Proteomic analysis in cardiovascular diseases

1. Cardiovascular diseases are a major cause of morbidity and mortality in western countries. The molecular mechanisms responsible for heart dysfunction are still largely unknown, except in cases of genetic defects or alteration of genes and proteins. 2. The publication of genome sequences from humans and other species has demonstrated the complexity of biology, including the finding that one gene does not encode for only one protein but for several, due to mRNA splicing and post-translational modifications. 3. Proteomic analysis can provide an overall understanding of changes in the levels of protein expression. Differential proteomics is a powerful tool for improving our understanding of integrated biochemical responses. The main techniques used are two-dimensional electrophoresis (2D-gel) and Surface-Enhanced Laser Desorption/Ionization Time of Flight (SELDI-TOF) to separate proteins associated with mass spectrometry. Bioinformatic tools make it possible to compare protein profiles obtained from diverse biological samples. 4. The combination of these approaches has proved to be particularly interesting for studying cardiovascular diseases and thereby improving our understanding of the mechanisms involved and identifying new biochemical factors and biomarkers involved in these diseases.     

Proteomic response analysis of endothelial cells of human coronary artery to stimulation with carbachol

AIM: To identify the molecular basis of the endothelial target for acetylcholine (ETA). METHODS: Proteomic methods were used to monitor changes in protein expression in the first 10h following the stimulation of human coronary endothelial cells with carbachol 100μmol/L. Thirty proteins showing the largest changes were identified by mass spectrometry. RESULTS: Based on analysis with Image Master 2-D Elite software, about 623 protein spots were detected in control cells and 825 protein spots in carbachol-treated cells, the matching rate was 68.1%. Among all the detected spots, 39 were up-regulated and 29 were down-regulated, showing detectable changes varied from 1.7-3.8 folds. Forty one spots in the peptide mass fingerprints were successfully obtained. The most interesting feature was that all the four newly synthesized proteins belonged to the heat shock protein family.CONCLUSION: These identified proteins played key roles in the molecular mechanism of ETA.     

Carbon and nitrogen isotopic analysis of morphine from opium and heroin samples originating in the four major heroin producing regions

The forensic analysis of stable isotopes is a valuable tool to geo-source natural or semisynthetic drugs such as cocaine and heroin. The present study describes a novel methodology to isolate morphine from opium for isotopic analysis. Furthermore, this isotopic data from regional sources is corroborated with morphine data obtained from seized heroin (deacetylated to morphine) from the same regions. All five primary alkaloids of opium, namely, morphine, codeine, thebaine, noscapine, and papaverine, were quantified using high performance liquid chromatography with photodiode array (PDA) detector before the preparative experiment to gather a complete major alkaloidal profile. Morphine fractions of authentic opium submissions from Mexico, South America, Southwest Asia, and Southeast Asia were isolated and collected using preparative high performance liquid chromatography, and the collected morphine samples were subsequently analyzed by isotope ratio mass spectrometry. Carbon and nitrogen isotope data are presented. The data demonstrate that nitrogen ratios are capable of differentiating samples from Mexico and South America while carbon ratios are able to distinguish Southwest Asian samples from other source regions. Analogous results have routinely been observed (as part of Heroin Signature Program analysis) for morphine obtained from deacetylated authentic heroin samples from the same source regions. The results suggest that the poppy growing region has a greater influence on the carbon and nitrogen isotope values than the heroin manufacturing processes employed. When utilized in conjunction with existing signature methodologies, carbon and nitrogen isotope ratio data can enhance the ability to geo-source heroin.     

Proteomic profiling and protein identification by 2-DE-MS in the liver of Carassius auratus exposed to Yongcheng coalmine subsidence area

Proteomic profile of the Carassius auratus liver was constructed to evaluate contamination degree of aquatic organism exposed to coalmine subsidence area. The results revealed that 55 proteins were up-regulated and 51 down-regulated among 160 differentially expressed proteins. These proteins were mainly involved in the physiological activities, such as granzyme B signaling, dermatan sulfate biosynthesis and cytotoxic T lymphocyte mediated apoptosis of target cells. Meanwhile, they were related to two network interaction modules. One was cell adhesion and migration, angiogenesis, DNA repair, and the other was small molecule metabolism, structural molecule activity and defense response. Ingenuity Pathway Analysis (IPA) was further utilized to predict differential proteins molecular activation, including tnf, tp53, Anxa3, fgf2 and mvp. The result sheds light on liver protein expression profiles of C. auratus, and provides important data for controlling and managing water pollution from heavy metals.     

Proteomic analysis of rat aorta during atherosclerosis induced by high cholesterol diet and injection of vitamin D3

1. Atherosclerosis (AS) in rats displays important clinical similarities to human AS. 2. After the experimental model of AS in rat was established and using a proteomic approach, we compared the protein profiling of aorta tissues from healthy and AS rats. 3. Using two-dimensional electrophoresis (2-DE), over 1878 protein species were separated; among them, 1239 protein spots were matched between different gels with average matching rate of approximately 66%. Gel analysis and protein characterization have identified 58 protein spots whose abundance is significantly altered in AS rats. 4. By using matrix-associated laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and NCBInr database, 46 proteins were successfully identified. Among them, 18 proteins were of increased abundance in diseased tissues including a group of oxidization-related enzymes such as peroxiredoxin2 and NADH dehydrogenase Fe-S protein 6, components of inflammatory pathways such as lamin A, while 28 proteins were of decreased abundance in the diseased state, including CaM-KII inhibitory protein, transferring, fructose-bisphosphate aldolase. 5. We believe that these results would give insights into the cellular and molecular mechanisms involved in AS development and might lead to the discovery of novel diagnostic markers and new therapeutic opportunities.     

超高效液相色谱-串联质谱法测定人血浆中安罗替尼浓度的方法学研究及应用

目的：建立一种快速测定人血浆中安罗替尼浓度的超高效液相色谱-串联质谱法(UPLC-MS/MS)并评估临床应用。方法：以泽布替尼为内标物,血浆用乙腈蛋白沉淀,使用Ultimate XB-C18色谱柱 (100mm × 2.1mm, 3.0μm) 分离,流动相为10mM醋酸胺+0.1%甲酸和乙腈进行梯度洗脱,流速为0.6 mL/min,进样量为5μL。应用电喷雾离子化,正离子模式下用多反应模式监测安罗替尼(m/z 408.1→339.1)和内标物(m/z 472.2→290.1)的浓度,考察该方法学的专属性、定量下限与标准曲线、精密度与回收率、基质效应与稳定性及临床应用。结果：安罗替尼在1.0-100.0ng/ml线性关系良好,标准曲线为y=0.515148x+0.0144461(R₂^=0.9984),精密度RSD小于9%,回收率和基质效应分别是104.81-107.32% 和102.54%-104.26%,该方法稳定性良好、血浆基质对安罗替尼测定结果影响较小。本研究考察了52名服用安罗替尼单药治疗的非小细胞肺癌患者,采集服用安罗替尼治疗第43天的谷浓度血浆,并对其血药浓度进行测定,发现所有患者安罗替尼的血药浓度均高于1.0 ng/mL (最低定量下限),血药浓度均值±标准差是11.38± 4.29ng/mL,变异系数是37.66%,表现出较大的个体间差异。结论：本方法灵敏度高、专属性强、定量准确,适用于人血浆中安罗替尼的浓度监测。     

Comparative proteomic analysis of cellular response of human airway epithelial cells (A549) to benzo(a)pyrene

This work aimed to investigate the cellular response of human airway epithelial cells (A549) to oxidative stress induced by benzo(a)pyrene [B(a)P]. Levels of intracellular reactive oxygen species (ROS) and lipid peroxidation were investigated in A549 cells treated with varying concentrations of B(a)P. A comparative proteomic analysis of total proteins was performed in cells treated with 1 µM B(a)P [B(a)P-1] and untreated cells. The expression of Mn superoxide dismutase (Mn SOD), one of the identified down-regulated proteins in B(a)P-1 cells, was then analyzed by Western blotting. The total antioxidant activity, total superoxide dismutase activity, catalase (CAT) activity, and glutathione reductase (GR) activity were all analyzed after B(a)P treatment. Our results demonstrated that 1 µM B(a)P could induce ROS generation and lead to lipid peroxidation in A549 cells, and 23 differentially expressed proteins were identified. The expression levels of Mn SOD and the total SOD were induced at 0.1 µM and suppressed at 1 µM and 10 µM. Up-regulation of CAT and GR activity resulted in an increase in total antioxidant activity in A549 after exposure to B(a)P. These findings provide a basis for understanding the mechanisms of mitochondrial dysfunction and perturbation of antioxidant status induced by B(a)P on airway epithelial cells.     

人椎间盘纤维环软骨细胞外基质小分子蛋白的双向电泳和质谱鉴定研究

目的初步研究双向电泳和质谱鉴定在人椎间盘纤维软骨的蛋白质组特性研究中的价值和作用,为进一步研究椎间盘疾病的治疗寻找有效途径。方法取人的正常椎间盘的纤维环组织,针对细胞外基质的小分子蛋白进行蛋白萃取,通过固相pH梯度(IPG)等电聚焦和梯度聚丙烯酰胺双向电泳分别分离纤维环的蛋白质,分析电泳图像,使用基质辅助激光解吸附飞行时间质谱仪(MALDI)对蛋白质点进行分析鉴定。结果双向电泳显示,纤维环中的胶原结合蛋白在pH3~10,相对分子质量在22000~250000范围内能够被很好地分离,对各蛋白质点进行质谱仪鉴定,确定了其中的17种蛋白质。结论双向电泳可有效地分离椎间盘纤维环组织中的细胞外基质小分子的胶原结合蛋白质,通过质谱仪鉴定可以确定其组成成分,为进一步研究椎间盘病变中胶原结合蛋白的改变及作用打下基础。     

A combined proteomics and metabolomics approach to assess the effects of gold nanoparticles in vitro

Omics technologies, such as proteomics or metabolomics, have to date been applied in the field of nanomaterial safety assessment to a limited extent. To address this dearth, we developed an integrated approach combining the two techniques to study the effects of two sizes, 5 and 30?nm, of gold nanoparticles (AuNPs) in Caco-2 cells. We observed differences in cells exposed for 72?h to each size of AuNPs: 61 responsive (up/down-regulated) proteins were identified and 35 metabolites in the cell extract were tentatively annotated. Several altered biological pathways were highlighted by integrating the obtained multi-omics data with bioinformatic tools. This provided a unique set of molecular information on the effects of nanomaterials at cellular level. This information was supported by complementary data obtained by immunochemistry, microscopic analysis, and multiplexed assays. A part from increasing our knowledge on how the cellular processes and pathways are affected by nanomaterials (NMs), these findings could be used to identify specific biomarkers of toxicity or to support the safe-by-design concept in the development of new nanomedicines.     

Optimization by factorial analysis of caprylic acid precipitation of non-immunoglobulins from hyperimmune equine plasma for antivenom preparation

Optimization of caprylic acid precipitation of equine plasma non-immunoglobulin proteins for antivenom preparation was achieved by regression analysis of the responses of three highly significant factors assayed by factorial design. The factors studied were caprylic acid concentration, plasma pH and temperature, and their response was assessed in terms of filtration speed, residual albumin, total protein content and turbidity. The results evidenced that the three variables are involved in the precipitation process. Moreover, the factors displayed significant interactions, indicating that their levels distinctly affect the optimization procedure. The best combination was 3% caprylic acid, 37 °C and plasma pH 4.9; under these conditions, all immunoglobulins and only 0.1% albumin remained in the supernatant, in a very fast and simple procedure. After formulation, the antivenom obtained by this procedure presented full lethality neutralizing activity and absence of protein aggregates.