Worm wounding increases levels of pollen-related food allergens in soybean (Glycine max)

The levels of food allergens in worm-wounded or non-wounded green soybeans (edamame) and mature soybeans were investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA), using allergen-specific antibodies. Non-wounded and worm-wounded soybeans showed similar total protein profiles after Coomassie brilliant blue staining, but some protein bands were observed to have been changed by worm wounding. Immunoblotting with specific antibodies for major soybean allergens (Gly m 5, Gly m 6, Gly m Bd 30 K, and Kunitz soybean trypsin inhibitor) revealed that protein band profiles and intensities were not significantly changed by worm wounding. In contrast, levels of the pollen-related soybean allergens Gly m 4 and Gly m 3 were strongly increased by worm wounding in both green and mature soybeans, as detected by immunoblotting and ELISA. These results suggested that the pollen-related food allergen risk (i.e., oral allergy syndrome; OAS) from soybeans might be enhanced by worm wounding of soybeans.     

Identification of cross‐reactive B‐cell epitopes between Bos d 9.0101(Bos Taurus) and Gly m 5.0101 (Glycine max) by epitope mapping MALDI‐TOF MS

Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross‐allergenicity described between soy and milk proteins. We have previously identified several cross‐reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1‐casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α‐casein‐specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross‐reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI‐TOF MS analysis. On a second approach, the peptide mixture was resolved by RP‐HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI‐TOF MS. This novel MS based approach led us to identify and characterize four peptides on α‐casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross‐reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross‐reactivity, to further develop new and more effective vaccines for food allergy.     

Identification of the 7S and 11S globulins as percutaneously sensitizing soybean allergens as demonstrated through epidermal application of crude soybean extract

Cutaneous exposure to food allergens can predispose individuals to food allergies. Soybean, a major allergenic food, is an ingredient in various cosmetic products. However, the types of soybean proteins that are percutaneously sensitizing in humans or animal models remain unknown. In this study, BALB/c mice were dorsally shaved and epicutaneously exposed to a crude soybean extract including sodium dodecyl sulfate or distilled water alone. Specific IgEs secreted in response to 7S globulin (Gly m 5), 11S globulin (Gly m 6), Gly m 3, and Gly m 4 were measured using enzyme-linked immunosorbent assays or immunoblots. Exposure to soybean extract elicited the secretion of soybean-specific IgEs. Of the soybean proteins, 7S and 11S globulins acted as percutaneous sensitizers in 6/9 mice (67%). Additionally, IgE bound specifically and preferentially to the 7S globulin β subunit. In conclusion, this is the first report to identify percutaneously sensitizing soybean allergens in a mouse model.     

Rapid direct detection of the major fish allergen, parvalbumin, by selected MS/MS ion monitoring mass spectrometry

Parvalbumins beta (β-PRVBs) are considered the major fish allergens. A new strategy for the rapid and direct detection of these allergens in any foodstuff is presented in this work. The proposed methodology is based on the purification of β-PRVBs by treatment with heat, the use of accelerated in-solution trypsin digestion under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of only nineteen β-PRVB peptide biomarkers by Selected MS/MS Ion Monitoring (SMIM) in a linear ion trap (LIT) mass spectrometer. The present strategy allows the direct detection of the presence of fish β-PRVBs in any food product in less than 2 hours.     

Food allergen detection by mass spectrometry: From common to novel protein ingredients

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.     

Cross-reactivity of pollen and food allergens: soybean Gly m 4 is a member of the Bet v 1 superfamily and closely resembles yellow lupine proteins

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes.     

大豆过敏蛋白与品种改良

食物过敏是一个全世界关注的公共卫生问题。大豆蛋白在食品加工业的广泛应用,对大豆敏感人群带来了潜在的威胁。如何降低大豆过敏原含量提升大豆食品安全已成为日益关注的问题。大豆种子过敏蛋白包括种子贮存蛋白、结构蛋白和防御相关蛋白,其中7S 伴球蛋白的多肽片段Gly m Bd 28K, Gly m Bd 30K和Gly m Bd 60K是三种主要的过敏原。目前通过对过敏蛋白的理化性质、过敏原性和基因结构的认识,运用食品加工工艺、传统育种及基因工程技术等方法,在减少大豆和大豆产品的过敏原性方面已取得一定的进展。本文拟从大豆过敏原的分类、主要过敏原Gly m Bd 28K和Gly m Bd 30K的理化性质及基因结构、大豆过敏蛋白在遗传改良中的应用对大豆过敏蛋白进行综述。     

Genetic modification removes an immunodominant allergen from soybean

The increasing use of soybean (Glycine max) products in processed foods poses a potential threat to soybean-sensitive food-allergic individuals. In vitro assays on soybean seed proteins with sera from soybean-sensitive individuals have immunoglobulin E reactivity to abundant storage proteins and a few less-abundant seed proteins. One of these low abundance proteins, Gly m Bd 30 K, also referred to as P34, is in fact a major (i.e. immunodominant) soybean allergen. Although a member of the papain protease superfamily, Gly m Bd 30 K has a glycine in the conserved catalytic cysteine position found in all other cysteine proteases. Transgene-induced gene silencing was used to prevent the accumulation of Gly m Bd 30 K protein in soybean seeds. The Gly m Bd 30 K-silenced plants and their seeds lacked any compositional, developmental, structural, or ultrastructural phenotypic differences when compared with control plants. Proteomic analysis of extracts from transgenic seed detected the suppression of Gly m Bd 30 K-related peptides but no other significant changes in polypeptide pattern. The lack of a collateral alteration of any other seed protein in the Gly m Bd 30 K-silenced seeds supports the presumption that the protein does not have a role in seed protein processing and maturation. These data provide evidence for substantial equivalence of composition of transgenic and non-transgenic seed eliminating one of the dominant allergens of soybean seeds.     

Fluorescent isotope-coded affinity tag (FCAT). I: Design and synthesis

A novel class of isotope-coded affinity tag is proposed possessing a fluorescent feature, referred to as fluorescent isotope-coded affinity tag (FCAT), to provide a new tool for quantitative proteomics. The label is designed to bind cysteine containing proteins or peptides. The FCAT reagent comprises four functional elements: a specific chemical reactivity group toward sulfhydryl groups; a linker that can incorporate the stable isotopes; a hydroxymethylbenzoic residue (base labile group) to cleave off a large part of the label before MS analysis; and a fluorescent tag for absolute quantification. The fluorescent part of the tag is also planned to be utilized to isolate the FCAT-labeled peptides via antibody based pull-down method. In this paper, we report on the solid phase organic synthesis of the light isotope containing FCAT molecule. The new labeling reagent showed good reactivity with model cysteine containing peptides. The fluorophore group was also effectively cleaved off from the labeled products to accommodate easier MS based analysis.     

Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry

After intake of food or herbal medicinal products containing quercetin glycosides, the systemic availability of the genuine glycoside, as well as the systemic occurrence of the aglycone or conjugates of this polyphenol has been a matter of dispute. Consequently, we designed this study to develop a reliable method for determination of quercetin and its metabolites. Following consumption of fried onions five different glucuronides of quercetin could be identified in human plasma samples by means of HPLC–UV–MS/MS. Selective determination of the target compounds was achieved by simultaneous UV (254 nm) and MS/MS detection with selected reaction monitoring experiments using positive mode electrospray ionisation. In contrast, neither the free flavonol nor the genuine glycoside could be detected in plasma. Identification of the quercetin glucuronides detected in vivo was confirmed by comparison with authentic reference compounds synthesised enzymatically using glucuronyl transferase from rabbit liver.     

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.     

Analysis of glucosinolates, isothiocyanates, and amine degradation products in vegetable extracts and blood plasma by LC-MS/MS

Dietary glucosinolates are under intensive investigation as precursors of cancer-preventive isothiocyanates. Quantitation of the dose and bioavailability of glucosinolates and isothiocyanates requires a comprehensive analysis of the major dietary glucosinolates, isothiocyanates, and related metabolites. We report a liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the comprehensive analysis of the seven major dietary glucosinolates, related isothiocyanates, and putative amine degradation products. The parent glucosinolates were sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin, and gluconasturtiin. The LC-MS/MS analysis method for these compounds was developed and validated; a standard addition analysis protocol was used generally to avoid the requirement for stable isotopic standards. Where stable isotopic standards were available, internal standardization with these gave estimates in agreement with those obtained by the standard addition analysis protocol. For glucosinolates, negative ion electrospray LC-MS/MS analysis was performed. Isothiocyanates and amines were prederivatized to the corresponding thiourea and N-acetamides, respectively, and were quantified by positive ion electrospray LC-MS/MS. The limits of detection were 0.5-2 pmol; the recoveries for glucosinolates, isothiocyanates, and amines were 85-90%, 50-85%, and 60-70%, respectively; and the intra- and interbatch coefficients of variation were 1-4% and 3-10%, respectively. These methods provide facile access to comprehensive analytical data on the major dietary glucosinolates and related metabolites to quantify inputs and metabolic formation of these compounds in cancer prevention and related studies.     

FragmentationAnalyzer: An open‐source tool to analyze MS/MS fragmentation data

A thorough understanding of the fragmentation processes in MS/MS can be a powerful tool in assessing the resulting peptide and protein identifications. We here present the freely available, open‐source FragmentationAnalyzer tool ( http://fragmentation‐analyzer.googlecode.com ) that makes it straightforward to analyze large MS/MS data sets for specific types of identified peptides, using a common set of peptide properties. This enables the detection of fragmentation pattern nuances related to specific instruments or due to the presence of post‐translational modifications.     

Identification of the 23-kDa peptide derived from the precursor of Gly m Bd 28K, a major soybean allergen, as a new allergen

One of the major soybean allergens, Gly m Bd 28K, is suggested to be biosynthesized as a preproprotein form, which would be composed of a signal peptide, Gly m Bd 28K and the C-terminal peptide (the 23-kDa peptide). However, the 23-kDa peptide has never been characterized. In the present study, we prepared a monoclonal antibody (mAb) against a recombinant 23-kDa peptide expressed in Escherichia coli to detect the 23-kDa peptide in soybean. Several proteins were detected by immunoblotting with the mAb. All of the proteins were shown to have the identical N-terminal amino acid sequence, suggesting that the proteins correspond to the C-terminal part of the Gly m Bd 28K precursor. Furthermore, Gly m Bd 28K and the 23-kDa peptide were observed to come out at the 21st day after flowering and to locate in the crystalloid part of protein storage vacuoles in growing cotyledons. Some of the 23-kDa peptides were shown to be glycoproteins with an N-linked glycan moiety and exhibited the binding to IgE antibodies in the sera of patients sensitive to soybean. The binding of the peptides to IgE antibodies was suggested to be predominantly dependent on their glycan moiety. This study proves the occurrence of the 23-kDa peptide in soybean and that it is a new allergen.     

A method for simple identification of signature peptides derived from polyUb-K48 and K63 by MALDI-TOF MS and chemically assisted MS/MS fragmentation

Summary. A simple method is described to identify signature peptides derived from polyubiquitin (polyUb) chains. The method is based on MALDI-TOF MS/MS analysis after chemically assisted fragmentation, and works on peptides isolated from polyacrylamide gels. PolyUb chains branched at K48 and K63 were chosen as models for Ub-protein conjugates. They were resolved by SDS-PAGE, and their tryptic peptides (in-gel-trypsinolysis) derivatized with 3-sulfopropinic acid NHSester to obtain chemically assisted fragmentation during the MS/MS analysis. PolyUb-K63 produced a single peptide identified as ⁵⁵TLSDYNIQK⁶³ (GG)ESTLHLVLR⁷². PolyUb-K48 produced two branched signature peptides identified as ⁴³LIFAGK⁴⁸(GG)QLEDGR⁵⁴ and ⁴³LIFAGK⁴⁸(LRGG)QLEDGR⁵⁴. The recovery of signature peptide with LRGG as branched chain underscores the need to take limited proteolysis into account in the search for detection of ubiquitinated peptides in proteomics studies. In conclusion, a simple method has been described allowing the identification of signature peptides, which are diagnostic markers of the majority of polyUb-conjugated proteins. In principle, the method should be applicable also for other more rare signature peptides.     

SoyProDB: A database for the identification of soybean seed proteins

Soybean continues to serve as a rich and inexpensive source of protein for humans and animals. A substantial amount of information has been reported on the genotypic variation and beneficial genetic manipulation of soybeans. For better understanding of the consequences of genetic manipulation, elucidation of soybean protein composition is necessary, because of its direct relationship to phenotype. We have conducted studies to determine the composition of storage, allergen and anti-nutritional proteins in cultivated soybean using a combined proteomics approach. Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) was implemented for the separation of proteins along with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS) for the identification of proteins. Our analysis resulted in the identification of several proteins, and a web based database named soybean protein database (SoyProDB) was subsequently built to house and allow scientists to search the data. This database will be useful to scientists who wish to genetically alter soybean with higher quality storage proteins, and also helpful for consumers to get a greater understanding about proteins that compose soy products available in the market. The database is freely accessible.

Availability

http://bioinformatics.towson.edu/Soybean_Seed_Proteins_2D_Gel_DB/Home.aspx     

Proteomics-driven identification of short open reading frame-encoded peptides

Accumulating evidence has shown that a large number of short open reading frames (sORFs) also have the ability to encode proteins. The discovery of sORFs opens up a new research area, leading to the identification and functional study of sORF encoded peptides (SEPs) at the omics level. Besides bioinformatics prediction and ribosomal profiling, mass spectrometry (MS) has become a significant tool as it directly detects the sequence of SEPs. Though MS-based proteomics methods have proved to be effective for qualitative and quantitative analysis of SEPs, the detection of SEPs is still a great challenge due to their low abundance and short sequence. To illustrate the progress in method development, we described and discussed the main steps of large-scale proteomics identification of SEPs, including SEP extraction and enrichment, MS detection, data processing and quality control, quantification, and function prediction and validation methods.     

Determination of bioactive compounds in fermented soybean products using GC/MS and further investigation of correlation of their bioactivities

The active ingredients and bioactivities (anti-oxidant, anti-tyrosinase, anti-proliferative and estrogenic activities) of soybean and soybean products (Cheonggukjang, Meju, Makjang, Doenjang and soy sauce) produced by different fermentation processes were compared. There were high correlations between active ingredients and bioactivities. Free phenolic acids extracted from soybean products were identified and quantified by gas chromatography/mass spectrometry (GC/MS). Overall, the components and activities in fermented soybean products were different than those in soybeans. Total phenolic content (TPC), protein content (PC) and anti-oxidant activity increased as fermentation time increased. TPC and PC showed strong negative correlations with anti-oxidant activity. Doenjang and soy sauce, two long-term fermented products, showed lower total flavonoid content (TFC) and estrogenic activities than short-term fermented soybean products. This might be explained by the decomposition and hydrolysis of flavonoids due to the long fermentation time and high temperature. Strong anti-proliferative activity against cancer cell lines, which was highly correlated with TFC, was found in Meju and Cheonggukjang. Soybean and all fermented products except Meju exhibited effective tyrosinase inhibitory activities. Fermented products showed stronger estrogenic activity than soybeans, which was highly correlated with syringic acid.     

Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data.Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared.This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.