应用SELDI-TOF-MS技术分析慢性乙肝患者血清差异表达蛋白

【目的】应用表面加强激光解析离子化飞行时间质谱(surface enhanced laser desorption-ionization time-of-flight mass spectrometry,SELDI-TOF-MS)技术分析慢性乙肝患者血清差异表达蛋白。【方法】选择临床明确诊断的慢性乙肝患者和正常健康体检者各12名,利用弱阳离子芯片CM10经质谱分析血清中差异表达蛋白。【结果】样品血清中共获得与芯片结合的67个有效蛋白质峰。其中相对分子质量4107、4980、5650、5923道尔顿的4个蛋白峰有差异,相对分子质量为4980道尔顿的蛋白峰在慢性乙肝组高表达。经检索,发现相对分子质量为4980道尔顿的蛋白与细胞色素B相对分子质量相符。【结论】利用SELDI-TOF-MS技术寻找慢性乙肝患者无创性血清学指标是一种可行的方法,细胞色素B可能在慢性乙肝形成中起一定作用。     

结肠癌组织蛋白质组成分的双向凝胶电泳分析

目的 分析结肠癌病人癌组织、癌旁组织及正常结肠黏膜组织双向凝胶电泳的蛋白质表达图谱,寻找差异表达的蛋白质.方法 采用固相pH梯度双向凝胶电泳分离结肠癌病人配对癌组织、癌旁组织及正常结肠黏膜组织的总蛋白,银染显色,PDQuest 2DE软件分析差异表达的蛋白质点.结果 获得了重复性较好的双向凝胶电泳银染图谱.图像分析显示,3组胶的平均匹配率分别为:癌组织87.3%、癌旁组织80.3%、正常结肠黏膜组织84.0%.等电聚焦方向上的平均偏差为(1.16±0.29)mm,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)方向上的平均偏差为(1.00±0.38)mm.差异分析共获得164个差异表达的蛋白质点.结论 双向凝胶电泳分离结肠癌组织的总蛋白可获得重复性较好的结果.获得的差异蛋白质点为寻找结肠癌早期诊断的分子标记物提供了理论依据.     

肺脏缺血预处理的蛋白质组学研究

目的 研究大鼠肺组织缺血预处理（IP）的蛋白质组变化。方法 20只SD大鼠，随机分为预处理组和对照组，每组10只。常规开胸后建立左上肺缺血再灌注模型，全部大鼠均遭受了缺血再灌注损伤（IRI），但预处理组在缺血再灌注前给予IP干预，对所有样本总蛋白质进行二维凝胶电泳分离，差异表达的蛋白质点进行基质辅助激光解吸电离飞行时间质谱分析后Mascot软件搜索SWISS—PROT和NCBI数据库进行鉴定。结果 本研究建立了分辨率较高、重复性较好的鼠肺缺血再灌注损伤的双向凝胶电泳参考图谱，45个蛋白质点表达存在差异，其中包括巯基特异性抗氧化荆（TSA）和6个热休克蛋白家族在内的30个蛋白质点得到了鉴定。结论 蛋白质组学是研究IRI和IP肺脏保护机制的很有前途的工具。TSA在IP对肺脏IRI的保护机制中起重要作用。     

卵巢癌患者血清中肿瘤特异标志物的鉴定研究

目的:通过分析卵巢癌患者血清中的蛋白质组分,寻找可能的肿瘤相关蛋白并对该蛋白鉴定,以期确定其作为卵巢癌血清学诊断的特异标志物。方法:采用双向凝胶电泳方法分离6例卵巢癌患者及2例正常健康妇女血清总蛋白,经考马斯亮蓝染色后对比找出差异蛋白斑点,该蛋白斑点经蛋白酶水解后进行液质联用串联质谱(LC-MS/MS)分析,用SEQUEST搜索软件查询Finngan公司提供的FASTA格式蛋白序列数据库。采用WesternBlotting、ELISA、斑点印迹及亲和层析等方法对目标蛋白作进一步的分析。结果:经双向电泳分离,卵巢癌患者血清中有3-4个组分显示含量高于正常健康妇女血清,选其中最明显的一个差异斑点进行质谱分析,对库检索确定为结合珠蛋白。进一步的分析证实,卵巢癌患者血清中结合珠蛋白总含量有高于正常健康妇女的趋势。此外,通过凝集素斑点印迹法检测发现,岩藻糖基化结合珠蛋白成分明显增加是该蛋白总量增高的主要特性。结论:检测血清结合珠蛋白总量以及特异的岩藻糖基化组分可能成为卵巢癌的血清诊断指标之一,可与其它检测方法相结合,提高卵巢癌早期诊断的有效性。     

蛋白质组学与中医药现代化研究

结合中医药的历史发展与现状,说明在探讨复杂生命现象时,蛋白质组学技术在对机体的整体性认识与中医理论上的认识有一定的趋同性,显示了两者结合的必然性和重要性,从蛋白质组学在揭示中医证候研究、中药作用机理研究、中药药效评价方面的作用,说明将蛋白质组学技术应用于中医药现代化研究,对于加快传统中医药与现代生命科学技术的结合,推动中医药现代化、国际化必将产生重大影响。     

平肝潜阳法对偏头痛肝阳上亢型大鼠模型肾上腺组织蛋白质组的影响

目的探讨平肝潜阳法对偏头痛肝阳上亢型大鼠模型作用的分子机制。方法用电刺激SD大鼠三叉神经节的方法并灌服附子汤制造偏头痛肝阳上亢型模型。采用双向凝胶电泳（2-DE）技术分离大鼠肾上腺组织的总蛋白，图像分析识别差异表达的蛋白质点，基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）获得相应的肽质量指纹图（PMF），搜索数据库鉴定蛋白质。结果得到分辨率及重复性较好的正常对照组、偏头痛肝阳上亢型大鼠、中药治疗组大鼠肾上腺组织的2-DE图谱，质谱分析鉴定得到其中的8个差异蛋白质点。结论平肝潜阳药物可能通过调节多种与能量代谢有关的蛋白和泛素的表达而起到治疗偏头痛肝阳上亢证的作用。     

结直肠癌原发灶和肝转移灶组织中蛋白质组差异表达及临床意义

目的探讨结直肠癌发生和肝转移相关蛋白及其与肝转移的关系。方法采用等电聚焦／SDS聚丙烯酰胺双向凝胶电泳法,对比分析结直肠癌原发灶、癌旁肠黏膜和肝转移灶中蛋白质组表达差异,经质谱分析、鉴定差异蛋白点;采用细胞转染方法,将差异蛋白基因导入到结直肠癌细胞,观察细胞生物学行为改变。结果结直肠癌原发灶、肝转移灶与正常肠黏膜蛋白质组分在PH7．0—9．5范围内有明显差别。分析13个差异蛋白点,其中2个蛋白点在癌原发灶组织中表达下调,5个蛋白点在原发灶组织中表达上调,6个蛋白点在肝转移组织中表达上调。经质谱鉴定,碳酸酐酶Ⅱ（CAⅡ）在癌组织中表达下调,磷酸甘油酸激酶Ⅰ、延胡索酸水合酶、醛缩酶A在原发灶中表达上调。精氨酸酶,谷胱甘肽S-转移酶A3在肝转移灶中表达上调。将碳酸酐酶ⅡcDNA克隆转染HR-8348细胞后,癌细胞侵袭、趋化运动能力及耐药性均明显减弱。结论结直肠癌原发灶和肝转移灶蛋白质组表达有显著差异,碳酸酐酶Ⅱ表达下调和精氨酸酶、谷胱甘肽S-转移酶A3增强表达可使结直肠癌细胞生物学行为发生改变并促进肝转移发生。     

SELDI-MS-based expression profiling of ductal invasive and lobular invasive human breast carcinomas

Expression profiling using proteomic techniques has a great potential to identify new biomarkers that might help to better diagnose and treat diseases such as breast cancer, which is one of the leading causes of cancer death in women. Surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS) combines chromatographic separation of peptides and proteins with mass spectrometry and is a fast, user-friendly tool to analyze protein and peptide profiles. SELDI-MS was employed for a comparative analysis of lobular invasive versus ductal invasive breast tumors to find differentially expressed proteins and peptides, and to validate this technique for biomarker identification using complex samples such as tissue. After optimization of sample preparation using HMEC and MCF-7 cell lines, 20 breast tumors were analyzed, and about 550 mass signals corresponding to an estimated 140 native peptides and proteins were detected in each tumor. Only 14% of the mass signals were present in more than six tumors of one subgroup or in more than 12 tumors of both groups showing a great overall heterogeneity of the peptide and protein profiles obtained. Peptide mass signals specific for each of the analyzed groups were identified. In addition, we detected peptides from laser-microdissected ductal invasive and intraductal tumor parts corresponding to peptides present in whole tumors. The low amount of identified peptides and proteins and the observed heterogeneity suggest that SELDI-MS is not well suited for biomarker identification of and profiling experiments on complex samples such as tumor tissue.     

Analytical assessment of MALDI-TOF Imaging Mass Spectrometry on thin histological samples. An insight in proteome investigation

BACKGROUND: The development of MALDI Imaging Mass Spectrometry is a promising technique in the investigation of biological molecular repertoire. We have pursued an analytical assessment of this technique in its application to proteome analysis. METHODS: A specific statistical method of analysis has been developed to enable data processing in the absence of internal standards, by defining similarity scores. RESULTS: The investigated linear mode MALDI-TOF set-up allows to obtain data variations comprised within the 30% of variation when assaying tissues samples from the same animal, while the 60% of variation was highlighted in the inter-mice series assaying syngenic animals. CONCLUSIONS: This analytical assessment represents the first step of a process that should validate the utilisation of this technique in the clinical practice.     

Proteomic identification of the involvement of the mitochondrial rieske protein in epilepsy

PURPOSE: Kindled seizures are widely used to model epileptogenesis, but the molecular mechanisms underlying the attainment of kindling status are largely unknown. Recently we showed that achievement of kindling status in the Sprague-Dawley rat is associated with a critical developmental interval of 25 +/- 1 days; the identification of this long, well-defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relation to normal and pathologic brain function. METHODS: By using proteomics on cerebral tissue from our new rat kindling model, we undertook a global analysis of protein expression in kindled animals. Some of the identified proteins were further investigated by using immunohistochemistry. RESULTS: We report the identification of a modified variant of the Rieske iron-sulfur protein, a component of the mitochondrial cytochrome bc1 complex, whose isoelectric point is shifted toward more alkaline values in the hippocampus of kindled rats. By immunohistochemistry, the Rieske protein is well expressed in the hippocampus, except in the CA1 subfield, an area of selective vulnerability to seizures in humans and animal models. We also noted an asymmetric, selective expression of the Rieske protein in the subgranular neurons of the dorsal dentate gyrus, a region implicated in neurogenesis. CONCLUSIONS: These results indicate that the Rieske protein may play a role in the response of neurons to seizure activity and could give important new insights into the molecular pathogenesis of epilepsy.