Label‐free quantitative proteome analysis of skeletal tissues under mechanical load

Skeletal tissue has the capability to adapt its mass and structure in response to mechanical stress. However, the molecular mechanism of bone and cartilage to respond to mechanical stress are not fully understood. A label‐free quantitative proteome approach was used for the first time to obtain a global perspective of the response of skeletal tissue to mechanical stress. Label‐free quantitative analysis of 1D‐PAGE‐LC/MS/MS based proteomics was applied to identify differentially expressed proteins. Differential expression analysis in the experimental groups and control group showed significant changes for 248 proteins including proteins related to proliferation, differentiation, regulation of signal transduction and energy metabolic pathways. Fluorescence labeling by incorporation of alizarin/calcein in newly formed bone minerals qualitatively demonstrated new bone formation. Skeletal tissues under mechanical load evoked marked new bone formation in comparison with the control group. Bone material apposition was evident. Our data suggest that 39 proteins were assigned a role in anabolic process. Comparisons of anabolic versus catabolic features of the proteomes show that 42 proteins were related to catabolic. In addition, some proteins were related to regulation of signal transduction and energy pathways, such as tropomyosin 4, fibronectin 1, and laminin, might be new molecular targets that are responsive to mechanical force. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the effects of mechanical force on skeletal tissue. J. Cell. Biochem. 108: 600–611, 2009. © 2009 Wiley‐Liss, Inc.     

Proteomic analysis of nasal cells from cystic fibrosis patients and non-cystic fibrosis control individuals: search for novel biomarkers of cystic fibrosis lung disease

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.     

Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs.     

Comparative Proteomics Analysis of Engineered Saccharomyces cerevisiae with Enhanced Biofuel Precursor Production

The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production.     

Combined effect of gender and caloric restriction on liver proteomic expression profile

We analyzed the combined effect of gender and CR on protein expression profile in liver. We identified 27 differentially expressed proteins involved in several cellular functions such as substrate metabolism, antioxidant systems, stress response, iron homeostasis and cardiovascular protection. This study reveals new cellular pathways liable to be similarly regulated in females and calorie restricted rats and which could be related with the greater longevity in these animals.     

蛋白质组学方法分析多T迷宫训练后的小鼠空间记忆蛋白

应用双向凝胶电泳结合质谱鉴定和数据库检索,分析比较C57BL/6J小鼠在多T迷宫(MTM)训练和记忆测试组与未训练组海马蛋白表达的差异,探讨与MTM空间记忆相关的蛋白质.C57BL/6J小鼠经MTM训练后,可对相应的空间线索保持记忆能力,其海马蛋白质表达存在明显差异,14个蛋白质与MTM空间记忆形成显著相关.其中,6个蛋白点表达显著上调,8个蛋白点表达水平显著降低.这些蛋白按功能可分为6类: 细胞骨架相关蛋白,物质运输相关蛋白,蛋白合成相关蛋白,能量和物质代谢相关蛋白,信号转导相关蛋白,通道蛋白. 这些空间记忆形成相关蛋白的研究深化了对空间记忆机制的认识,为研究和治疗认知相关疾病提供了新靶标.     

磷高效水稻根系对低磷胁迫响应的差异蛋白分析

以IR71331为材料,通过水培试验,采用双向电泳分离不同磷浓度下（低磷浓度为0.5 mg·L^-1,对照为10 mg·L^-1）水稻生长3 d和6 d根系差异蛋白.结果表明：与对照相比,低磷胁迫下共有29个蛋白,其中3 d时间点有17个蛋白上调、11个下调、1个新增,6 d时间点有8个上调、19个下调、1个抑制表达、1个无明显变化 .经鉴定,其中的10个差异表达蛋白可归为信号转导相关蛋白、基因表达相关蛋白、代谢相关蛋白离子转运相关蛋白4个功能类群.信号转导相关蛋白分别为富含甘氨酸RNA结合蛋白和类似参与磷酸盐饥饿反应调控子;基因表达相关蛋白分别为推定的mRNA前体剪接因子SF2和推定的AAA蛋白酶家族FtsH;代谢相关蛋白分别为腺苷酸琥珀酸裂解酶、丝氨酸蛋白酶抑制剂(serpin)、S-腺苷蛋氨酸合成酶（SAM）和类似MYB类转录因子;离子转运相关蛋白分别为阳离子转运ATP酶和肌浆网膜蛋白.这些蛋白分别参与了信号识别、信号调控、mRNA 的剪接、信号传递、蛋白质降解、细胞体内离子转运和平衡等生理过程.其中serpin、SAM和MYB类转录因子是水稻响应低磷胁迫的关键蛋白.水稻根系对低磷胁迫存在着一个复杂的抗逆信号应答和代谢调控网络,其作用机理可以通过差异表达的蛋白质得以体现.     

Modulation of endoperoxide product levels and cyclophosphamide-induced injury by glutathione repletion

Glutathione is a tripeptide important in a number of diverse cellular functions including enzymatic reactions involved in prostaglandin endoperoxide metabolism. We have previously reported that cyclophosphamide administration to rats results in acute lung injury manifested by increased bronchoalveolar lavage albumin concentrations. In the current study we examine whether cyclophosphamide treatment affects pulmonary glutathione stores or bronchoalveolar endoperoxide metabolic product levels and whether these effects may be related to acute lung injury caused by the drug. We show that cyclophosphamide treatment causes a dose-dependent reduction in pulmonary glutathione stores 4 h after drug administration. In addition, acute lung injury as the result of cyclophosphamide can be abrogated by coadministration of oxothiazolidine carboxylate, an intracellular cysteine delivery system that also reverses pulmonary glutathione depletion induced by cyclophosphamide in our study. Finally, cyclophosphamide treatment reduces prostaglandin E2 concentrations in bronchoalveolar lavage and alveolar macrophage culture supernatant in a dose-dependent fashion and increases bronchoalveolar thromboxane concentrations in low dose-treated animals. These effects are reversed to a variable degree by coadministration of oxothiazolidine carboxylate. Our study suggests in vivo pulmonary arachidonic acid metabolism and cyclophosphamide-induced acute lung injury are modulated by cellular glutathione stores. These findings may have important implications for the treatment of acute lung injury.     

家蚕精巢蛋白质的双向电泳及质谱分析

精巢是雄性家蚕Bombyx mori的生殖腺,它的主要功能是产生精子,全面检测和鉴定精巢器官的蛋白分布将为分析家蚕雄性个体的发育和繁殖奠定基础。本研究利用双向聚丙烯酰胺凝胶电泳和蛋白硝酸银染色技术对家蚕5龄第5天幼虫的精巢组织进行了蛋白检测,利用基质辅助激光解析质量飞行时间质谱（MALDI-TOF-MS）对表达量较高的蛋白点进行了肽质量指纹图谱鉴定。结果表明：家蚕精巢蛋白质可以检测出1 000个以上的蛋白点,这些蛋白点主要集中在分子量为15～90 kD区域,等电点3.5～9之间,其中60个蛋白点得到了成功鉴定,按照已知或推测的蛋白功能,将其分为8类,包括：细胞骨架和细胞结构蛋白,膜蛋白或信号相关蛋白,大量应激反应蛋白（伴侣蛋白）,线粒体和能量产生相关蛋白,转录调控和翻译及DNA/RNA结合相关蛋白,酶和少量血液组成蛋白。其中很多蛋白可能在鞭毛形成、能量代谢及减数分裂过程中有重要作用。这些结果为进一步认识家蚕精子形成过程提供了重要的生物学信息。     

Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves

Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.     

Temporal expression of hypoxia-regulated genes is associated with early changes in redox status in irradiated lung

The development of normal lung tissue toxicity after radiation exposure results from multiple changes in cell signaling and communication initiated at the time of the ionizing event. The onset of gross pulmonary injury is preceded by tissue hypoxia and chronic oxidative stress. We have previously shown that development of debilitating lung injury can be mitigated or prevented by administration of AEOL10150, a potent catalytic antioxidant, 24h after radiation. This suggests that hypoxia-mediated signaling pathways may play a role in late radiation injury, but the exact mechanism remains unclear. The purpose of this study was to evaluate changes in the temporal expression of hypoxia-associated genes in irradiated mouse lung and determine whether AEOL10150 alters expression of these genes. A focused oligo array was used to establish a hypoxia-associated gene expression signature for lung tissue from sham-irradiated or irradiated mice treated with or without AEOL10150. Results were further verified by RT-PCR. Forty-four genes associated with metabolism, cell growth, apoptosis, inflammation, oxidative stress, and extracellular matrix synthesis were upregulated after radiation. Elevated expression of 31 of these genes was attenuated in animals treated with AEOL10150, suggesting that expression of a number of hypoxia-associated genes is regulated by early development of oxidative stress after radiation. Genes identified herein could provide insight into the role of hypoxic signaling in radiation lung injury, suggesting novel therapeutic targets, as well as clues to the mechanism by which AEOL10150 confers pulmonary radioprotection.     

Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics-based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, alpha-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.     

Proteomic analysis of the lungs of mice infected with different pathotypes of H5N1 avian influenza viruses

The virulence of influenza virus is determined by viral and host factors. Data on the genetic basis of the virulence of H5N1 influenza viruses have increased over the past decade; however, the contributions of host factors to the outcomes of H5N1 infection remain largely unknown. Here, we tested two chicken H5N1 viruses in mice and found that A/chicken/VN1214/2007 was nonlethal in mice and only replicated in the lung, whereas A/chicken/VN1180/2006 was highly lethal and replicated systemically in mice. To investigate the host response against these two different virus infections, we performed proteomic analysis by using 2D DIGE on the lung tissues of mice collected on days 1 and 3 postinoculation with different viruses or PBS as a control. Thirty-nine differentially expressed (DE) proteins related to "immune and stimulus response," "macromolecular biosynthesis and metabolism," and "cellular component and cytoskeleton" were identified in the virus-inoculated groups. Moreover, 13 DE proteins were identified between the two virus-inoculated groups, implying that these proteins may play important roles in the different outcomes of infection with these two viruses. Our data provide important information regarding the host response to mild and lethal H5N1 influenza virus infection.     

硫酸镁对脑源性肺损伤综合征的影响

目的探讨不同剂量的硫酸镁(MgSO4)对大鼠脑源性肺损伤后神经源性肺水肿、血浆炎性因子TNF-α及肺组织病理形态学变化的影响。方法将30只SD雄性大鼠按随机数字法分为假手术组(A组)、模型组(B组)及硫酸镁50mg/kg干预组(C组)、硫酸镁100mg/kg干预组(D组)、硫酸镁200mg/kg干预组(E组),每组6只。建立大鼠颅脑损伤模型后,硫酸镁干预组即刻按50mg/kg 25%MgSO4腹腔注射,C组注射1次、D组注射2次、E组注射4次,每8h注射1次。A组及B组的大鼠注射相同剂量生理盐水作对照,C组大鼠注射1次及D组大鼠注射2次MgSO4后给予注射相同剂量的生理盐水作对照,注射方法及间隔时间同E组。伤后48h测定大鼠肺组织含水量、血浆TNF-α浓度,肺组织常规HE染色,光镜观察肺组织病理形态学变化。结果大鼠颅脑创伤后肺组织含水量均高于假手术组,以C组最明显(P0.05),差异有统计学意义。B、C、D、E组大鼠之间肺组织含水量比较差异无统计学意义。B、C、D、E组大鼠TNF-α浓度均明显高于假手术组(P0.05),D组血浆TNF-α浓度明显低于B组(P0.05),E组血浆TNF-α浓度明显低于B组(P0.01),其他各组间差异无统计学意义。假手术组肺组织形态正常,肺血管无扩张,无炎症细胞浸润;B、C、D、E组与假手术组比较均可见终末支气管腔内充满炎症细胞,周围肺组织的肺泡腔内可见炎细胞浸润,肺血管扩张、充血。B、C、D、E组在炎症细胞浸润及肺毛细血管扩张方面无明显差异。结论脑外伤可导致脑源性肺损伤综合征,可导致神经源性肺水肿;硫酸镁可降低大鼠脑损伤后血浆TNF-α浓度,对肺水肿无明显影响。     

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT‐ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress‐induced, nine proteins with inflammatory stress‐induced, and 14 proteins with endoplasmic reticulum stress‐induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c‐Jun amino‐terminal kinase‐dependent manner. In addition, three proteins, 14–3‐3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD‐95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.