Proteomic Characterization of Bradyrhizobium diazoefficiens Bacteroids Reveals a Post-Symbiotic, Hemibiotrophic-Like Lifestyle of the Bacteria within Senescing Soybean Nodules.

The form and physiology of Bradyrhizobium diazoefficiens after the decline of symbiotic nitrogen fixation has been characterized. Proteomic analyses showed that post-symbiotic B. diazoefficiens underwent metabolic remodeling as well-defined groups of proteins declined, increased or remained unchanged from 56 to 119 days after planting, suggesting a transition to a hemibiotrophic-like lifestyle. Enzymatic analysis showed distinct patterns in both the cytoplasm and the periplasm. Similar to the bacteroid, the post-symbiotic bacteria rely on a non-citric acid cycle supply of succinate and, although viable, they did not demonstrate the ability to grow within the senescent nodule.

The Bacteroid Periplasm in Soybean Nodules Is an Interkingdom Symbiotic Space.

The functional role of the periplasm of nitrogen-fixing bacteroids has not been determined. Proteins were isolated from the periplasm and cytoplasm of Bradyrhizobium diazoefficiens bacteroids and were analyzed using liquid chromatography tandem mass spectrometry proteomics. Identification of bacteroid periplasmic proteins was aided by periplasm prediction programs. Approximately 40% of all the proteins identified as periplasmic in the B. diazoefficiens genome were found expressed in the bacteroid form of the bacteria, indicating the periplasm is a metabolically active symbiotic space. The bacteroid periplasm possesses many fatty acid metabolic enzymes, which was in contrast to the bacteroid cytoplasm. Amino acid analysis of the periplasm revealed an abundance of phosphoserine, phosphoethanolamine, and glycine, which are metabolites of phospholipid metabolism. These results suggest the periplasm is a unique space and not a continuum with the peribacteroid space. A number of plant proteins were found in the periplasm fraction, which suggested contamination. However, antibodies to two of the identified plant proteins, histone H2A and lipoxygenase, yielded immunogold labeling that demonstrated the plant proteins were specifically targeted to the bacteroids. This suggests that the periplasm is an interkingdom symbiotic space containing proteins from both the bacteroid and the plant.

Proteomic mapping for legume nodule organogenesis.

While genetic screens have identified mutants of the model legume Lotus japonicus that can nodulate in the absence of rhizobia, the lack of a proteome map is a major hindrance to understanding the functional protein networks associated with this nodulation process. In this issue of Proteomics, Dam et al. (Proteomics 2014, 14, 230-240) developed 2D gel-based reference maps of nodules and roots of Lotus and a spontaneous nodule formation mutant (snf1). Comparative proteomic analysis of roots and two developmental stages of nodules provide useful insights into tissue-specific mechanisms underlying nodule organogenesis. Additionally, a comparison of interspecies nodule proteomes displays that overlapping and individual mechanisms are associated with legume nodulation.

Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry.

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay-a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay-to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

Quantitation of soybean allergens using tandem mass spectrometry.

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 µg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity.

The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase-client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase-substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH(292)SMSDPGSTYR derived from the pyruvate dehydrogenase E1alpha subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.

Evolution of seed allergen quantification--from antibodies to mass spectrometry.

Development of accurate, high-throughput approaches for protein allergen quantification is important for the seed industry as a means to monitor natural variability in expression and ensure introduced transgenes do not collaterally alter the expression of any known allergen. Analytical approaches for protein quantification have undergone a renaissance in recent years with the emergence of soft-ionization approaches and advanced mass spectrometers capable of achieving low attomolar sensitivity. These advances coupled with bioinformatic tools to mine mass spectral data are collectively referred to as proteomics, and allow for the large-scale study of proteins with high precision and quantitative accuracy. In this review, we discuss differential and quantitative proteomics workflows that proceed from discovery profiling to targeted, quantitative analysis of specific proteins using stable isotopically-labeled, synthetic peptide doping standards. These synthetic peptide standards, also referred to as AQUA peptides, are synthetic mimics to proteotypic peptides and allow for absolute quantification of proteins in complex biological mixtures. The approaches discussed herein are ideal for the analysis of prominently expressed proteins such as protein allergens from plant seed, as no gels or sample pre-fractionation is required. We discuss these new techniques in the context of traditional, antibody-based technologies for allergen detection and quantification.

Variation in Seed Allergen Content From Three Varieties of Soybean Cultivated in Nine Different Locations in Iowa, Illinois, and Indiana.

Soybean (Glycine max) is an important food stock, and also considered an allergenic food with at least eight well characterized allergens. However, it is a less prevalent allergen source than many other foods and is rarely life-threatening. Soybean is incorporated into commonly consumed foods, and therefore, the allergens pose a potential concern for individuals already sensitized. The protein profile of soybean can be affected by several factors including genetic and environmental. To investigate how soybean allergen content may be affected by genetics and/or environment, nine soy allergens were quantified from three commercial soybean varieties grown at nine locations in three states within a single climate zone in North America; Iowa, Illinois, and Indiana, United States. Quantitation was achieved using liquid chromatography-selected reaction monitoring (LC-SRM) tandem mass spectrometry with AQUA peptide standards specific to the nine target allergens. Quantitation of allergen concentration indicated that both genetics and location affected specific allergen content. Seven of the nine allergens were significantly influenced by genetics, with the exceptions of glycinin G4 and KTI 3. The allergens P34, Gly m Bd 28k, glycinin G3, and KTI 1 showed statistically significant impact from location as well, but at a lower threshold of significance compared with genetics (cultivar/variety). This dataset contributes to our understanding of the natural variation of endogenous allergens, as it represents a sampling of soybeans grown in a controlled, distributed plot design under agronomic conditions common for commercial soybean food and feed production. The aim was to build upon our recent understanding of how allergens are expressed as part of the overall soybean proteome.

Development of an isoform-specific tandem mass spectrometry assay for absolute quantitation of maize lipid transfer proteins.

Precise and accurate quantitation of maize grain allergens is important for seed and food industries. The major allergen in maize grain is Zea m 14, a lipid transfer protein (LTP). The B73 maize genome encodes for at least six LTPs sharing 15%-87% sequence identity to Zea m 14. Phylogenetic analysis of the maize LTP family revealed one gene that corresponds to Zea m 14 (denoted as LTPa) and two other genes sharing 43% (LTPc) and 74% (LTPb) identity with Zea m 14 that are putative homologues. Using stable isotope peptide mimics as internal standards for LTPs, we present a multiple reaction monitoring mass spectrometry approach for multiplexed, absolute quantitation of all three LTP proteins and alternative transcript models therein. To validate quantitative accuracy, a redundant peptide, simultaneously representing the two most abundant LTPs, was included. Analysis of 21 maize varieties revealed LTPa was most prominently expressed in maize grain, ranging from 9 to 32 µg LTP/mg protein. Proteins belonging to the LTPb and LTPc gene models were also expressed but at approximately 10- and 100-fold lower levels than LTPa, respectively. The quantitative results provided by the redundant peptide show around 95% agreement with the sum of the two unique peptides, thus providing support for the LTP gene models and validating the accuracy of this method. Though not all Zea m 14-related LTPs are abundant in grain, their high sequence homology and detectable expression in maize grain signify that LTPb and LTPc are putative allergens and should be accounted for in any quantitation strategy for maize LTP allergens.

Environmental effects on allergen levels in commercially grown non-genetically modified soybeans: assessing variation across north america.

Soybean (Glycinemax) is a hugely valuable soft commodity that generates tens of billions of dollars annually. This value is due in part to the balanced composition of the seed which is roughly 1:2:2 oil, starch, and protein by weight. In turn, the seeds have many uses with various derivatives appearing broadly in processed food products. As is true with many edible seeds, soybeans contain proteins that are anti-nutritional factors and allergens. Soybean, along with milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, and wheat, elicit a majority of food allergy reactions in the United States. Soybean seed composition can be affected by breeding, and environmental conditions (e.g., temperature, moisture, insect/pathogen load, and/or soil nutrient levels). The objective of this study was to evaluate the influence of genotype and environment on allergen and anti-nutritional proteins in soybean. To address genetic and environmental effects, four varieties of non-GM soybeans were grown in six geographically distinct regions of North America (Georgia, Iowa, Kansas, Nebraska, Ontario, and Pennsylvania). Absolute quantification of proteins by mass spectrometry can be achieved with a technique called multiple reaction monitoring (MRM), during which signals from an endogenous protein are compared to those from a synthetic heavy-labeled internal standard. Using MRM, eight allergens were absolutely quantified for each variety in each environment. Statistical analyses show that for most allergens, the effects of environment far outweigh the differences between varieties brought about by breeding.

Validation of gel-free, label-free quantitative proteomics approaches: applications for seed allergen profiling.

Plant seeds provide a significant portion of the protein present in the human diet, but are also the major contributors of allergenic proteins that cause a majority of the reported cases of food-induced anaphylaxis. New varieties of grains and nuts as well as other seeds could be screened for allergen content before they are introduced as cultivars for food production using mass spectrometry-based quantitation approaches. Here, we present a practical comparison of gel-free and label-free methods, peak integration and spectral counting, using a linear trap mass spectrometer. The results show that both methods are linear and reproducible with protein standards from 5-200 ng, however, bioinformatic analysis for spectral counting is much simpler and therefore more amenable to high-throughput sample processing. We therefore applied spectral counting towards the analysis of transgenic peanut lines targeting the reduction of a prominent allergen. Spectral count analysis of an Ara h 2 (conglutin-7) RNA-silenced line confirmed reduction of this allergen as well as Ara h 6 (conglutin), which was further confirmed by quantitative immunoblotting. Other collateral changes include an increase in Ara h 10 (oleosin 1) in one of the three lines, a decrease in conarachin as well as increased 13-lipoxygenase and Ahy-3 (arachin) in two of three lines.

Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation.

Although recent advances in broad-scale gene expression analysis have dramatically increased our knowledge of the repertoire of mRNAs present in multiple cell types, it has become increasingly clear that examination of the expression, localization, and associations of the encoded proteins will be critical for determining their functional significance. In particular, many signaling receptors, transducers, and effectors have been proposed to act in higher-order complexes associated with physically distinct areas of the plasma membrane. Adult muscle stem cells (satellite cells) must, upon injury, respond appropriately to a wide range of extracellular stimuli: the role of such signaling scaffolds is therefore a potentially important area of inquiry. To address this question, we first isolated detergent-resistant membrane fractions from primary satellite cells, then analyzed their component proteins using liquid chromatography-tandem mass spectrometry. Transmembrane and juxtamembrane components of adhesion-mediated signaling pathways made up the largest group of identified proteins; in particular, neural cell adhesion molecule (NCAM), a multifunctional cell-surface protein that has previously been associated with muscle regeneration, was significant. Immunohistochemical analysis revealed that not only is NCAM localized to discrete areas of the plasma membrane, it is also a very early marker of commitment to terminal differentiation. Using flow cytometry, we have sorted physically homogeneous myogenic cultures into proliferating and differentiating fractions based solely upon NCAM expression.

Reduction of IgE binding and nonpromotion of Aspergillus flavus fungal growth by simultaneously silencing Ara h 2 and Ara h 6 in peanut.

The most potent peanut allergens, Ara h 2 and Ara h 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies of the transgene. Ara h 2 expression was significantly suppressed in all three lines, whereas Ara h 6 was reduced in two lines. Expression of peanut allergens Ara h 1 and Ara h 3 was not noticeably affected. Significant reduction of human IgE binding to Ara h 2 and Ara h 6 also was observed. Seed weight and germination data from transgenic and nontransgenic segregants showed no significant differences. Data collected from in vitro Aspergillus flavus infection indicate no significant difference in fungal growth between the transgenic lines and the nontransgenic controls. These data suggest that silencing Ara h 2 and Ara h 6 is a feasible approach to produce hypoallergenic peanut.