Environmental enrichment alters protein expression as well as the proteomic response to cocaine in rat nucleus accumbens.

Prior research demonstrated that environmental enrichment creates individual differences in behavior leading to a protective addiction phenotype in rats. Understanding the mechanisms underlying this phenotype will guide selection of targets for much-needed novel pharmacotherapeutics. The current study investigates differences in proteome expression in the nucleus accumbens of enriched and isolated rats and the proteomic response to cocaine self-administration using a liquid chromatography mass spectrometry (LCMS) technique to quantify 1917 proteins. Results of complementary Ingenuity Pathways Analyses (IPA) and gene set enrichment analyses (GSEA), both performed using protein quantitative data, demonstrate that cocaine increases vesicular transporters for dopamine and glutamate as well as increasing proteins in the RhoA pathway. Further, cocaine regulates proteins related to ERK, CREB and AKT signaling. Environmental enrichment altered expression of a large number of proteins implicated in a diverse number of neuronal functions (e.g., energy production, mRNA splicing, and ubiquitination), molecular cascades (e.g., protein kinases), psychiatric disorders (e.g., mood disorders), and neurodegenerative diseases (e.g., Huntington's and Alzheimer's diseases). Upregulation of energy metabolism components in EC rats was verified using RNA sequencing. Most of the biological functions and pathways listed above were also identified in the Cocaine X Enrichment interaction analysis, providing clear evidence that enriched and isolated rats respond quite differently to cocaine exposure. The overall impression of the current results is that enriched saline-administering rats have a unique proteomic complement compared to enriched cocaine-administering rats as well as saline and cocaine-taking isolated rats. These results identify possible mechanisms of the protective phenotype and provide fertile soil for developing novel pharmacotherapeutics. Proteomics data are available via ProteomeXchange with identifier PXD000990.

Proteomic identification of carbonylated proteins in the kidney of trichloroethene-exposed MRL+/+ mice.

Trichloroethene (TCE), a common environmental and occupational pollutant, is associated with multiorgan toxicity. Kidney is one of major target organs affected as a result of TCE exposure. Our previous studies have shown that exposure to TCE causes increased protein oxidation (protein carbonylation) in the kidneys of autoimmune-prone MRL+/+ mice, and suggested a potential role of protein oxidation in TCE-mediated nephrotoxicity. To assess the impact of chronic TCE exposure on protein oxidation, particularly to identify the carbonylated proteins in kidneys, female MRL+/+ mice were treated with TCE at the dose of 2 mg/ml via drinking water for 36 weeks and kidney protein extracts were analyzed for protein carbonyls and carbonylated proteins identified using proteomic approaches (2D gel, Western blot, MALDI TOF/TOF MS/MS, etc.). TCE treatment led to significantly increased protein carbonyls in the kidney protein extracts (20 000 g pellet fraction). Interestingly, among 18 identified carbonylated proteins, 10 were found only in the kidneys of TCE-treated mice, whereas other 8 were present in the kidneys of both control and TCE-treated mice. The identified carbonylated proteins represent skeletal proteins, chaperones, stress proteins, enzymes, plasma protein and proteins involved in signaling pathways. The findings provide a map for further exploring the role of carbonylated proteins in TCE-mediated nephrotoxicity.

Src activation by ß-adrenoreceptors is a key switch for tumour metastasis.

Noradrenaline can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here we identify Src as a key regulator of phosphoproteomic signalling networks activated in response to beta-adrenergic signalling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumour cell migration, invasion and growth. In human ovarian cancer samples, high tumoural noradrenaline levels were correlated with high pSrc(Y419) levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signalling in the tumour microenvironment.

Model studies on iTRAQ modification of peptides: sequence-dependent reaction specificity.

A multiplexed peptide quantification strategy using the iTRAQ reagent has been described for relative measurements of peptides in digested protein mixtures. To validate the chemical specificity of the iTRAQ reaction, we have performed a detailed study of iTRAQ reactivity with two sets of synthetic peptides. The first set of peptides had sequences of Tyr-Xaa-Ser-Glu-Gly-Leu-Ser-Lys and Tyr-Xaa-Ser-Glu-Tyr-Leu-Ser-Lys where Xaa = Ala, Pro, Trp, Tyr, or Glu and was designed to study the extent of O-acylation by iTRAQ, especially hydroxyl-containing residues in different positions. The second set of peptides included Ala-Ser-Glu-His-Ala-Xaa-Tyr-Gly where Xaa = Ser, Thr, or Tyr and was selected to investigate the effect of histidyl residues separated by one amino acid residue from seryl, tyrosyl, or threonyl residues. Our findings indicated that, in addition to variable levels of O-acylation of nonsequence-specific hydroxyl-containing residues, significant sequence-specific O-acylation of seryl, threonyl, and tyrosyl hydroxyls occurred when separated one residue removed from a histidyl residue, that is, (Tyr/Ser)-Xaa-His or His-Xaa-(Tyr/Ser/Thr). This behavior was verified by a separate spiking experiment of one of the first set of peptides into Escherichia coli protein extracts, followed by retention time targeted LC-MS/MS to demonstrate the occurrence of modifications in a complex mixture. These sequence-dependent O-acylation modifications can be confounding factors to accurate MS quantification. Reversal of peptide O-acylation by the iTRAQ reagent can be accomplished by reaction with hydroxylamine with virtually no cleavage of N-acylation and is a recommended modification of the iTRAQ protocol for many applications.

Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins.

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP(6)). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP(6)- and inositol pyrophosphate (InsP(7))-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP(6) enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.

High-throughput liquid-liquid fractionation of multiple protein post-translational modifications.

Post-translational protein modifications have contributed significantly to the identification of macromolecular biomarkers of biological processes. We have modified a two-dimensional HPLC system (Beckman Coulter PF2D ProteomeLab) to create proteome maps of post-translational protein modifications. This system resolves complex protein mixtures by anion exchange chromatofocusing in the first dimension and hydrophobicity (reverse phase chromatography) in the second dimension. The simultaneous identification of multiple protein modifications, accomplished by incorporating a photo diode array (PDA) detector into the PF2D system, facilitates the simultaneous production of three-dimensional proteome maps and visualization of both unmodified and post-translationally modified (PTM) proteins at their signature wavelengths within the proteome. We describe procedures for the simultaneous resolution of proteome maps, the identification of proteins modified by nitration, carbonylation, and phosphorylation, and proteins with unique spectra such as the heme containing proteins.

Toward the Proteome of the Human Peripheral Blood Eosinophil.

Eosinophils are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood eosinophils from normal human donors primarily employing 2-dimensional gel electrophoresis with protein spot identification by matrix-assisted laser desorption/ionization mass spectrometry. Protein subfractionation methods employed included isoelectric focusing (Zoom(®) Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3,141 proteins which had Mascot expectation scores of 10(-3) or less. Of these 426 were unique and non-redundant of which 231 were novel proteins not previously reported to occur in eosinophils. Ingenuity Pathway Analysis showed that some 70% of the non-redundant proteins could be subdivided into categories that are clearly related to currently known eosinophil biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the eosinophil. This dataset of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

Tandem time-of-flight mass spectrometry with a curved field reflectron.

The unique focusing properties of the curved-field reflectron provide a simple solution to the problem of compensating for the broad range of energies of product ions produced postsource in a time-of-flight mass spectrometer. This has been shown previously for the technique known as postsource decay, but in this report we demonstrate its use for tandem time-of-flight mass spectrometry using a high-performance MALDI time-of-flight instrument modified by the addition of a collision chamber to enable the recording of mass-selected product ions formed by collision-induced dissociation (CID). In particular, the curved-field reflectron enables the use of the full 20-keV kinetic energy provided by the ion source extraction voltage as the collision energy in the laboratory frame and obviates the need to reaccelerate the product ions, using a second "source" or "lift" cell. Results are presented for the collision-induced dissociation of fullerenes over a range of collision gas pressures and precursor ion attenuation. In addition, CID tandem mass spectra are obtained for several peptides.

Bacillus spore identification via proteolytic peptide mapping with a miniaturized MALDI TOF mass spectrometer.

An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer. A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder. To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases. Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications.

A miniaturized matrix-assisted laser desorption/ionization time of flight mass spectrometer with mass-correlated acceleration focusing.

Mass correlated acceleration (MCA) has now been integrated into a 4 in (10.2 cm) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer to achieve high resolving power across a broader mass range, without sacrificing detection of higher mass ions. The goal was to combine MCA with a custom-built miniaturized instrument such as those that might be used for field-portable applications. Unlike other pulsed extraction methods, MCA is not mass dependent and mass spectra can be achieved with a single tuning of instrument parameters. Additionally, the multi-channel recording advantage is better realized because ions of all masses can be brought into focus simultaneously. The MCA dual-stage ion source compensates dynamically for the mass dependence by incorporating an extraction pulse region followed by an acceleration region that contains a time-dependent waveform correlated with mass. The technique was validated with applications in peptide mixtures and protein digestions. Diagnostic studies for the instrument include m/z range and limits of detection.

Mass correlated acceleration in a reflectron MALDI TOF mass spectrometer: an approach for enhanced resolution over a broad mass range.

Compared to continuous extraction, pulsed extraction (PE) of ions formed by matrix-assisted laser desorption/ionization (MALDI) in time-of-flight (TOF) mass spectrometers significantly improves mass resolution. Parameters such as extraction voltage, delay time, and correction pulse must be varied, however, to achieve optimum mass resolution over a broad mass range because the PE method is mass dependent. We previously reported a novel method, mass correlated acceleration (MCA), which we have now combined with a reflectron MALDI TOF mass spectrometer to further enhance mass resolution over a broader mass range. Unlike the PE method, MCA is not mass dependent and high resolution mass spectra can be achieved with a single tuning of instrument parameters. The ions may be brought into focus simultaneously, i.e., the multi-channel recording advantage can be more fully realized. The MCA dual-stage ion source design includes an extraction pulse region and an acceleration region that contains a time-dependent waveform correlated with mass. We demonstrate the validity of this novel technique with applications in peptide mixture analysis and protein digests of lysozyme and bovine serum albumin.