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Summary This study revealed activation-dependent and coordinated relocation of Medicago sativa SIMKK and SIMK after salt stress. Arabidopsis seedlings stably overexpressing YFP-tagged SIMKK showed altered salt sensitivity and proteome changes., Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)–SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK–yellow fluorescent protein (YFP) fusions. SIMKK–YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK–YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK–YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.

Actin cytoskeleton is a vital cellular structure primarily known for controlling cell integrity, division and expansion. Here we present a proteomic dissection of Arabidopsis roots treated by actin depolymerizing agent latrunculin B. Pharmacological disintegration of the actin cytoskeleton by latrunculin B caused downregulation of several proteins involved in the actin organization and dynamics. Moreover, this approach helped to identify new protein candidates involved in gene transcription, due to the altered abundance of proteins involved in mRNA nuclear export. Finally, latrunculin B negatively affected the abundance of abscisic acid (ABA) responsive proteins. Significance This article substantially contributes to the current knowledge about the importance of actin organization and dynamics in proteome remodelling. We employed gel based and gel free proteomic analyses and identified several new protein candidates and protein networks linking actin dynamics to the gene transcription and to the ABA response in Arabidopsis.

Fragmentation mechanisms of phytoalexin analogs, including brassitin and brassinin and their glucosylated analogs, have been studied by electrospray (ESI) ion trap (IT) multistage (MS n, n = 1–4) mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF/ToF) and ESI-Q/ToF tandem mass spectrometry techniques. At the fragmentation of sodium adducts a hitherto not described process has been elucidated The proposed mechanism of this process includes cyclization of the brassitin and brassinin cationized adducts through a six-membered cycle of the molecules and the elimination of isocyanate or isothiocyanate from the thio- or dithiocarbamate moiety, giving rise to [M + Na – 43]+ or [M + Na – 59]+ adducts. The elimination of NH=C=O or NH=C=S molecules has been confirmed by the high resolution measurement of the elemental composition of the ions produced and quantum-chemical calculations of the six-membered transition state. Other fragmentation routes include cleavage of an alkane linker, while numerous characteristic hexopyranose pathways are taking place in the glucosylated compounds. The presented theoretical data on the ESI and MALDI behavior of the saccharidic, as well as of the indole aglycon parts, can facilitate structural elucidation of the analogous compounds.

The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn(2+) or Zn(2+)) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

Oligosaccharides (mono- to hexamers) that mimic the terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba, have been studied by electrospray ion trap (ESI IT) mass spectrometry. Sodium or potassium-cationized adducts are characteristic ions under the conditions of ESI-MS analysis. The tentative pathways of fragmentation have been proven by multistage ion trap MS (MS(n), n = 1-3). The predominant pathway of fragmentation of the oligomers is the neutral loss of monosaccharide residue shortening the length of the oligosaccharide. In this way, conversion of the Ogawa to Inaba fragments takes place under the conditions of measurement. ESI MS/MS provided sufficient information about molecular mass, the number of saccharide residues, and the structure of saccharides, about the C (4)-amide of 3-deoxy-L-glycero-tetronic acid (DGT) of the compounds investigated, and allows to distinguish between Ogawa and Inaba serotypes.

A convergent synthesis of 1-(β-D-glucopyranosyl)-, 1-(α-D-mannopyranosyl)- and 1-(β-D-ribofuranosyl)benzocamalexin was elaborated as an alternative route to the linear approach based on the indoline-indole method. 1,2-Anhydrosaccharides and 1,2-cyclic sulfites as saccharide donors were used in the key glycosylation step. Coupling with benzocamalexin resulted in moderate to excellent yields of nucleoside analogs, depending on the saccharide donor, catalyst and solvent used.

Fragmentation mechanisms of electrospray ionization (ESI) mass spectrometry of aldobiuronic and pseudoaldobiuronic acid derivatives were elucidated by multistage mass spectrometric (MS(n), n = 2-5) measurements of selected ions. Characteristic under the conditions of ESI-MS analysis is the production of alkali metal (Na and K) cationized adducts. The probability the of locations of Na cations in per-O-methylated compounds was proved by quantum chemical calculations, using the Jaguar program. The most probably position of alkali metal attachment is the carboxy group of the methoxycarbonyl C-5 group of the uronic acid unit. Characteristic cleavages vary according the kind of O-derivatization. In most cases they take place on the acidic part of the dimer and at the interglycosidic oxygen atom. As a result, the criteria for the differentiation of aldobiouronic and pseudoaldobiouronic acids derivatives were elucidated.

The linear synthesis of 1-(β-D-glucopyranosyl)-, 1-(β-D-galactopyranosyl)-, 1-(β-D-mannopyranosyl)- and 1-(β-D-ribofuranosyl)benzocamalexin was elaborated from indoline as a starting compound and corresponding pentaacetylhexoses or 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose as suitable glycosyl donors.

Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.

Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.

The amount of clinically relevant, allergy-related proteins in wheat grain is still largely unknown. The application of proteomics may create a platform not only for identification and characterization, but also for quantitation of these proteins. The aim of this study was to evaluate the data-independent quantitative mass spectrometry (MS(E)) approach in combination with 76 wheat allergenic sequences downloaded from the AllergenOnline database ( www.allergenonline.org ) as a starting point. Alcohol soluble extracts of gliadin and glutenin proteins were analyzed. This approach has resulted in identification and quantification of 15 allergenic protein isoforms that belong to amylase/trypsin inhibitors, γ-gliadins, and high or low molecular weight glutenins. Additionally, several peptides carrying four previously discovered epitopes of γ-gliadin B precursor have been detected. These data were validated against the UniProt database, which contained 11764 Triticeae protein sequences. The identified allergens are discussed in relation to Baker's asthma, food allergy, wheat dependent exercise induced anaphylaxis, atopic dermatitis, and celiac disease (i.e., gluten-sensitive enteropathy). In summary, the results showed that the MS(E) approach is suitable for quantitative analysis and allergens profiling in wheat varieties and/or other food matrices.

A series of mono- or disaccharide-protein conjugates were prepared by amide bond formation. Incorporation efficiency of the aminosaccharides on bovine serum albumin was determined by colorimetry and matrix assisted laser desorption/ionization mass spectrometry. Study compares two amide bond coupling agents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. Paper demonstrates a practical usefulness of natural bovine serum albumin succinylation. Large number of its carboxyl groups results in its superior reactivity towards simple aminosaccharides.

Precise content of gliadin (Glia) and glutenin (Glu) proteins in wheat grain are largely unknown despite their association with celiac disease, various allergies, and physical processing properties of wheat. Developing methods to quantitatively measure clinically relevant proteins could support advancement in understanding exposure thresholds and clinical study design. The aim of this study was to use a data-independent mass spectrometry (MS(E)) approach for quantifying gliadin and glutenin proteins in wheat grain. The biologically replicated analysis yielded concentrations for 34 gliadin and 22 glutenin proteins. The primary focus of this survey was on measuring celiac disease proteins and baker's asthma associated proteins along with the proteins associated with viscoelastic properties of wheat flour and grain texture. The technical coefficients of variation ranged from 0.12 to 1.39 and indicate that MS(E) proteomics is a reproducible quantitative method for the determination of gliadin and glutenin content in the highly complex matrix of protein extracts from wheat grain. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Partially O-acetylated xylo-oligosaccharides (DXO) isolated from almond shells by autohydrolysis as well as their de-acetylated form (DeXO) were subjected to chemical, molecular, and structural analyses. They represent a mixture of neutral and acidic oligomers and low-molecular weight polymers related to (4-O-methyl-D-glucurono)-D-xylan. DXO and DeXO showed direct mitogenic activity and enhancement of the T-mitogen-induced proliferation of rat thymocytes, indicating the immunostimulatory potential of the almond shell xylo-oligosaccharides.

Saccharides (mono- to hexamers) that mimic the terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba have been studied by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI ToF/ToF) mass spectrometry (MS). Cationized adducts are the characteristic ions formed through the capture of sodium or potassium cations under MALDI MS conditions. Three characteristic pathways dominate in the fragmentation of model substances under MALDI ToF/ToF collision-induced dissociation MS/MS conditions of measurement. The first is the elimination which shortens the length of the oligosaccharide. In this way, conversion of the Ogawa to Inaba fragments takes place under the conditions of measurement. In the second, the conjugated transfer of electrons in the upstream unit of oligosaccharides takes place. The third route brings about the elimination of one α-hydroxy-γ-butyrolactone molecule from the 4-(3-deoxy-L- glycero-tetronamido) group. The MALDI ToF/ToF MS/MS provided sufficient information about molecular mass, the number of saccharide residues, the structure of saccharides, the C(4)- amide of 3-deoxy-L- glycero-tetronic acid (DGT) of the compounds investigated and allows Ogawa and Inaba serotypes to be distinguished.

A series of 2-glycosyl-benzimidazoles with alpha-d-arabinopyranosyl, beta-d-galactopyranosyl, beta-d-glucopyranosyl, beta-d-mannopyranosyl, and beta-l-rhamnopyranosyl configurations were obtained in 52-73% yields from the corresponding C-glycosylmethanal dimethyl acetals and o-phenylenediamine under catalysis with hydrogen chloride or a strongly acidic cation-exchange resin. Intermediate benzimidazolines were spontaneously oxidized by air to produce the final products in the one-pot procedure. The prepared compounds did not show any inhibitory effect on the growth of 12 strains of five different species of pathogenic yeasts.

Saccharides (mono through hexasaccharides) that mimic the terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba, were studied by electrospray ion trap (ESI IT) mass spectrometry (MS) in the negative mode. Anionized adducts are the characteristic ions formed by the capture of H3O2− under the condition of ESI MS analysis. The reactive species are produced by reaction of hydroxyl anions with the molecule of water. Thus the [M + H3O2]− have the highest m/z value in the ESI IT negative mass spectra. After dissociation of adducts by loss of 2H2O the [M – H]− ions are produced. The fragmentation pathways were confirmed by multistage measurements (MS n). The predominant pathway of fragmentation of the mono- and oligomers is the elimination of a molecule of α-hydroxy-γ-butyrolactone from the 4-(3-deoxy-l-glycero-tetronamido) group. The other characteristic pathway occurs by shortening the length of oligosaccharides. In this way, conversion of the Ogawa to Inaba fragments takes place under the conditions of measurement. Negative ESI MS/MS provided sufficient information about molecular mass, the number of saccharide residues, basic structure of saccharides, about the tetronamide part of the compounds investigated and allowed Ogawa and Inaba serotypes to be distinguished.

A linear synthesis of substituted 1-(β-D-glucopyranosyl)benzocamalexins starting from indoline and penta-O-acetyl-β-D-glucopyranose was elaborated. Jacobson cyclization of corresponding 4-substituted peracetylated β-D-glucopyranosylindole-3-carbothioanilides employing potassium ferricyanide under basic conditions was a key synthetic step.

A new four-step synthesis of substituted 2-(1H-indol-3-yl)benzothiazoles is described, using N3-phenyl-1H-indole-3-carbothioamides as key intermediates. The structure of the obtained products was determined by IR, 1H, 13C NMR and MS spectral methods.