Determination of Allergen Levels, Isoforms, and Their Hydroxyproline Modifications Among Peanut Genotypes by Mass Spectrometry.

The recently published reference genome of peanuts enables a detailed molecular description of the allergenic proteins of the seed. We used LC-MS/MS to investigate peanuts of different genotypes to assess variability and to better describe naturally occurring allergens and isoforms. Using relative quantification by mass spectrometry, minor variation of some allergenic proteins was observed, but total levels of Ara h 1, 2, 3, and 6 were relatively consistent among 20 genotypes. Previously published RP-HPLC methodology was used for comparison. The abundance of three Ara h 3 isoforms were variable among the genotypes and contributed to a large proportion of total Ara h 3 where present. Previously unpublished hydroxyproline sites were identified in Ara h 1 and 3. Hydroxylation did not vary significantly where sites were present. Peanut allergen composition was largely stable, with only some isoforms displaying differences between genotypes. The resulting differences in allergenicity are of unknown clinical significance but are likely to be minor. The data presented herein allow for the design of targeted MS methodology to allow the quantitation and therefore control of peanut allergens of clinical relevance and observed variability.

Purification and Initial Characterization of Ara h 7, a Peanut Allergen from the 2S Albumin Protein Family.

2S albumins are important peanut allergens. Within this protein family, Ara h 2 and Ara h 6 have been described in detail, but Ara h 7 has received little attention. We now describe the first purification of Ara h 7 and its characterization. Two Ara h 7 isoforms were purified from peanuts. Mass spectrometry revealed that both the isoforms have a post-translation cleavage, a hydroxyproline modification near the N-terminus, and four disulfide bonds. The secondary structure of both Ara h 7 isoforms is highly comparable to those of Ara h 2 and Ara h 6. Both Ara h 7 isoforms bind IgE, and Ara h 7 is capable of inhibiting the binding between Ara h 2 and IgE, suggesting at least partially cross-reactive IgE epitopes. Ara h 7 was found in all main market types of peanut, at comparable levels. This suggests that Ara h 7 is a relevant allergen from the peanut 2S albumin protein family.

Shellfish Tropomyosin IgE Cross-Reactivity Differs Among Edible Insect Species.

SCOPE: Insects are a potentially environmentally friendly alternative dietary protein source to supplement mammalian and fish sources, but potential allergenic risks are a concern. Consumption of insects may result in anaphylaxis and has been implicated in cross-reactivity with shellfish. Many allergenic proteins may be involved in cross-reactivity, including tropomyosin (TM). The uniformity of TM cross-reactivity among edible insects is unknown. Candidate edible insects for variability in shellfish IgE cross-reactivity are investigated. METHODS AND RESULTS: Selected insects and known related sources of allergens are extracted and probed by immunoblot with sera/plasma from patients sensitized to insects or shellfish. Quantification of TM in these extracts is performed using mass spectrometry. A comparison of the quantity of TM and the IgE reactivity of TM from these insects is performed. Distinct patterns of IgE cross-reactivity are observed with three insect species showing diminished reactivity. This pattern is not consistent with the amount of TM present in these insects, or with overall sequence homology. CONCLUSION: Insects display a diversity of TM-associated IgE reactivity. It is likely that minor sequence features and/or structural effects are primarily responsible. Additionally, it is demonstrated that some insect species may present significantly less IgE cross-reactivity to shrimp than do others.

Thermal processing of peanut impacts detection by current analytical techniques.

Recalls of spice containing products due to undeclared peanut have highlighted the importance of analytical methods in these foods. We examined the performance of peanut detection methods in cumin and garlic, focusing on quantitative ELISA. Although suitable for qualitative detection, accurate quantitation proved difficult. Roasting of peanut contaminants influenced ELISA results, with raw peanut over-detected (3.9-fold) and roasted peanut under-detected (3.5-fold). Further investigation demonstrated the importance of protein targets for ELISA. The kit which gave the least variable results was based on detection of 2S albumin proteins. Additionally, we show that these proteins are more efficiently extracted from roasted peanut. We conclude that current methods are largely suitable for the qualitative detection of peanut in cumin and garlic. Quantitation relies on assumptions as to the state of thermal processing of peanut. We suggest that analytical method providers address robust detection by target selection, including identifying targets by MS.

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.

Determination of the N-glycosylation patterns of seed proteins: applications to determine the authenticity and substantial equivalence of genetically modified (GM) crops.

Methods have been developed to determine the N-glycosylation pattern of proteins at the single-seed level in two different biological systems. These were the well-characterized and widely consumed storage protein phaseolin from several species of Phaseolus (bean) and the α-amylase inhibitor from the same Phaseolus species expressed transgenically in pea. The N-glycosylation pattern of the α-amylase inhibitor expressed transgenically in pea was different from that of the inhibitor present in common bean (P. vulgaris), the species of origin of the gene. However, multivariate analysis showed that the differences in N-glycan patterns between the α-amylase inhibitors from common bean and pea were less than those between the inhibitors from common bean and two related bean species, lima bean (Phaseolus lunatus) and tepary bean (Phaseolus acutifolius).

Distinct patterns of control and expression amongst members of the PEP carboxylase kinase gene family in C4 plants.

We have examined the complexity of the phosphoenolpyruvate carboxylase kinase (PPCK) gene family in the C(4) monocots maize and sorghum. Maize contains at least four PPCK genes. The encoded proteins are similar to other phosphoenolpyruvate carboxylase (PEPC) kinases, in that they comprise a protein kinase domain with minimal extensions, except that two of the proteins contain unusual acidic insertions. The spatial and temporal expression patterns of the genes provide information about their presumed functions. Expression of ZmPPCK1 in leaves is mesophyll cell-specific and light-induced, indicating that it encodes the PEPC kinase that is responsible for the phosphorylation of leaf PEPC during C(4) photosynthesis. Surprisingly, ZmPPCK2 is expressed in leaf bundle sheath cells, preferentially in the dark. This suggests that a main function of the ZmPPCK2 gene product is to allow PEPC to function anaplerotically in bundle sheath cells in the dark without interfering with the C(4) cycle. ZmPPCK2, ZmPPCK3 and ZmPPCK4 are all induced by exposure of tissue to cycloheximide, whereas ZmPPCK1 is not. This suggests that the ZmPPCK2, ZmPPCK3 and ZmPPCK4 genes share the property that their expression is controlled by a rapidly turning over repressor. Sequence and expression data show that sorghum contains orthologues of ZmPPCK1 and ZmPPCK2.

Comparison of repetitive sequences derived from high molecular weight subunits of wheat glutenin, an elastomeric plant protein.

A strategy has been developed to create repetitive peptides incorporating substitutions in the PGQGQQGYYPTSLQQ consensus repeat sequence of high molecular weight subunits in order to investigate natural sequence variations in elastomeric proteins of wheat gluten. After introduction of glutamic and aspartic acid residues, the peptide behaved similarly to the unmodified form at low pH, but became readily water soluble at pH > 6. Substitution of Gln for Leu at position 13 resulted in only small changes to the secondary structure of the water-insoluble peptides, as did Tyr8His and Thr11Ala. The effects of proline substitutions depended on their location: Leu13Pro substitution had little effect on solubility and structure, but Gln6Pro substitution resulted in dramatic changes. Peptides with two Gln6Pro substitutions had similar properties to the water-insoluble parental peptide, but those with 6 or 10 substitutions were readily soluble. The results indicated that specific sequences influence noncovalent intermolecular interactions in wheat gluten proteins.

Structure and expression of phosphoenolpyruvate carboxylase kinase genes in solanaceae. A novel gene exhibits alternative splicing.

Phosphorylation of phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) plays an important role in the control of central metabolism in higher plants. Two PPCK (PEPc kinase) genes have been identified in tomato (Lycopersicon esculentum cv Alicante), hereafter termed LePPCK1 and LePPCK2. The function of the gene products has been confirmed by transcription of full-length cDNAs, translation, and in vitro assay of kinase activity. Previously studied PPCK genes contain a single intron. LePPCK2 also contains a novel second intron that exhibits alternative splicing. The correctly spliced transcript encodes a functional PEPc kinase, whereas unspliced or incorrectly spliced transcripts encode a truncated, inactive protein. The relative abundance of the transcripts depends on tissue and conditions. Expression of LePPCK2 was markedly increased during fruit ripening. In ripe Alicante fruit, the locule and seeds contained only the correctly spliced LePPCK2 transcripts, whereas in ripe fruit of the tomato greenflesh mutant, they contained correctly and incorrectly spliced transcripts. Potato (Solanum tuberosum) contains genes that are very similar to LePPCK1, and LePPCK2; StPPCK2 exhibits alternative splicing. Aubergine (Solanum melongena) and tobacco (Nicotiana tabacum) also contain a PPCK2 gene; the sequence of the alternatively spliced intron is highly conserved between all four species. The data suggest that the two PPCK genes have different roles in tissue-specific regulation of PEPc and that the alternative splicing of PPCK2 transcripts is functionally significant.