Combining proteomics and lipid analysis to unravel Confidor stress response in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model for studying the influence of different stress factors on eukaryotic cells. In this work we used the pesticide imidacloprid, in the Confidor formulation, as the stress factor and analyzed its influence on the metabolic activity, proteome and lipid content and composition of Saccharomyces cerevisiae yeast. During the cultivation of yeast, the lowest recommended application dose of Confidor (0.025%, v/v) was added to the growth media and its influence on the mitochondria, cytosol with microsomes, and the whole yeast cells was monitored. The results show that under the stress provoked by the toxic effects of Confidor, yeast cells density significantly decreased and the percentage of metabolically disturbed cells significantly increased comparing with untreated control. Also, there was a downregulation of majority of glycolytic, gluconeogenesis, and TCA cycle enzymes (Fba1, Adh1, Hxk2, Tal1, Tdh1,Tdh3, Eno1) thus providing enough acetyl‐CoA for the lipid restructuring and accumulation mechanism since we have found the changes in the cell and mitochondrial lipid content and FA composition. This data suggest that lipids could be the molecules that orchestrate the answer of the cells in the stress response to the Confidor treatment.     

用双向凝胶电泳分析低浓度烷化剂N-甲基-N′-硝基-N-亚硝胍引起的人羊膜FL细胞蛋白质的表达差异

目的　为了研究哺乳类细胞受到低浓度烷化剂攻击后蛋白表达谱的变化。方法　用双向凝胶电泳结合相应的 2 DE分析软件比较烷化剂N 甲基 N′ 硝基 N 亚硝基胍 (MNNG)处理组和二甲基亚砜对照组的FL细胞的蛋白质组的表达差异。结果MNNG处理后的FL细胞中检测到 10个新出现的蛋白点 ,同时有 5个蛋白点在MNNG处理后消失 ;有30个点在表达量上有显著变化 ,其中 16个点在MNNG处理后表达升高 ,另 14个点则表达量降低。结论　在低浓度烷化剂攻击的FL细胞中有一系列蛋白质表达水平的改变 ,提示这些发生改变的蛋白质可能参与了哺乳类细胞非定标性突变的发生。     

Protein expression changes in a cell system of beta-cell maturation reflect an acquired sensitivity to IL-1β

Aim/hypothesis Type 1 diabetes mellitus (T1DM) is caused by specific destruction of the pancreatic beta cells in the islets of Langerhans. Increased sensitivity to cytokines, in particular to interleukin-1 (IL-1) seems to be an acquired trait during beta-cell maturation. In response to cytokines both protective and deleterious mechanisms are induced in beta cells, and when the deleterious prevail, T1DM develops. The aims of this study were to identify perturbation in protein patterns (PiPP) associated with beta-cell maturation, and compare these changes to previous analyses of IL-1 exposed rat islets. For this purpose, proteome analyses were carried out using a cell-line, which matures from a glucagon-producing pre-beta-cell phenotype (NHI-glu) to an insulin-producing beta-cell phenotype (NHI-ins). We have previously shown that this maturation is accompanied by acquired sensitivity to the toxic effects of IL-1.Methods 2D-gel electrophoresis was used to separate the proteins and MALDI-MS and database searches were performed to identify the proteins.Results During beta-cell maturation 135 protein spots out of 2239 detectable changed expression levels. Of these, 74 were down-regulated, 44 up-regulated, 16 were suppressed and 1 was expressed de novo. Using MALDI-MS, positive identification was obtained for 93 out of the 135 protein-spots revealing 97 different proteins. Of these, 22 proteins were in common with changes identified in previous proteome analysis of perturbation in protein pattern in IL-1 exposed rat islets. Several of the proteins were present in more than one spot suggesting post-translational modification.Conclusion/interpretation Several proteins and protein modifications were identified that could be critically involved in beta-cell maturation, insulin-gene expression and the acquired IL-1 sensitivity.Abbreviations T1DM Type 1 diabetes mellitus - PiPP perturbation in protein pattern - NO nitric oxide - iNOS inducible nitric oxide synthase - 2D-GE 2 dimensional gel electrophoresis - IEF isoelectric focusing - NEPHGE non-equilibrium pH-gradient electrophoresis - WF Wistar Furth - BB Bio Breeding - PDX-1 pancreatic duodenum homeobox 1 - ASS argininosuccinate synthetase - HSP heat shock protein - Picot PKC-interacting cousin of thioredoxin - JNK Jun N-terminal kinase - VDAC voltage-dependent anion channel - GST glutathione-S-transferase - Mw molecular weight - pI isoelectric point     

Urinary proteome analysis for prostate cancer diagnosis: cost-effective application in routine clinical practice in Germany

Objectives: Capillary electrophoresis mass spectrometry urinary proteome analysis for prostate cancer has been shown to be highly accurate in the detection of prostate cancer. The aim of the present study was to report our experience with routine application of this test in clinical practice and its cost‐effectiveness. Methods: The urinary proteome analysis for prostate cancer test was carried out in 211 patients in outpatient centers. In 184 of them, data about their follow up and the test results were available for analysis. Prostate cancer was detected in 49 cases. Results: The test correctly recognized 42 out of 49 tumor patients, showing a sensitivity of 86% (95% confidence interval 73–94). Of 135 prostate cancer‐negative patients, 79 had a negative urinary proteome analysis for prostate cancer test (specificity 59% [79/135 95% confidence interval 50–66]). Negative and positive predictive values were 92% (95% confidence interval 84–96) and 43% (95% confidence interval 33–53), respectively. A statistically significant (P < 0.0005) improvement in terms of diagnostic accuracy was observed in comparison with serum prostate‐specific antigen and percent‐free prostate‐specific antigen. Whereas the urinary proteome analysis for prostate cancer test results agreed in 65.7% with follow‐up reference results, prostate‐specific antigen achieved 33.3% and percent‐free prostate‐specific antigen achieved 42.7%. Cost‐effectiveness analysis showed that the urinary proteome analysis for prostate cancer strategy outperformed the biopsy approach as well as prostate‐specific antigen tests. Conclusions: The non‐invasive urinary proteome analysis for prostate cancer test appears to be a helpful addition to prostate cancer diagnostics for patients with suspicious prostate‐specific antigen and/or digital‐rectal examination.     

海洛因成瘾者血浆蛋白质组分析

目的比较鉴定海洛因成瘾者与正常人血浆蛋白质组差异,为研究海洛因成瘾相关血浆蛋白提供线索。方法采集海洛因成瘾者(n=5)和正常对照者(n=5)血浆,剔除白蛋白和免疫球蛋白IgG,以pH4~7胶条等电聚焦为第l向,十二烷基磺酸钠(SDS)聚丙烯酰胺凝胶电泳为第2向,进行蛋白双向电泳。图像分析软件I mage MasterElit5.0分析蛋白质双向电泳图谱。选取组间吸光度体积百分比相差1.5倍以上的蛋白斑点,串联质谱分析鉴定。结果每张图谱平均检测到(563±23)个蛋白(亚基)斑点,其中5个蛋白点在2组图谱中差异1.5倍以上,鉴定结果分别为γ纤维蛋白原(fibrinogen gamma),人α-1B糖蛋白(α-1-Bglycoprotein-human),α-抗胰蛋白酶原(uncleavedα-1-Antitrypsin),视黄醇结合蛋白载体蛋白四聚体单体(chain of transthyretin),铜蓝蛋白(ceruloplasmin)。结论海洛因成瘾者血浆蛋白质组与正常人血浆对比存在差异。某些差异蛋白可能与海洛因成瘾造成的神经损伤相关。     

血清LGT蛋白质组和急性生理学与慢性健康状况评分系统Ⅱ对危重病患者预后评估的临床意义

目的 探讨危重病患者血清LGT蛋白质组的变化规律,分析其对疾病预后评估的临床意义.方法 采用蛋白芯片技术检测96例危重病患者和30例健康对照者的血清蛋白质组变化.测量LGT蛋白质组的丰度值,结合急性生理学与慢性健康状况评分系统Ⅱ(APACHE Ⅱ)分值,分析LGT蛋白质组对危重病患者预后评估的临床意义.结果 危重病患者血清指纹图谱存在LGT蛋白质组表达谱,APACHE Ⅱ评分≥15分组(35例)LGT蛋白质组丰度[(9.26±7.51)%]明显高于APACHE Ⅱ评分＜15分组(61例)的丰度[(4.19±4.07)%],且两组丰度明显高于健康对照组[(1.52±0.47)%],差异有统计学意义(均P＜0.01);患者LGT蛋白质组丰度与APACHE Ⅱ评分呈明显正相关(r=0.317,P=0.002).死亡组(23例)LGT蛋白质组丰度[(10.14±9.23)%]明显高于存活组(73例)的丰度[(5.83±3.57)%,P＜0.01];且LGT蛋白质组丰度≥5%组的病死率[68.0%(17/25)]明显高于丰度＜5%组[8.5%(6/71),P＜0.01].用LGT蛋白质组预测预后的阳性预测率为68.0%,阴性预测率为91.5%;假阳性率为32.0%,假阴性率为8.5%.结论 LGT蛋白质组与病情的严重程度及预后密切相关,可能成为危重病患者预后评估的重要指标;结合APACHE Ⅱ评分系统可为临床早期评估危重病患者预后提供更可靠的依据.

Abstract：

Objective To investigate the expression of serum lost goodwill target(LGT)proteome,and to analyze its clinical significance in evaluating prognosis of patient with critical illness on the basis of acute physiology and chronic health evaluation Ⅱ(APACHE Ⅱ)score. Methods The serum samples were collected from 96 patients with critical illness and 30 healthy volunteers as healthy control. The expression of serum LGT proteome was detected by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry(SELDI-TOF-MS)protein-chip technology. The abundance value of LGT proteome in patients at admission was measured, and at the same time APACHE Ⅱ score was estimated, in order to analyze its clinical significance in patients with critical illness. Results The amount of LGT proteome in APACHE Ⅱ≥15 group[n= 35,(9.26 ± 7. 51)%]was significantly higher than that of APACHE Ⅱ＜ 15 group[n= 61,(4. 19 ± 4.07)%], and the LGT proteome amount in both groups was significantly higher than that of the healthy control group[(1.52± 0.47)%, both P＜0.01]. Spearman correlation analysis showed that there was significant positive correlation between the abundance of LGT proteome and the APACHE Ⅱ score (r=0. 317, P=0. 002). The abundance of LGT proteome in death group[n=23,(10. 14±9. 23)%]was significantly higher than that in survival group[n=73,(5. 83±3.57)%, P＜0. 01]. The fatality rate of the LGT proteome group with average abundance exceeding 5%[68.0%(17/25)]was significantly higher than that of the LGT proteome group with average abundance lower than 5%[8.5%(6/71), P＜0.01].According to the LGT proteome abundance to evaluate the prognosis of the patients, the positive predict rate was 68.0 %, the negative predict rate was 91.5 %, the false positive rate was 32. 0%, the false negative rate was 8.5%. Conclusion The LGT proteome was intimately correlated with the severity degree of disease condition and prognosis in patients with critical illness. The determination of LGT proteome combined with APACHE Ⅱ score evaluation can probably be an important indicator in evaluating the prognosis of patient with critical illness. Further research on LGT proteome is warranted to facilitate the prognostication and clinical decision-making.     

蛋白质组研究中双向聚丙烯酰胺凝胶电泳图谱的计算机分析

目的：建立蛋白质组研究中双向聚丙烯酰胺凝胶电泳（ｔｗｏ－ｄｉｍｅｎｓｉｏｎａｌｐｏｌｙａｃｒｙｌａｍｉｄｅｇｅｌｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ，２－ＤＰＡＧＥ）图谱的计算机分析方法。方法：利用ＩｍａｇｅＭａｓｔｅｒ２－Ｄ分析软件。结果：我们对人肝癌细胞株ＨｅｐＧ２蛋白质组的２－ＤＰＡＧＥ图谱进行了计算机分析，识别了１０００以上的蛋白斑点（其中肉眼可视斑点为４００左右），同时获得了所识别斑点的等电点、相对分子质量、斑点面积、Ｄ值、Ｄ％、斑点体积及相对体积等参数。结论：建立了蛋白质组研究中图像分析体系，为今后蛋白质组２－ＤＰＡＧＥ数据的分析与比较、数据库的建立、蛋白质的功能预测以及大规模斑点的模式识别奠定了基础。