereward) showed an increased number of identifications when using the GluPro v1.0, 1.1 and 1.2 databases compared to the limited number of verified sequences bread wheat sequences in reviewed UniProt. This family of databases will provide a basis for proteomic profiling of gluten proteins from all the gluten containing cereals and support identification of specific peptide markers for use in development of new methods for gluten quantitation based on coeliac toxic motifs found in all relevant cereal species.

With an ever-increasing allergic population and an emerging market for allergen-free foods, accurate detection of allergens in foods has never been more important. Although ELISA-based methods are the most widely used for detection of allergens in food, there is a need for the development of orthogonal approaches. A commercial ELISA detected a relatively high concentration of peanut and almond in an allergen-free product. However, another commercial ELISA declared a low peanut concentration and was negative for almond. Further testing using a commercial almond lateral-flow device confirmed the results from the second ELISA kit and demonstrated that the positive detection of almond was due to cross-reactivity. An MS method was used for final confirmation that the reported results were negative for both almond and peanut.