39 research outputs found

    Capillary Electrophoresis–Nanoelectrospray Ionization–Selected Reaction Monitoring Mass Spectrometry via a True Sheathless Metal-Coated Emitter Interface for Robust and High-Sensitivity Sample Quantification

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    A new sheathless transient capillary isotachophoresis (CITP)/capillary zone electrophoresis (CZE)–MS interface, based on a commercially available capillary with an integrated metal-coated ESI emitter, was developed in this study aiming at overcoming the reproducibility and ruggedness problems suffered to a certain degree by almost all the available CE–MS interfaces, and pushing the CE–MS technology suitable for routine sample analysis with high sensitivity. The new CITP/CZE–MS interface allows the electric contact between ESI voltage power supply and the CE separation liquid by using a conductive liquid that comes in contact with the metal-coated surface of the ESI emitter, making it a true sheathless CE–MS interface. Stable electrospray was established by avoiding the formation of gas bubbles from electrochemical reaction inside the CE capillary. Crucial operating parameters, such as sample loading volume, flow rate, and separation voltage, were systematically evaluated for their effects on both CITP/CZE separation efficiency and MS detection sensitivity. Around one hundred CITP/CZE–MS analyses can be easily achieved by using the new sheathless CITP/CZE interface without a noticeable loss of metal coating on the ESI emitter surface, or degrading of the ESI emitter performance. The reproducibility in analyte migration time and quantitative performance of the new interface was experimentally evaluated to demonstrate a LOQ below 5 attomole

    Effective coupling of CE with nanoESI MS via a true sheathless metal-coated emitter interface for robust and high sensitivity sample quantification (ASMS 2016)

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    <p>Capillary electrophoresis (CE) coupled with mass spectrometry (MS) is a promising alternative to conventional liquid chromatography (LC) MS in chemical and biological sample analysis due to its high resolving power and fast separation speed. Reproducibility and ruggedness problems, suffered to a certain degree by almost all the CE-MS interfaces, limit its broad applications. We present the development of a new sheathless CE-MS interface aiming at overcoming these problems and pushing CE-MS suitable to routine sample analysis with high sensitivity. A systematic evaluation of the new interface was performed using a hybrid capillary isotachophoresis (CITP) and capillary zone electrophoresis (CZE) separation coupled with electrospray ionization (ESI) MS for its achievable sensitivity and reproducibility in sample quantification. </p

    Proteomic Analysis of Mouse Testis Reveals Perfluorooctanoic Acid-Induced Reproductive Dysfunction via Direct Disturbance of Testicular Steroidogenic Machinery

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    Perfluorooctanoic acid (PFOA) is a ubiquitous environmental pollutant suspected of being an endocrine disruptor; however, mechanisms of male reproductive disorders induced by PFOA are poorly understood. In this study, male mice were exposed to 0, 0.31, 1.25, 5, and 20 mg PFOA/kg/day by oral gavage for 28 days. PFOA significantly damaged the seminiferous tubules and reduced testosterone and progesterone levels in the testis in a dose-dependent manner. Furthermore, PFOA exposure reduced sperm quality. We identified 93 differentially expressed proteins between the control and the 5 mg/kg/d PFOA treated mice using a quantitative proteomic approach. Among them, insulin like-factor 3 (INSL3) and cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1) as Leydig-cell-specific markers were significantly decreased. We examined in detail the expression patterns of CYP11A1 and associated genes involved in steroidogenesis in the mouse testis. PFOA inhibited the mRNA and protein levels of CYP11A1 and the mRNA levels of 17β-hydroxysteroid dehydrogenase (17β-HSD) in a dose-dependent manner. Moreover, <i>in vitro</i> study showed the reduction in progesterone levels was accompanied by decreased expression of CYP11A1 in cAMP-stimulated mLTC-1 cells. Our findings indicate that PFOA exposure can impair male reproductive function, possibly by disturbing testosterone levels, and CPY11A1 may be a major steroidogenic enzyme targeted by PFOA

    Comparison of the incidence of apoptosis in placenta from pregnant women with ICP compared to that in placenta from healthy pregnant women (Ă—400).

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    <p>TUNEL assay demonstrated the incidence of apoptosis in placenta from pregnant women with ICP was higher than that in placenta from healthy pregnant women (A and B). Semi-quantitative analysis indicated there was significantly higher incidence of apoptosis in placenta from pregnant women with ICP (<i>P</i> = 0.007) (C).</p

    Differentially expressed proteins in the placenta tissue from pregnant women with ICP and healthy pregnant women identified by iTRAQ labeling-based proteomics.

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    <p>The table contains quantitative information for proteins which were at least > 1.5-fold upregulated or at least <0.67-fold downregulated in pregnant women with ICP (P) compared with healthy pregnant women (C), as defined in the experimental procedures. The key proteins verified by Western blot and immunohistochemisty analysis were highlighted in bold. The corresponding average ratios between the two groups (P:C) were given.</p

    Immunohistochemical staining for PRDX6, ERp29 and MPO in the placental tissue from pregnant women with ICP and healthy pregnant women (Ă—400).

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    <p>Immunohistochemstry images demonstrated higher expression of PRDX6 (B) and ERp29 (D), and lower expression of MPO (F) in cytoplasm and/or nucleus of trophoblasts in the placenta from pregnant women with ICP than those in placenta from healthy pregnant women (A, C, E).</p

    Bioinformatic analysis of differentially expressed proteins in the placental tissue from pregnant women with ICP and healthy pregnant women predicted by PathwayStudio™.

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    <p>Analysis of the cellular pathways affected by the differentially expressed proteins was performed using the PathwayStudio software. Proteins are shown as red ovals, regulated processes are represented by yellow squares. Regulation events are displayed with arrows and documented by literature citations.</p

    Cluster analysis of differentially expressed proteins in the placental tissue from pregnant women with ICP and healthy pregnant women.

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    <p>Hierarchical cluster analysis for the 38 differentially expressed proteins displaying significantly altered expression levels in the placental tissue from pregnant women with ICP and healthy pregnant women. “N” represents healthy pregnant women and “P” represents pregnant women with ICP. The protein expression levels are shown as colored boxes; red indicates a high expression level and green indicates a low expression level.</p

    mUbiSiDa: A Comprehensive Database for Protein Ubiquitination Sites in Mammals

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    <div><p>Motivation</p><p>Protein ubiquitination is one of the important post-translational modifications by attaching ubiquitin to specific lysine (K) residues in target proteins, and plays important regulatory roles in many cell processes. Recent studies indicated that abnormal protein ubiquitination have been implicated in many diseases by degradation of many key regulatory proteins including tumor suppressor, oncoprotein, and cell cycle regulator. The detailed information of protein ubiquitination sites is useful for scientists to investigate the mechanism of many cell activities and related diseases.</p><p>Results</p><p>In this study we established mUbiSida for mammalian Ubiquitination Site Database, which provides a scientific community with a comprehensive, freely and high-quality accessible resource of mammalian protein ubiquitination sites. In mUbiSida, we deposited about 35,494 experimentally validated ubiquitinated proteins with 110,976 ubiquitination sites from five species. The mUbiSiDa can also provide blast function to predict novel protein ubiquitination sites in other species by blast the query sequence in the deposit sequences in mUbiSiDa. The mUbiSiDa was designed to be a widely used tool for biologists and biomedical researchers with a user-friendly interface, and facilitate the further research of protein ubiquitination, biological networks and functional proteomics. The mUbiSiDa database is freely available at <a href="http://reprod.njmu.edu.cn/mUbiSiDa" target="_blank">http://reprod.njmu.edu.cn/mUbiSiDa</a>.</p></div
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