Study of Potential Synergistic Effect of Probiotic Formulas on Acrylamide Reduction.

Acrylamide (AA) is a food processing contaminant commonly found in fried and baked food products. In this study, the potential synergistic effect of probiotic formulas in reducing AA was studied. Five selected probiotic strains (Lactiplantibacillus plantarum subsp. plantarum ATCC14917 (L. Pl.), Lactobacillus delbrueckii subsp. bulgaricus ATCC11842 (L. B.), Lacticaseibacillus paracasei subsp. paracasei ATCC25302 (L. Pa), Streptococcus thermophilus ATCC19258, and Bifidobacterium longum subsp. longum ATCC15707) were selected for investigating their AA reducing capacity. It was found that L. Pl. (108 CFU/mL) showed the highest AA reduction percentage (43-51%) when exposed to different concentrations of AA standard chemical solutions (350, 750, and 1250 ng/mL). The potential synergistic effect of probiotic formulas was also examined. The result demonstrated a synergistic AA reduction effect by the probiotic formula: L. Pl. + L. B., which also showed the highest AA reduction ability among the tested formulas. A further study was conducted by incubating selected probiotic formulas with potato chips and biscuit samples followed by an in vitro digestion model. The findings demonstrated a similar trend in AA reduction ability as those found in the chemical solution. This study firstly indicated the synergistic effect of probiotic formulas on AA reduction and its effect was also highly strain-dependent.

rPTMDetermine: A Fully Automated Methodology for Endogenous Tyrosine Nitration Validation, Site-Localization, and Beyond.

We present herein rPTMDetermine, an adaptive and fully automated methodology for validation of the identification of rarely occurring post-translational modifications (PTMs), using a semisupervised approach with a linear discriminant analysis (LDA) algorithm. With this strategy, verification is enhanced through similarity scoring of tandem mass spectrometry (MS/MS) comparisons between modified peptides and their unmodified analogues. We applied rPTMDetermine to (1) perform fully automated validation steps for modified peptides identified from an in silico database and (2) retrieve potential yet-to-be-identified modified peptides from raw data (that had been missed through conventional database searches). In part (1), 99 of 125 3-nitrotyrosyl-containing (nitrated) peptides obtained from a ProteinPilot search were validated and localized. Twenty nitrated peptides were falsely assigned because of incorrect monoisotopic peak assignments, leading to erroneous identification of deamidation and nitration. Five additional nitrated peptides were, however, validated after performing nonmonoisotopic peak correction. In part (2), an additional 236 unique nitrated peptides were retrieved and localized, containing 113 previously unreported nitration sites; 25 endogenous nitrated peptides with novel sites were selected and verified by comparison with synthetic analogues. In summary, we identified and confidently validated 296 unique nitrated peptides-collectively representing the largest number of endogenously identified 3-nitrotyrosyl-containing peptides from the cerebral cortex proteome of a Macaca fascicularis model of stroke. Furthermore, we harnessed the rPTMDetermine strategy to complement conventional database searching and enhance the confidence of assigning rarely occurring PTMs, while recovering many missed peptides. In a final demonstration, we successfully extended the application of rPTMDetermine to peptides featuring tryptophan oxidation.

Evidence for the Prerequisite Formation of Phenoxyl Radicals in Radical-Mediated Peptide Tyrosine Nitration In Vacuo.

The elementary mechanism of radical-mediated peptide tyrosine nitration, which is a hallmark of post-translational modification of proteins under nitrative stress in vivo, has been elucidated in detail by using an integrated approach that combines the gas-phase synthesis of prototypical molecular tyrosine-containing peptide radical cations, ion-molecule reactions, and isotopic labeling experiments with DFT calculations. This reaction first involves the radical recombination of . NO2 towards the prerequisite phenoxyl radical tautomer of a tyrosine residue, followed by proton rearrangements, finally yielding the stable and regioselective 3-nitrotyrosyl residue product. In contrast, nitration with the π-phenolic radical cation tautomer is inefficient. This first direct experimental evidence for the elementary steps of the radical-mediated tyrosine nitration mechanism in the gas phase provides a fundamental insight into the regioselectivity of biological tyrosine ortho-nitration.

Formation of n → π+ interaction facilitating dissociative electron transfer in isolated tyrosine-containing molecular peptide radical cations.

Long-range electron transfer in proteins can be rationalized as a sequential short-distance electron-hopping processes via amino acid residues having low ionization energy as relay stations. Tyrosine residues can serve as such redox-active intermediates through one-electron oxidation to form a π-radical cation at its phenol side chain. An electron transfer from a vicinal functional group to this π-electron hole completes an elementary step of charge migration. However, transient oxidized/reduced intermediates formed at those relay stations during electron transfer processes have not been observed. In this study, formation of analog reactive intermediates via electron donor-acceptor coupling is observed by using IRMPD action spectroscopy. An elementary charge migration at the molecular level in model tyrosine-containing peptide radical cations [M]˙+ in the gas phase is revealed with its unusual Cα-Cß bond cleavage at the side chain of the N-terminal residue. This reaction is induced by the radical character of the N-terminal amino group (-NH2˙+) resulting from an n → π+ interaction between the nonbonding electron pair of NH2 (n) and the π-electron hole at the Tyr side chain (π+). The formation of -NH2˙+ is supported by the IRMPD spectrum showing a characteristic NH2 scissor vibration coupled with Tyr side-chain stretches at 1577 cm-1. This n → π+ interaction facilitates a dissociative electron transfer with NH2 as the relay station. The occurrence of this side-chain cleavage may be an indicator of the formation of reactive conformers featuring the n → π+ interaction.

Dissociative electron transfer of copper(ii) complexes of glycyl(glycyl/alanyl)tryptophan in vacuo: IRMPD action spectroscopy provides evidence of transition from zwitterionic to non-zwitterionic peptide structures.

We report herein the first detailed study of the mechanism of redox reactions occurring during the gas-phase dissociative electron transfer of prototypical ternary [CuII(dien)M]˙2+ complexes (M, peptide). The two final products are (i) the oxidized non-zwitterionic π-centered [M]˙+ species with both the charge and spin densities delocalized over the indole ring of the tryptophan residue and with a C-terminal COOH group intact, and (ii) the complementary ion [CuI(dien)]+. Infrared multiple photon dissociation (IRMPD) action spectroscopy and low-energy collision-induced dissociation (CID) experiments, in conjunction with density functional theory (DFT) calculations, revealed the structural details of the mass-isolated precursor and product cations. Our experimental and theoretical results indicate that the doubly positively charged precursor [CuII(dien)M]˙2+ features electrostatic coordination through the anionic carboxylate end of the zwitterionic M moiety. An additional interaction exists between the indole ring of the tryptophan residue and one of the primary amino groups of the dien ligand; the DFT calculations provided the structures of the precursor ion, intermediates, and products, and enabled us to keep track of the locations of the charge and unpaired electron. The dissociative one-electron transfer reaction is initiated by a gradual transition of the M tripeptide from the zwitterionic form in [CuII(dien)M]˙2+ to the non-zwitterionic M intermediate, through a cascade of conformational changes and proton transfers. In the next step, the highest energy intermediate is formed; here, the copper center is 5-coordinate with coordination from both the carboxylic acid group and the indole ring. A subsequent switch back to 4-coordination to an intermediate IM1, where attachment to GGW occurs through the indole ring only, creates the structure that ultimately undergoes dissociation.

A tricyclic pyrrolobenzodiazepine produced by Klebsiella oxytoca is associated with cytotoxicity in antibiotic-associated hemorrhagic colitis.

Cytotoxin-producing Klebsiella oxytoca is the causative agent of antibiotic-associated hemorrhagic colitis (AAHC). Recently, the cytotoxin associated with AAHC was identified as tilivalline, a known pentacyclic pyrrolobenzodiazepine (PBD) metabolite produced by K. oxytoca Although this assertion of tilivalline's role in AAHC is supported by evidence from animal experiments, some key aspects of this finding appear to be incompatible with toxicity mechanisms of known PBD toxins. We therefore hypothesized that K. oxytoca may produce some other uncharacterized cytotoxins. To address this question, we investigated whether tilivalline alone is indeed necessary and sufficient to induce cytotoxicity or whether K. oxytoca also produces other cytotoxins. LC-MS- and NMR-based metabolomic analyses revealed the presence of an abundant tricyclic PBD, provisionally designated kleboxymycin, in the supernatant of toxigenic K. oxytoca strains. Moreover, by generating multiple mutants with gene deletions affecting tilivalline biosynthesis, we show that a tryptophanase-deficient, tilivalline-negative K. oxytoca mutant induced cytotoxicity in vitro similar to tilivalline-positive K. oxytoca strains. Furthermore, synthetic kleboxymycin exhibited greater than 9-fold higher cytotoxicity than tilivalline in TC50 cell culture assays. We also found that the biosynthetic pathways for kleboxymycin and tilivalline appear to overlap, as tilivalline is an indole derivative of kleboxymycin. In summary, our results indicate that tilivalline is not essential for inducing cytotoxicity observed in K. oxytoca-associated AAHC and that kleboxymycin is a tilivalline-related bacterial metabolite with even higher cytotoxicity.

Tautomerization and Dissociation of Molecular Peptide Radical Cations.

Radical-mediated dissociations of peptide radical cations have intriguing unimolecular gas phase chemistry, with cleavages of almost every bond of the peptide backbone and amino acid side chains in a competitive and apparently "stochastic" manner. Challenges of unraveling mechanistic details are related to complex tautomerizations prior to dissociations. Recent conjunctions of experimental and theoretical investigations have revealed the existence of non-interconvertible isobaric tautomers with a variety of radical-site-specific initial structures, generated from dissociative electron transfer of ternary metal-ligand-peptide complexes. Their reactivity is influenced by the tautomerization barriers, perturbing the nature, location, or number of radical and charge site(s), which also determine the energetics and dynamics of the subsequent radical-mediated dissociatons. The competitive radical- and charge-induced dissociations are extremely dependent on charge density. Charge sequesting can reduce the charge densities on the peptide backbone and hence enhance the flexibility of structural rearrangement. Analysing the structures of precursors, intermediates and products has led to the discovery of many novel radical migration prior to peptide backbone and/or side chain fragmentations. Upon these successes, scientists will be able to build peptide cationic analogues/tautomers having a variety of well-defined radical sites.

Loss of water from protonated polyglycines: interconversion and dissociation of the product imidazolone ions.

Collision-induced dissociation of isotopically labelled protonated pentaglycine produced two abundant [b5]+ ions, the products of the loss of water from the first and second amide groups, labelled [b5]+I and [b5]+II. IRMPD spectroscopy and DFT calculations show that these two [b5]+ ions feature N1-protonated 3,5-dihydro-4H-imidazol-4-one structures. 15N-Labelling established that some interconversion occurs between these two ions but dissociations are preferred. For both ions, DFT calculations show that the barrier to interconversion is slightly higher than those to dissociation. Dehydration of protonated hexaglycine produced three imidazolone ions. Ions [b6]+I and [b6]+II exhibit analogous CID spectra to those from [b5]+I and [b5]+II; however, the spectrum of the [b6]+III ion was dramatically different, showing losses predominantly of a further water molecule or cleavage of the second amide bond to give the glycyloxazolone (a deprotonated [b2]+ ion, labelled GlyGlyox (114 Da)) from the N-terminus. Protonated polyglycines [Glyn + H]+, where n = 7-9, all readily lose at least one water molecule. The corresponding [bn]+ ions lose either a further water molecule, an oxazolone from the N-terminus or a truncated peptide from the C-terminus. The number of amino acid residues in the latter two eliminated neutral molecules provides insight into the location of the imidazolone in the peptide chain and which oxygen was lost in the initial dehydration reaction. From this analysis, it appears that water loss from the longer protonated polyglycines is predominantly from the central residues.

Characterization of the Molecular Mechanisms Underlying the Chronic Phase of Stroke in a Cynomolgus Monkey Model of Induced Cerebral Ischemia.

Stroke is one of the main causes of mortality and long-term disability worldwide. The pathophysiological mechanisms underlying this disease are not well understood, particularly in the chronic phase after the initial ischemic episode. In this study, a Macaca fascicularis stroke model consisting of two sample groups, as determined by MRI-quantified infarct volumes as a measure of the stroke severity 28 days after the ischemic episode, was evaluated using qualitative and quantitative proteomics analyses. By using multiple online multidimensional liquid chromatography platforms, 8790 nonredundant proteins were identified that condensed to 5223 protein groups at 1% global false discovery rate (FDR). After the application of a conservative criterion (5% local FDR), 4906 protein groups were identified from the analysis of cerebral cortex. Of the 2068 quantified proteins, differential proteomic analyses revealed that 31 and 23 were dysregulated in the elevated- and low-infarct-volume groups, respectively. Neurogenesis, synaptogenesis, and inflammation featured prominently as the cellular processes associated with these dysregulated proteins. Protein interaction network analysis revealed that the dysregulated proteins for inflammation and neurogenesis were highly connected, suggesting potential cross-talk between these processes in modulating the cytoskeletal structure and dynamics in the chronic phase poststroke. Elucidating the long-term consequences of brain tissue injuries from a cellular prospective, as well as the molecular mechanisms that are involved, would provide a basis for the development of new potentially neurorestorative therapies.

Isomerization versus dissociation of phenylalanylglycyltryptophan radical cations.

Four isomers of the radical cation of tripeptide phenylalanylglycyltryptophan, in which the initial location of the radical center is well defined, have been isolated and their collision-induced dissociation (CID) spectra examined. These ions, the π-centered [FGWπ˙]+, α-carbon- [FGα˙W]+, N-centered [FGWN˙]+ and ζ-carbon- [Fζ˙GW]+ radical cations, were generated via collision-induced dissociation (CID) of transition metal-ligand-peptide complexes, side chain fragmentation of a π-centered radical cation, homolytic cleavage of a labile nitrogen-nitrogen single bond, and laser induced dissociation of an iodinated peptide, respectively. The π-centered and tryptophan N-centered peptide radical cations produced almost identical CID spectra, despite the different locations of their initial radical sites, which indicated that interconversion between the π-centered and tryptophan N-centered radical cations is facile. By contrast, the α-carbon-glycyl radical [FGα˙W]+, and ζ-phenyl radical [Fζ˙GW]+, featured different dissociation product ions, suggesting that the interconversions among α-carbon, π-centered (or tryptophan N-centered) and ζ-carbon-radical cations have higher barriers than those to dissociation. Density functional theory calculations have been used to perform systematic mechanistic investigations on the interconversions between these isomers and to study selected fragmentation pathways for these isomeric peptide radical cations. The results showed that the energy barrier for interconversion between [FGWπ˙]+ and [FGWN˙]+ is only 31.1 kcal mol-1, much lower than the barriers to their dissociation (40.3 kcal mol-1). For the [FGWπ˙]+, [FGα˙W]+, and [Fζ˙GW]+, the barriers to interconversion are higher than those to dissociation, suggesting that interconversions among these isomers are not competitive with dissociations. The [z3 - H]˙+ ions isolated from [FGα˙W]+ and [Fζ˙GW]+ show distinctly different fragmentation patterns, indicating that the structures of these ions are different and this result is supported by the DFT calculations.

Label-Free Quantitative Proteomics Reveals Survival Mechanisms Developed by Hypertrophic Chondrocytes under ER Stress.

Emerging evidence implicates ER stress caused by unfolded mutant proteins in chondrocytes as the underlying pathology of chondrodysplasias. ER stress is triggered in hypertrophic chondrocytes (HCs) in a mouse model (13del) of metaphyseal chondrodysplasia type Schmid (MCDS) caused by misfolded mutant collagen X proteins, but the HCs do not undergo apoptosis; rather chondrocyte differentiation is altered, causing skeletal abnormality. How 13del HCs can escape from apoptosis and survive ER stress is not understood. Here we compared the proteomes of HCs isolated from 13del growth plates with normal HCs using a label-free quantitative mass spectrometry approach. Pathway enrichment analyses of differentially expressed proteins showed significant changes in glycolysis and ER-mitochondria pathways in 13del HCs as well as in ATDC5 cell lines expressing wt and 13del collagen X. In vivo, we showed expression of mitochondrial calcium channels was reduced while mitochondrial membrane polarity was maintained in 13del chondrocytes, while in vitro, glucose uptake was maintained. We propose 13del HCs survive by a mechanism whereby changes in ER-mitochondria communication reduce import of calcium coupled to maintenance of mitochondrial membrane polarity. These findings provide the initial insights into our understanding of growth plate changes caused by protein misfolding in the pathogenesis of chondrodysplasias.

Discovery, Synthesis, and Functional Characterization of a Novel Neuroprotective Natural Product from the Fruit of Alpinia oxyphylla for use in Parkinson's Disease Through LC/MS-Based Multivariate Data Analysis-Guided Fractionation.

Herein we report the discovery of a novel lead compound, oxyphylla A [(R)-4-(2-hydroxy-5-methylphenyl)-5-methylhexanoic acid] (from the fruit of Alpinia oxyphylla), which functions as a neuroprotective agent against Parkinson's disease. To identify a shortlist of candidates from the extract of A. oxyphylla, we employed an integrated strategy combining liquid chromatography/mass spectrometry, bioactivity-guided fractionation, and chemometric analysis. The neuroprotective effects of the shortlisted candidates were validated prior to scaling up the finalized list of potential neuroprotective constituents for more detailed chemical and biological characterization. Oxyphylla A has promising neuroprotective effects: (i) it ameliorates in vitro chemical-induced primary neuronal cell damage and (ii) alleviates chemical-induced dopaminergic neuron loss and behavioral impairment in both zebrafish and mice in vivo. Quantitative proteomics analyses of oxyphylla A-treated primary cerebellar granule neurons that had been intoxicated with 1-methyl-4-phenylpyridinium revealed that oxyphylla A activates nuclear factor-erythroid 2-related factor 2 (NRF2)-a master redox switch-and triggers a cascade of antioxidative responses. These observations were verified independently through western blot analyses. Our integrated metabolomics, chemometrics, and pharmacological strategy led to the efficient discovery of novel bioactive ingredients from A. oxyphylla while avoiding the nontargeting, labor-intensive steps usually required for identification of bioactive compounds. Our successful development of a synthetic route toward oxyphylla A should lead to its availability on a large scale for further functional development and pathological studies.

Nucleophilic substitution by amide nitrogen in the aromatic rings of [zn - H]˙⁺ ions; the structures of the [b2 - H - 17]˙⁺ and [c1 - 17]⁺ ions.

Peptide radical cations that contain an aromatic amino acid residue cleave to give [zn - H]˙⁺ ions with [b2 - H - 17]˙⁺ and [c1 - 17](+) ions, the dominant products in the dissociation of [zn - H]˙⁺, also present in lower abundance in the CID spectra. Isotopic labeling in the aromatic ring of [Yπ˙GG](+) establishes that in the formation of [b2 - H - 17]˙⁺ ions a hydrogen from the δ-position of the Y residue is lost, indicating that nucleophilic substitution on the aromatic ring has occurred. A preliminary DFT investigation of nine plausible structures for the [c1 - 17](+) ion derived from [Y(π)˙GG](+) shows that two structures resulting from attack on the aromatic ring by oxygen and nitrogen atoms from the peptide backbone have significantly better energies than other isomers. A detailed study of [Y(π)˙GG](+) using two density functionals, B3LYP and M06-2X, with a 6-31++G(d,p) basis set gives a higher barrier for attack on the aromatic ring of the [zn - H]˙⁺ ion by nitrogen than by the carbonyl oxygen. However, subsequent rearrangements involving proton transfers are much higher in energy for the oxygen-substituted isomer leading to the conclusion that the [c1 - 17](+) ions are the products of nucleophilic attack by nitrogen, protonated 2,7-dihydroxyquinoline ions. The [b2 - H - 17]˙⁺ ions are formed by loss of glycine from the same intermediates involved in the formation of the [c1 - 17](+) ions.

Fully automated multidimensional reversed-phase liquid chromatography with tandem anion/cation exchange columns for simultaneous global endogenous tyrosine nitration detection, integral membrane protein characterization, and quantitative proteomics mapping in cerebral infarcts.

Protein tyrosine nitration (PTN) is a signature hallmark of radical-induced nitrative stress in a wide range of pathophysiological conditions, with naturally occurring abundances at substoichiometric levels. In this present study, a fully automated four-dimensional platform, consisting of high-/low-pH reversed-phase dimensions with two additional complementary, strong anion (SAX) and cation exchange (SCX), chromatographic separation stages inserted in tandem, was implemented for the simultaneous mapping of endogenous nitrated tyrosine-containing peptides within the global proteomic context of a Macaca fascicularis cerebral ischemic stroke model. This integrated RP-SA(C)X-RP platform was initially benchmarked through proteomic analyses of Saccharomyces cerevisiae, revealing extended proteome and protein coverage. A total of 27 144 unique peptides from 3684 nonredundant proteins [1% global false discovery rate (FDR)] were identified from M. fascicularis cerebral cortex tissue. The inclusion of the S(A/C)X columns contributed to the increased detection of acidic, hydrophilic, and hydrophobic peptide populations; these separation features enabled the concomitant identification of 127 endogenous nitrated peptides and 137 transmembrane domain-containing peptides corresponding to integral membrane proteins, without the need for specific targeted enrichment strategies. The enhanced diversity of the peptide inventory obtained from the RP-SA(C)X-RP platform also improved analytical confidence in isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses.

A versatile reversed phase-strong cation exchange-reversed phase (RP-SCX-RP) multidimensional liquid chromatography platform for qualitative and quantitative shotgun proteomics.

An automatable, robust, high-performance online multidimensional liquid chromatography (MDLC) platform comprising of pH 10 reversed-phase (RP), strong cation exchange (SCX), and pH 2 RP separation stages has been integrated into a modified commercial off-the-shelf LC instrument with a simple rewiring, enabling accelerated routine qualitative and quantitative proteomics analyses. This system has been redesigned with a dual-trap column configuration to improve the throughput by greatly decreasing the system idle time. The performance of this new design has been benchmarked through analysis of the total lysate of S. cerevisiae, in comparison with that of the former tailor-made system featuring more complicated components; the total run time per "load-and-go" LC/MS analysis was approximately 24 h, with minimal idle time and no labor-intensive steps. This platform features high-resolution fractionations, ease of use and a high degree of user programmability in the first two chromatographic dimensions, allowing flexible and effective sampling with (RP-SCX-RP) or without (RP-RP) the inclusion of SCX sub-fractionation; good proteome coverage and reproducibility was demonstrated through the analyses of bacterial, cell culture, and monkey brain tissue proteomes. The viability of the 3D RP-SCX-RP has been proven in proteome-wide studies of STO fibroblasts and yeast tryptic digests, resulting in extended proteome and protein coverages with high reproducibility-in particular, discovering extra-hydrophilic peptides-at the expense of the acquisition time. The identified inventory of the rat pheochromocytoma PC12 cell proteome-a total of 6345 proteins and 97 309 unique peptides is the most comprehensive dataset to date-provides an example of the value of the 3D RP-SCX-RP. The use of orthogonal chromatographic dimensions in the 3D RP-SCX-RP also circumvents the issues of isobaric interference of mass-tagging background contaminations, while significantly improving the accuracy of isobaric tags for relative and absolute quantitation (iTRAQ)-based protein quantitation experiments.

Proteomic evidence for genetic epistasis: ClpR4 mutations switch leaf variegation to virescence in Arabidopsis.

Chloroplast development in plants is regulated by a series of coordinated biological processes. In this work, a genetic suppressor screen for the leaf variegation phenotype of the thylakoid formation 1 (thf1) mutant combined with a proteomic assay was employed to elucidate this complicated network. We identified a mutation in ClpR4, named clpR4-3, which leads to leaf virescence and also rescues the var2 variegation. Proteomic analysis showed that the chloroplast proteome of clpR4-3 thf1 is dominantly controlled by clpR4-3, providing molecular mechanisms that cause genetic epistasis of clpR4-3 to thf1. Classification of the proteins significantly mis-regulated in the mutants revealed that those functioning in the expression of plastid genes are oppositely regulated while proteins functioning in antioxidative stress, protein folding, and starch metabolism are changed in the same direction between thf1 and clpR4-3. The levels of FtsHs including FtsH2/VAR2, FtsH8, and FtsH5/VAR1 are greatly reduced in thf1 compared with those in the wild type, but are higher in clpR4-3 thf1 than in thf1. Quantitative PCR analysis revealed that FtsH expression in clpR4-3 thf1 is regulated post-transcriptionally. In addition, a number of ribosomal proteins are less expressed in the clpR4-3 proteome, which is in line with the reduced levels of rRNAs in clpR4-3. Furthermore, knocking out PRPL11, one of the most downregulated proteins in the clpR4-3 thf1 proteome, rescues the leaf variegation phenotype of the thf1 and var2 mutants. These results provide insights into molecular mechanisms by which the virescent clpR4-3 mutation suppresses leaf variegation of thf1 and var2.

Online two-dimensional porous graphitic carbon/reversed phase liquid chromatography platform applied to shotgun proteomics and glycoproteomics.

A novel fully automatable two-dimensional liquid chromatography (2DLC) platform has been integrated into a modified commercial off-the-shelf LC instrument, incorporating porous graphitic carbon (PGC) separation and conventional low-pH reversed-phase (RP) separation for both proteomics and N-glycomics analyses; the dual-trap column configuration of this platform offers desirable high-throughput analyses with almost no idle time, in addition to a miniaturized setup and simplified operation. The total run time per analysis was only 19 h when using eight PGC fractions for unattended large-scale qualitative and quantitative proteomic analyses; the identification of 2678 nonredundant proteins and 11,984 unique peptides provided one of the most comprehensive proteome data sets for primary cerebellar granule neurons (CGNs). The effect of pH on the PGC column was investigated for the first time to improve the hydrophobic peptide coverage; the performance of the optimized system was first benchmarked using tryptic digests of Saccharomyces cerevisiae cell lysates and then evaluated through duplicate analyses of Macaca fascicularis cerebral cortex lysates using isobaric tags for relative and absolute quantitation (iTRAQ) technology. An additional plug-and-play PGC module functioned in a complementary manner to recover unretained hydrophilic solutes from the low-pH RP column; synchronization of the fractionations between the PGC-RP system and the PGC module facilitated simultaneous analyses of hydrophobic and hydrophilic compounds from a single sample injection event. This methodology was applied to perform, for the first time, detailed glycomics analyses of Macaca fascicularis plasma, resulting in the identification of a total 130 N-glycosylated plasma proteins, 705 N-glycopeptides, and 254 N-glycosylation sites.

Formation, isomerization, and dissociation of ε- and α-carbon-centered tyrosylglycylglycine radical cations.

The fragmentation products of the ε-carbon-centered radical cations [Y(ε)˙LG](+) and [Y(ε)˙GL](+), made by 266 nm laser photolysis of protonated 3-iodotyrosine-containing peptides, are substantially different from those of their π-centered isomers [Y(π)˙LG](+) and [Y(π)˙GL](+), made by dissociative electron transfer from ternary metal-ligand-peptide complexes. For leucine-containing peptides the major pathway for the ε-carbon-centered radical cations is loss of the side chain of the leucine residue forming [YG(α)˙G](+) and [YGG(α)˙](+), whereas for the π-radicals it is the side chain of the tyrosine residue that is lost, giving [G(α)˙LG](+) and [G(α)˙GL](+). The fragmentations of the product ions [YG(α)˙G](+) and [YGG(α)˙](+) are compared with those of the isomeric [Y(ε)˙GG](+) and [Y(π)˙GG](+) ions. The collision-induced spectra of ions [Y(ε)˙GG](+) and [YGG(α)˙](+) are identical, showing that interconversion occurs prior to dissociation. For ions [Y(ε)˙GG](+), [Y(π)˙GG](+) and [YG(α)˙G](+) the dissociation products are all distinctly different, indicating that dissociation occurs more readily than isomerization. Density functional theory calculations at B3LYP/6-31++G(d,p) gave the relative enthalpies (in kcal mol(-1) at 0 K) of the five isomers to be [Y(ε)˙GG](+) 0, [Y(π)˙GG](+) -23.7, [YGG(α)˙](+) -28.7, [YG(α)˙G](+) -31.0 and [Y(α)˙GG](+) -38.5. Migration of an α-C-H atom from the terminal glycine residue to the ε-carbon-centered radical in the tyrosine residue, a 1-11 hydrogen atom shift, has a low barrier, 15.5 kcal mol(-1) above [Y(ε)˙GG](+). By comparison, isomerization of [Y(ε)˙GG](+) to [YG(α)˙G](+) by a 1-8 hydrogen atom migration from the α-C-H atom of the central glycine residue has a much higher barrier (50.6 kcal mol(-1)); similarly conversion of [Y(ε)˙GG](+) into [Y(π)˙GG](+) has a higher energy (24.4 kcal mol(-1)).

Goldfish spexin: solution structure and novel function as a satiety factor in feeding control.

Spexin (SPX) is a neuropeptide identified recently by bioinformatic approach. At present not much is known about its biological actions, and comparative studies of SPX in nonmammalian species are still lacking. To examine the structure and function of SPX in fish model, SPX was cloned in goldfish and found to be highly comparable with its mammalian counterparts. As revealed by NMR spectroscopies, goldfish SPX is composed of an α-helix from Gln(5) to Gln(14) with a flexible NH2 terminus from Asn(1) to Pro(4), and its molecular surface is largely hydrophobic except for Lys(11) as the only charged residue in the helical region. In goldfish, SPX transcripts were found to be widely expressed in various tissues, and protein expression of SPX was also detected in the brain. In vivo feeding studies revealed that SPX mRNA levels in the telencephalon, optic tectum, and hypothalamus of goldfish brain could be elevated by food intake. However, brain injection of goldfish SPX inhibited both basal and NPY- or orexin-induced feeding behavior and food consumption. Similar treatment also reduced transcript expression of NPY, AgRP, and apelin, with concurrent rises in CCK, CART, POMC, MCH, and CRH mRNA levels in different brain areas examined. The differential effects of SPX treatment on NPY, CCK, and MCH transcript expression could also be noted in vitro in goldfish brain cell culture. Our studies for the first time unveil the solution structure of SPX and its novel function as a satiety factor through differential modulation of central orexigenic and anorexigenic signals.