Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells

We present a fully defined culture system (adapted Essential8^TM [E8^TM] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8^TM medium was modified by adding (1) l ‐proline, (2) l ‐ornithine, (3) N^ω‐hydroxy‐nor‐l ‐arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l ‐ornithine, followed by 3.5 mM l ‐proline and by lowering the arginine concentration in the medium to 99.5 μM. No major changes in pluripotency and cell amount could be observed for the adapted E8^TM media with ornithine and proline. However, our subsequent ion mobility assisted data‐independent acquisition (high‐definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion.     

Sample preparation project for the subcellular proteome of mouse liver

Organelle proteome has become one of the most important fields of proteomics, and the subcellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project (HLPP). The liver of a C57BL/6J mouse was chosen as the model to find the optimum method for subcellular preparation. The method we selected could obtain the multiple fractions including plasma membrane, mitochondria, nucleus, ER, and cytosol from a single homogenate. With the same procedure, it is for the first time that the preparation method of frozen homogenized livers was compared with that of the fresh livers and frozen livers. We systematically evaluated the purity, efficiency, and integrity by protein yield, immunoblotting, and transmission electron microscopy. Taken together, the method of multiple fractions from a single tissue is effective enough for subcellular fractionation of mouse liver. We give a selective sample preparation method for frozen homogenized livers, for rare clinical samples, which cannot easily be used for subcellular separation immediately. But the frozen livers are not recommended for organelles isolation. This result is especially useful for sample preparation of human liver for subcellular fractionation of HLPP.     

Mass spectrometric immunoassay for quantitative determination of transthyretin and its variants

Transthyretin (TTR, or prealbumin) is a tetrameric protein found in plasma and cerebrospinal fluid. Its major role is to transport thyroid hormones (thyroxin-T4) and retinol (through association with retinol-binding protein). TTR has been studied extensively due to the great number of point mutations that result in sequence heterogeneity. Many of these variants are associated with pathological conditions that result in extracellular deposition of amyloid fibers in tissues. In this work, we have developed a rapid mass spectrometric immunoassay for determination and quantification of TTR and its variants from human serum and plasma samples. The assay was fully characterized in terms of its precision, linearity and recovery characteristics. The new assay was also compared with a conventional TTR ELISA. Furthermore, we have applied the optimized method to analyze TTR and its modifications in 44 human plasma samples, and in the process optimized a method for TTR proteolytic digestion and identification of point mutations.     

A characterization of cuprizone-induced giant mouse liver mitochondria

Cuprizone affects the liver of treated mice in a random manner, causing no appreciable change in some cases and inducing the formation of megamito-chondria with altered properties in others. Lack of a full appreciation of this variability may be at the origin of some discrepancies in published work dealing with the properties of cuprizone mouse liver mitochondria (CMLM). CMLM from fully affected livers were remarkably labile and difficult to isolate in a coupled state by homogenization and centrifugation techniques. The integral respiratory chain proteins of CMLM were functionally normal, with the exception of succinic dehydrogenase which showed considerable inhibition. Coupled morphological and functional analysis provided evidence that these properties were independent of CMLM size, a matter which had remained doubtful thus far and bears on the validity of literature reports.Abbreviations MLM mouse liver mitochondria - CMLM cuprizone mouse liver mitochondria - PMS phenazine methosulfate - Mops 4-morphoinopropanesulfonic acid - TMPD tetramethyl-p-phenylenediamine - AA Antimycin A - RCR respiratory control ratio - BSA bovine serum albumin     

A gene affecting liver activities of three glycosidases in the house mouse

A gene locus is described controlling liver activities in the house mouse of three glycosidases, i.e., -galactosidase, -glucuronidase, and N-acetyl--hexosaminidase. An allele conferring low activity is present in the inbred strain LIS/A, and an allele for high activity is present in A/Br Af mice. The three enzyme activities are correlated with each other. The possible linkage between this gene and the Bgs locus on chromosome 9 is discussed.     

四氯化碳诱导小鼠肝脏纤维化的差异蛋白质组学

以四氯化碳(Carbon tetrachloride,CCl4)诱导小鼠肝组织纤维化为研究模型,发现并探讨纤维化肝组织与正常肝组织在蛋白质组水平上的差异。实验小鼠(C57 BL/6)随机分为两组,由橄榄油和四氯化碳诱导15周,并分别对这两组肝组织的全蛋白表达谱进行质谱检测,应用GO(Gene Ontology)功能分类分析和KEGG(Kyoto Enyoolpedia of Genes and Genomes)信号通路的富集分析方法对鉴定到的全蛋白表达谱进行差异表达分析。在对照组和实验组中,我们分别鉴定到17 382和20 486条特异性肽段,图谱平均利用率大于50%,共计鉴定到蛋白4 991种(蛋白特异性肽段个数至少为1),其中差异表达蛋白有2 135种(差异倍数大于或等于2),表达上调蛋白1 264种,下调蛋白871种。纤维化肝脏组织中与细胞外基质组成(Extracellular matrix organization)、细胞骨架组成(Cytoskeleton organization)、有机磷酸代谢(Organophosphate metabolic process)、细胞定位(Cellular localization)和细胞组分调节(Regulation of cellular component organization)相关蛋白的表达是上调的;另外,与小分子代谢(Small molecule metabolic process)、蛋白质转运(Protein transport)和有机氮化合物的代谢(Organonitrogen compound metabolic process),以及四吡咯的合成过程(Tetrapyrrole biosynthetic process)有关蛋白的表达是下调的。信号通路富集分析结果表明,纤维化与VEGF和T细胞受体信号调节通路密切相关。结果提示,纤维化的形成不仅是一个复杂的信号转导过程,更是一个炎症与免疫相互促成的结果;增强肝实质细胞的存活,降低相关信号的传递及接收都有可能对纤维化的发生和发展起到抑制效果。     

Effect of alcohol on the interleukin 6-mediated inflammatory response in a new mouse model of acute-on-chronic liver injury

Background and Aims

ACLF is usually associated with a precipitant in the setting of a chronically damaged liver. We aim to combine a mouse model with a pre-injured liver (Abcb4/Mdr2^?/?) with a recently standardized ethanol feeding model to dissect alcohol-related inflammatory responses in this model.

New approach to evaluating the effects of a drug on protein complexes with quantitative proteomics,using the SILAC method and bioinformatic approach

ABSTRACT

Protein–protein interactions (PPIs) lead the formation of protein complexes that perform biochemical reactions that maintain the living state of the living cell. Although therapeutic drugs should influence the formation of protein complexes in addition to PPI network, the methodology analyzing such influences remain to be developed. Here, we demonstrate that a new approach combining HPLC (high performance liquid chromatography) for separating protein complexes, and the SILAC (stable isotope labeling using amino acids in cell culture) method for relative protein quantification, enable us to identify the protein complexes influenced by a drug. We applied this approach to the analysis of thalidomide action on HepG2 cells, assessed the identified proteins by clustering data analyses, and assigned 135 novel protein complexes affected by the drug. We propose that this approach is applicable to elucidating the mechanisms of actions of other therapeutic drugs on the PPI network, and the formation of protein complexes.     

Systematic research on the pretreatment of peptides for quantitative proteomics using a C18 microcolumn

Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC–MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic‐binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic‐binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides.     

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell,and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.     

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell,and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.     

A systems biology-led insight into the role of the proteome in neurodegenerative diseases

Introduction: Multifactorial disorders are the result of nonlinear interactions of several factors; therefore, a reductionist approach does not appear to be appropriate. Proteomics is a global approach that can be efficiently used to investigate pathogenetic mechanisms of neurodegenerative diseases.

Areas covered: Here, we report a general introduction about the systems biology approach and mechanistic insights recently obtained by over-representation analysis of proteomics data of cellular and animal models of Alzheimer’s disease, Parkinson’s disease and other neurodegenerative disorders, as well as of affected human tissues.

Expert commentary: As an inductive method, proteomics is based on unbiased observations that further require validation of generated hypotheses. Pathway databases and over-representation analysis tools allow researchers to assign an expectation value to pathogenetic mechanisms linked to neurodegenerative diseases. The systems biology approach based on omics data may be the key to unravel the complex mechanisms underlying neurodegeneration.     

A new insight into the pathogenesis of filarial disease

Filariasis is a major public health problem throughout many regions of the tropics. The disease is caused by several species of filarial nematode including Wuchereria bancrofti and Brugia malayi, the agents of lymphatic filariasis, and Onchocerca volvulus, the cause of 'riverblindness'. Disease caused by these worms varies depending on the tissue location of the parasite, and is associated with episodes of acute and chronic inflammation. These pathologies, including elephantiasis and blindness, rank among the most disabling in the world. Studies aimed at characterizing the molecular nature of the inflammatory stimuli derived from filarial nematodes uncovered a long forgotten secret, their symbiont Wolbachia. LPS-like molecules from these intracellular bacteria are responsible for potent inflammatory responses from macrophages and in animal models of filarial disease. Wolbachia has also been associated with severe inflammatory reactions to filarial chemotherapy, being released into the blood following the death of the parasite. Recent studies in animal models even implicate Wolbachia in the onset of lymphodema and blindness. Taken together these studies suggest a major role for Wolbachia in the pathogenesis of filarial disease. It may be possible, through the use of antibiotic therapy, to clear worms of their bacteria, in the hope that this will prevent the onset and development of filarial pathology.     

A second gene affecting the sialylation of lysosomal α-mannosidase in mouse liver

We have previously reported on a mouse liver-specific genetic polymorphism associated with altered sialylation of lysosomal a-mannosidase. A second electrophoretic polymorphism for liver lysosomal a-mannosidase has now been found and characterized. This variation, between SWR/J and SM/JCv inbred mice, is determined by a single genetic locus (Map-2) on chromosome 17 and appears to be the result of further differences in sialylation of the lysosomal enzyme. The Map-2 gene appears to affect the processing of liver, spleen, and lung lysosomal a-mannosidase, whereas the Map-1 gene appears to be specific to the processing of liver lysosomal a-mannosidase (Dizik and Elliott, 1977). The more negatively charged electrophoretic liver phenotype (MA-A) characteristic of the SM/JCv strain is recessive to the phenotype (MA-B) characteristic of the SWR/J strain. In contrast, at the Map-1 locus, the more negatively charged phenotype is dominant. The electrophoretic pattern of development of the liver enzyme from SM/JCv mice is described.This work was supported in part by Grant GM-19521 from the U.S. Public Health Service. One of the authors (M. D.) was supported in part by USPHS Grant TAO-CA05016.In partial fulfillment of the requirements for the Ph.D. degree.     

A new insight into the gelatinization process of native starches

The gelatinization characteristics of seven different food starches (regular corn, high-amylose corn, waxy corn, wheat, rice, potato, and tapioca) were investigated. Each starch sample type was heated to 35, 40, 45, etc. up to 85 °C at 5 °C intervals, and freeze-dried. The treated samples were analyzed using light microscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and high-performance size exclusion chromatography (HPSEC). When heated, granules underwent structural changes prior to the visible morphological changes that took place during gelatinization. The nature of these structural changes depended on starch type. These results indicate that the starch gelatinization process is more complex than a simple granular order-to-disorder transition.     

Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of ±1.5% of full scale in 65 536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 μm when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 · 10^?4 torr^?1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 · 10^?4 torr^?1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficent of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.     

A new method for quantitative analysis of cell surface glycoproteome

As the altered glycosylation expressions of cell surface proteins are associated with many diseases, glycoproteomics approach has been widely applied to characterization of surface glycosylation alteration. In general, the abundances of proteolytic glycopeptides derived from corresponding glycoproteins can be measured to determine the abundances of glycoproteins. However, this quantification strategy cannot distinguish whether the changes are results from changes of protein abundance or changes in glycosite occupancy. For the accurate and specific quantification of the cell surface glycosylation profile, we proposed a modified cell surface‐capturing strategy where the glycopeptides were submitted to LC‐MS/MS analysis directly for identification of glycoproteins and the non‐glycopeptides were isotopically labelled for quantification of glycoproteins. This strategy was applied to comparatively analyze cell surface glycoproteins of two human cell lines, i.e. Chang Liver and HepG2 cells. Totally 341 glycoproteins were identified with 82.4% specificity for cell membrane proteins and 33 glycoproteins were quantified with significant expression change between the two cell lines. The differential expressions of two selected proteins (EMMPRIN and BCAM) were validated by Western blotting. This method enables specific and accurate analysis of the cell surface glycoproteins and may have broad application in the field of biomarker and drug target discovery.