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We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry (LC–MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED’s database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results.

Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. SIGNIFICANCE STATEMENT Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance current strategies for addiction treatment.

The Zaire Ebola virus (EBOV) protein VP35 is multifunctional; it inhibits IFN-α/β production and functions as a cofactor of the viral RNA polymerase. Mass spectrometry identified the double stranded RNA binding protein 76 (DRBP76/NFAR-1/NF90) as a cellular factor that associates with the VP35 C-terminal interferon inhibitory domain (IID). DRBP76 is described to regulate host cell protein synthesis and play an important role in host defense. The VP35-IID-DRBP76 interaction required the addition of exogenous dsRNA, but full-length VP35 associated with DRBP76 in the absence of exogenous dsRNA. Cells infected with a Newcastle disease virus (NDV)–expressing VP35 redistributed DRBP76 from the nucleus to the cytoplasm, the compartment in which EBOV replicates. Overexpression of DRBP76 did not alter the ability of VP35 to inhibit type I IFN production but did impair the function of the EBOV transcription/replication complex. These data suggest that DRBP76, via its association with VP35, exerts an anti-EBOV function.

NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl(-)]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl(-)-dependent (86)Rb(+) uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl(-) accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO(2) enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling.

Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.

The Ebola virus (EBOV) protein VP24 inhibits type I and II interferon (IFN) signaling by binding to NPI-1 subfamily karyopherin α (KPNA) nuclear import proteins, preventing their interaction with tyrosine-phosphorylated STAT1 (phospho-STAT1). This inhibits phospho-STAT1 nuclear import. A biochemical screen now identifies heterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2) nuclear import as an additional target of VP24. Co-immunoprecipitation studies demonstrate that hnRNP C1/C2 interacts with multiple KPNA family members, including KPNA1. Interaction with hnRNP C1/C2 occurs through the same KPNA1 C-terminal region (amino acids 424-457) that binds VP24 and phospho-STAT1. The ability of hnRNP C1/C2 to bind KPNA1 is diminished in the presence of VP24, and cells transiently expressing VP24 redistribute hnRNP C1/C2 from the nucleus to the cytoplasm. These data further define the mechanism of hnRNP C1/C2 nuclear import and demonstrate that the impact of EBOV VP24 on nuclear import extends beyond STAT1.

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34 + subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34 − cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.

Although most prokaryotic proteins have free NH2-termini and therefore can be directly sequenced, most eukaryotic proteins have blocked NH2-termini which precludes Edman degradation (1,2). In these instances, one of the most direct approaches to obtaining partial amino acid sequences is via enzymatic or chemical cleavage followed by peptide fractionation and sequencing. Although several different approaches may be taken to cleave proteins, one of the most common is to digest the protein enzymatically with a relatively specific protease such as trypsin or lysyl endopeptidase. Since final purification is often dependent on SDS-PAGE, cleavage procedures that can either be carried out in the polyacrylamide gel matrix (3, 4) or that may be used on samples that have been blotted from SDS polyacrylamide gels onto PVDF (5) or nitrocellulose (5,6) membranes are extremely useful. In both instances, the proteins are usually stained with Coomassie blue or Ponceau S prior to excision, and proteolytic digestion and the resulting peptides are separated by reverse-phase HPLC. Relatively straightforward solution and in-gel digestion procedures that have been used extensively in the Keck Foundation Biotechnology Resource Laboratory at Yale University will be described in this chapter, whereas a procedure suitable for in situ digestion of SDS-PAGE blotted proteins is described in Chapter 70.

A catalytic microreactor coupled with a multiphoton ionization time-of-flight mass spectrometer (MPI TOF) is used to directly observe stable and unstable reaction products from oxidative coupling of methane. Production of gas-phase methyl radicals from 10:1 mole ratio methane/oxygen mixture over CaO/Al{sub 2}O{sub 3} and 1% Sr/La{sub 2}O{sub 3} catalysts as a function of temperature is studied. At low methane conversion, ethane production can be accounted for by gas-phase coupling of methyl radicals produced by the catalytic reaction. Apparent energies of activation for methyl radical production of 41.6 and 30.0 kcal/mol for CaO/Al{sub 2}O{sub 3} and 1% Sr/La{sub 2}O{sub 3} catalysts, respectively, were determined by Arrhenius analysis of relative methyl radical production rates measured at low methane conversion.

Isotope-coded affinity tags (ICAT) is a labeling technique that provides insights into quantitative molecular changes. In this paper, we propose a new protocol to identify and analyze ICAT labeled peak pairs in high-resolution LC-MS data. Our major contributions are: (1) we use isotope distance constraint, ICAT distance constraint, and LC-span constraint to identify ICAT labeled peak pairs and (2) we propose to trigger tandem MS/MS scanning based on the ratio estimation value of identified ICAT peak pairs instead of the peak intensity values. Compared with current approaches that choose peaks with high intensity values for tandem MS/MS scanning, the new protocol can improve the scanning efficiency and accuracy.

Mass spectrometry has become a major tool in the study of proteomes. The analysis of proteolytic peptides and their fragment ions by this technique enables the identification and quantitation of the precursor proteins in a mixture. However, deducing chemical structures and then protein sequences from mass-to-charge ratios is a challenging computational task. Software tools incorporating powerful algorithms and statistical methods improved our ability to process the large quantities of proteomics data. Repositories of spectral data make both data analysis and experimental design more efficient. New approaches in quantitative and statistical proteomics make possible a greater coverage of the proteome, the identification of more post-translational modifications, and a greater sensitivity in the quantitation of targeted proteins.

Relative signal response differences are shown between the continuous and the discrete dynode‐type detectors for time‐of‐flight mass spectrometry at increasing pulse repetition rates.

Abstract 4778 Normal human CD34 positive precursor cells can be expanded in vitro and converted with high efficiency into maturing erythroid cells or myeloid cells by treating the cells with erythropoietin (EPO) or with G-CSF, respectively. The maturation of megakaryocytic cells can also be induced from human CD34 positive precursor cells by treatment with thrombopoietin (TPO) with lower efficiency. The induction time varies from 9 to 12 days depending on the lineages. Total RNAs were extracted every other day during the induction period for the purpose of gene profiling at intermediate steps of the differentiation. The samples were analyzed with the “Whole Transcriptome Shotgun Sequencing (RNA-seq)” method. Comparison of the gene expression among these three lineages at different stages of differentiation shows complex changes of a substantial number of genes in categories including transcription factors, cytokines etc. Basically, many genes change similarly in both erythroid and megakaryocytic lineage differentiation, the number of this shared genes are much less when comparing myeloid cells with one of these lineages. Initially during erythropoietin treatment, a large number of megakaryocyte mRNAs are upregulated to levels comparable to those of the erythroid specific genes, or to those seen in precursor cells treated with thrombopoietin. After several days of erythropoietin treatment, most of the megakryocyte related genes are down regulated, while there is continued upregulation of erythroid genes. Unlike many other megakaryocytic genes, the level of mRNA for the thrombopoietin receptor MPL is rapidly down-regulated suggesting that erythroid lineage commitment occurs prior to silencing of many megakaryocytic genes. Comparison of the linage specific genes identified groups of cell surface proteins which may be applied as new markers for sorting various cell populations. Some of these include DARC, LEPR, TFRC, BMPR2, TFR2, AXL, EPHB4, IL15RA, TNFRSF19, FZD5, ULBP1, CELSR2, RYK, MRGPRE, CRY1 which are specific for erythroid lineage; FPR1, EVI2A, CD14, and others totaling 50 proteins for myeloid lineage and more than 80 highly expressed surface markers such as GP1BA, F2RL3, GP1BB, ADRA2A, GABRE unique for megakaryocytic lineage. In parallel, proteomic analysis was carried out with an Isotope coded affinity tag (ICAT)-based protein profiling and Multiplexed Isobaric Tagging Technology (iTRAQ™). Statistical analysis shows good correlation between the relative changes of RNA and protein expression levels during the erythropoiesis, although there were large discrepancies in the relative levels of mRNA and protein at any one time point. Only those genes with very low or very high RNA values show the poor correlations. CTCF and its associated factor Rad21 are involved in the establishment of enhancer boundary elements and chromosomal conformation. We have begun genome wide analysis of these factors during erythroid and myeloid cell growth and differentiation. In this project, we also aimed to investigate the modulation mechanisms of CTCF during the CD34 positive cells differentiation into different lineages. Taking the advantage of the ChIP-Sequencing (ChIP-Seq) technology, we found that progressive changes in the sites of binding of the DNA binding protein CTCF occurs, and are not limited to the regions embedding erythroid genes, suggesting that there is a lineage specific global reorganization of chromatin. In addition to transcriptional controls, there are prominent post-transcriptional effects regulating the expression of key transcription factors including CTCF. Through this study, the regulation mechanisms of the chromatin remodeling, signal pathways and other, molecular events during hematopoiesis will be described in detail. Disclosures: No relevant conflicts of interest to declare.

Purpose of review Maintenance of cellular water and solute homeostasis is critical for survival of the erythrocyte. Inherited or acquired disorders that perturb this homeostasis jeopardize the erythrocyte, leading to its premature destruction. This study reviews recent progress in our understanding the determinants of erythrocyte hydration and its related disorders. Recent findings The molecular and genetic bases of primary disorders of erythrocyte hydration are poorly understood. Recent studies have implicated roles for the anion transporter, SLC4A1, and the Rh-associated glycoprotein, RhAG. The most common secondary disorder associated with perturbed hydration of the erythrocyte is sickle cell disease, in which dehydration contributes to disease pathology and clinical complications. Advances in understanding the mechanisms regulating erythrocyte solute and water content, particularly associated with KCl cotransport and Gardos channel activation, have revealed novel signaling mechanisms controlling erythrocyte hydration. These signaling pathways may provide innovative strategies to prevent erythrocyte dehydration in sickle cell disease. Summary Clinical, translational and biologic studies all contribute to our knowledge of erythrocyte hydration. Understanding the mechanisms controlling erythrocyte water and solute homeostasis will serve as a paradigm for other cells and may reveal new therapeutic targets for disease prevention and treatment.

Na+-activated potassium channels (KNa channels), which are encoded by the Slack and Slick genes contribute to neuronal adaptation during sustained stimulation, and, in auditory brainstem neurons, may regulate the accuracy of timing of action potentials. These channels have been found to be modulated very potently by activation of protein kinase C (PKC) and by receptors linked to activation of this kinase. Activators of PKC increase the amplitude of Slack-B currents and slow their rate of activation, and in contrast, activation of PKC decreases the amplitude of Slick currents. Heteromeric Slack/Slick channels are regulated by PKC to a greater extent than either Slack-B or Slick heteromers (90% decrease in amplitude). Previous experiments using Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) have identified three serine residues in Slack-B that are phosphorylated under basal conditions, but are not within consensus sites for PKC. In order to study the mechanisms of regulation of Slack and Slick channels by phosphorylation, we have begun to identify the specific residues that undergo phosphorylation by protein kinase C. Consensus sequence analysis predicts that there are 13 potential sites of possible PKC phosphorylation in the Slack-B sequence. We have constructed individual site mutants for each of these sites in which the serine/threonines have been mutated to alanines to prevent phosphorylation at these residues. These mutants were expressed in Xenopus oocytes and their response to a PKC-activating phorbol ester (TPA) was characterized by two-electrode whole cell clamp electrophysiology. Of the 13 consensus site mutants, only one generated currents that matched wild-type Slack-B currents in their amplitude and kinetic behavior, but completely failed to respond to application of TPA, suggesting that the phosphorylation state of a single residue regulates Slack-B current amplitude and rate of activation.

A catalytic microreactor coupled with a multiphoton ionization time-of-flight mass spectrometer (MPI TOF) is used to directly observe stable and unstable reaction products from oxidative coupling of methane. Production of gas-phase methyl radicals from 10:1 mole ratio methane/oxygen mixture over CaO/Al{sub 2}O{sub 3} and 1% Sr/La{sub 2}O{sub 3} catalysts as a function of temperature is studied. At low methane conversion, ethane production can be accounted for by gas-phase coupling of methyl radicals produced by the catalytic reaction. Apparent energies of activation for methyl radical production of 41.6 and 30.0 kcal/mol for CaO/Al{sub 2}O{sub 3} and 1% Sr/La{sub 2}O{sub 3} catalysts, respectively, were determined by Arrhenius analysis of relative methyl radical production rates measured at low methane conversion.

A new variable angle and incident energy electron energy loss spectrometer coupled to a supersonic jet expansion microwave discharge source has been constructed to study the electron scattering cross sections of stable and unstable atmospheric species. This apparatus was specifically designed to operate at low incident electron energies and at high scattering angles to observe forbidden transitions. In this paper the authors report the first measurements of the absolute differential and integral cross sections of the atomic oxygen (2p{sup 4} {sup 3}P-3p {sup 3}P), 10.99 eV, and (2p{sup 4} {sup 3}P-{sup 3}p{sup 5}P), 10.76 eV, transitions at a single electron impact energy of 30 eV. The direct excitation cross section ratio for the {sup 3}P and {sup 5}P states was found to be 6:1 which confirms previous suggestions that electron impact on O I is responsible for the observed 8446 {angstrom} and 7774 {angstrom} O I emissions in the dayside cusp region. Additional sources of O I 3p {sup 5}P are necessary to explain the nightside auroral 8446 {angstrom}/7774 {angstrom} ratio. The absolute cascade-free direct excitation integral cross sections for O I({sup 3}P-{sup 3}P) and O I ({sup 3}P-{sup 5}P) at 30 eV are 4.0 {times} 10{sup {minus}18} cm{sup 2}more » and 0.67 {times} 10{sup {minus}18} cm{sup 2}, respectively.« less