Proteomic analysis shows differential protein expression in endothelial progenitor cells between healthy subjects and ischemic stroke patients

Abstract

Objective: An increase in the circulating concentration of endothelial progenitor cells (EPCs) is associated with a better outcome in patients with acute ischemic stroke. Likewise, EPCs are heterogeneous cells, with functional differences and different protein expressions. Our objective was to compare protein expressions of EPCs from ischemic stroke patients and healthy subjects.

Methods: Eleven ischemic stroke patients and 11 healthy subjects, matched by age and gender, were included in this study. EPC colonies were defined as early outgrowth colony forming unit-endothelial cell. Cells were lysed and proteins were purified and separated on two-dimensional gels. Gel images were analyzed using the PDQuest software and protein differences between EPCs from ischemic stroke patients and healthy subjects were identified by mass spectrometry. Results were finally validated by western blot.

Results: Proteomic analysis revealed three qualitative differences between EPCs from healthy subjects and ischemic stroke patients. Two of them, endoplasmatic reticulum protein-29 and CdC-42, were only expressed in EPCs from healthy subjects, whereas elongation factor-2 was only identified in EPCs from ischemic stroke patients. Furthermore, we identified one protein, peroxiredoxin-1, whose expression was 10 times stronger in ischemic stroke patients than in healthy subjects. Western blot analysis showed greater expression of endoplasmatic reticulum protein-29 in EPCs from healthy subjects and elongation factor-2 and peroxiredoxin-1 in EPCs from ischemic stroke patients.

Conclusion: Proteomic analysis showed differences in protein expressions of EPCs from ischemic stroke patients and healthy subjects that may be involved in mechanisms related to functional impairment.     

吡那地尔后处理大鼠缺血再灌注损伤心肌线粒体的蛋白质组学研究

 目的: 运用蛋白质组学研究方法探讨吡那地尔后处理对缺血再灌注损伤大鼠心肌的保护作用。方法: 建立Langendorff大鼠离体心脏缺血再灌注损伤模型,随机分为吡那地尔后处理组(Pina组)和缺血再灌注损伤组(I/R组),每组9只。提取心肌线粒体蛋白质行双向凝胶电泳,应用质谱鉴定差异大于2倍的蛋白质点。结果: Pina组与I/R组比较,共发现7个差异蛋白质:Pina组NADH脱氢酶1α亚复合体10亚基(NDUFA10)﹑NADH脱氢酶铁硫蛋白2(NDUFS2)和NADH脱氢酶黄素蛋白2(NDUFV2)表达低于I/R组;Pina组异柠檬酸脱氢酶α亚基(IDHA)和Δ^3,5-Δ^2,4-二烯酰辅酶A异构酶(ECH1)表达高于I/R组;另有2个蛋白质点均被鉴定为ATP合酶δ亚基,一个蛋白质点表达升高,而另一个表达降低。结论:吡那地尔后处理可能抑制了复合体Ⅰ的亚基(NDUFA10﹑NDUFS2和NDUFV2)代偿性增加,但促进了IDHA和ECH1表达并引发了ATP合酶δ亚基发生磷酸化,这些改变可能均与吡那地尔后处理保护心肌的作用有关。     

寻常型银屑病（湿热证）的血清蛋白组学表达研究

【目的】应用同位素相对标记与绝对定量技术（iTRAQ技术）对寻常型银屑病（湿热证）患者的血清蛋白进行定量分析,探求其与正常人的差异性表达蛋白质,从蛋白组学角度揭示湿热证的部分内涵。【方法】取寻常型银屑病（湿热证）患者（15例）及正常人（10例）血清,予纯化、去丰度等处理后,十二烷基硫酸钠－聚丙烯酰胺凝胶（SDS-PAGE）电泳分离,经iTRAQ特异性标记酶解后的肽段,采用串联质谱分析鉴定其差异蛋白。【结果】共鉴定出787个蛋白质组,具有生物信息学分析（gene ontology , GO）功能注释的蛋白质共有718个;与正常人比较发现了651个显著性差异蛋白（P<0.05）,其中有强显著性差异的蛋白418个（P<0.01）。【结论】寻常型银屑病（湿热证）患者血清中存在与正常人有差异性表达蛋白,但哪种蛋白在辨别寻常型银屑病“湿热证”中处于关键地位,以及湿热症状与蛋白之间的关系等,还有待进一步研究。     

细粒棘球蚴囊液蛋白组分分析

目的　利用鸟枪法分析细粒棘球蚴（Echinococcus granulosus）囊液蛋白（hydatid cyst fluid protein,HCFP）组分,寻找其中具有潜在调控免疫失调性疾病功能的活性组分。方法　无菌收集细粒棘球蚴病患者肝囊肿中的细粒棘球蚴囊液,采用鸟枪法进行液相色谱⁃串联质谱鉴定,采用MaxQuant 1.6.1.0软件进行数据库检索。利用基因本体论（Gene Ontology,GO）对已鉴定的蛋白质从细胞组分、分子功能和生物学功能三个方面进行分类。结果　经十二烷基磺酸钠⁃聚丙烯酰胺凝胶电泳（SDS⁃PAGE）分离的细粒棘球蚴囊液蛋白条带相对分子量为25～70 kDa,质谱分析共获得37个蛋白,其中已鉴定蛋白32种、未知蛋白5种。已初步发现至少有4种蛋白具有潜在调节免疫失调性疾病作用,即抗原B、谷胱甘肽S⁃转移酶、硫氧还蛋白过氧化物酶和苹果酸脱氢酶。GO富集分析显示,已鉴定蛋白具有149种分子功能,参与341种生物学过程。结论　细粒棘球蚴囊液蛋白成分复杂多样,从已知蛋白中初步筛选出4种与调节免疫失调性疾病潜在相关的蛋白。     

Undifferentiated pelvic adenocarcinomas: diagnostic potential of protein profiling and multivariate analysis

Background and aims Despite improved techniques, the determination of tumor origin in poorly differentiated adenocarcinomas still remains a challenge for the pathologist. Here we report the use of protein profiling combined with principal component analysis to improve diagnostic decision-making in tumor samples, in which standard pathologic investigations cannot present reliable results. Materials and methods A poorly differentiated adenocarcinoma of unknown origin located in the pelvis, infiltrating the sigmoid colon as well as the ovary, served as a model to evaluate our proteomic approach. Firstly, we characterized the protein expression profiles from eight advanced colon and seven ovarian adenocarcinomas using two-dimensional gel electrophoresis (2-DE). Qualitative and quantitative patterns were recorded and compared to the tumor of unknown origin. Based on these protein profiles, match sets from the different tumors were created. Finally, a multivariate principal component analysis was applied to the entire 2-DE data to disclose differences in protein patterns between the different tumors. Results Over 89% of the unknown tumor sample spots could be matched with the colon standard gel, whereas only 63% of the spots could be matched with the ovarian standard. In addition, principal component analysis impressively displayed the clustering of the unknown case within the colon cancer samples, whereas this case did not cluster at all within the group of ovarian adenocarcinomas. Conclusion These results show that 2-DE protein expression profiling combined with principal component analysis is a sensitive method for diagnosing undifferentiated adenocarcinomas of unknown origin. The described approach can contribute greatly to diagnostic decision-making and, with further technical improvements and a higher throughput, become a powerful tool in the armentarium of the pathologist. UJ Roblick and FG Bader contributed equally to this work and should be recognized as first authors.     

The future of proteomics in the study of alcoholism

This article represents the proceedings of a workshop at the 2003 annual meeting of the Research Society on Alcoholism in Fort Lauderdale, FL. The workshop organizers/chairpersons were Chinnaswamy Kasinathan and Paul Manowitz. The presentations were (1) Introduction to the field of proteomics, by Kent Vrana; (2) Use of proteomics in the identification of urinary biomarkers for alcohol intake, by Chinnaswamy Kasinathan, Paul Thomas, and Paul Manowitz; (3) Proteomics screening illuminates ethanol-mediated induction of HDL proteins in macaques, by Kent Vrana, Randy Gooch, Travis Worst, Stephen Walker, Aaron Xu, Peter Pierre, Heather Green, and Kathleen Grant; and (4) Proteomics applied to the study of the liver, by Laura Beretta.