联合应用激光捕获显微切割技术及乙酰化稳定同位素标记定量技术在结直肠癌蛋白质组学研究中的价值

目的应用蛋白质组学技术,建立结直肠癌蛋白质组差异表达谱,筛选结直肠癌发生发展相关差异蛋白质。方法应用激光捕获显微切割技术,获取20例结直肠腺癌及配对的正常黏膜、20例绒毛状腺瘤、8例结直肠癌肝转移灶的靶细胞;然后应用基于乙酰化稳定同位素标记的定量蛋白质组学策略寻找差异蛋白。结果获得具有定量信息的高可信度蛋白873个。其中腺癌与正常黏膜、腺瘤及肝转移灶之间差异显著的蛋白质(定量比值≥2或≤0.5)分别为137、119和77个。结直肠癌与正常黏膜之间的差异蛋白在定位上几乎分布于细胞的各个部位。结论联合应用激光捕获显微切割技术及乙酰化稳定同位素标记定量技术进行结直肠癌蛋白质组学研究,发现的差异蛋白功能上参与多种生物学过程,表明本研究所采取的技术策略有很好的蛋白鉴定覆盖率,可成为研究肿瘤蛋白质组学的有效策略。     

SELDI—TOF—MS在结直肠癌血清蛋白质组学中的应用

蛋白质组学为结直肠癌的许多研究领域开拓了新的视野,表面增强激光解析电离飞行时间质谱（SELDI—TOF—MS）技术推动了其在结直肠癌血清蛋白质组学研究中的应用。在针对结直肠癌早期诊断、判断分期、监测治疗效果等方面进行了积极的探索。随着这一技术的成熟,它对结直肠癌的诊断、治疗具有广阔的应用前景。     

SELDI-TOF-MS在结直肠癌血清蛋白质组学中的应用

蛋白质组学为结直肠癌的许多研究领域开拓了新的视野,表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术推动了其在结直肠癌血清蛋白质组学研究中的应用.在针对结直肠癌早期诊断、判断分期、监测治疗效果等方面进行了积极的探索.随着这一技术的成熟,它对结直肠癌的诊断、治疗具有广阔的应用前景.     

结直肠癌化疗敏感性相关蛋白的蛋白质组学初步研究

目的:本研究应用蛋白质组学技术建立化疗敏感性不同的结直肠癌组织总蛋白双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)图谱,并鉴定部分差异表达蛋白,以发现与结直肠癌化疗敏感性有关的蛋白.方法:收集临床诊断为晚期结直肠癌的病例,肠镜活检获取新鲜结直肠癌标本后液氮保存备用,根据肿瘤药物敏感实验分为化疗高敏感组和化疗低敏感组.提取组织总蛋白,采用双向凝胶电泳技术得到各组凝胶图谱;采用PD-quest 7.3软件进行图像的合成、对比和差异分析,识别化疗高敏感组和化疗低敏感组之间差异表达的蛋白斑点;选取差异蛋白质点,胶内酶解后行肽指纹图分析及网上数据库检索,鉴定差异蛋白质;应用Western印迹法检测部分差异蛋白的表达情况.结果: 建立了化疗高敏感组和化疗低敏感组的双向凝胶电泳图谱,多数蛋白质点集中于pH 4～8、相对分子质量为(20～100)×10~3.高敏感组和低敏感组的电泳图谱中平均蛋白质点数分别为(842±23)个和(793±19)个,2组平均匹配率为90.7%,2组间差异表达蛋白质点数为(79.00±13.56)个;选择30个差异蛋白质点进行质谱分析,经数据库查询鉴定出9个差异表达蛋白.结论:在化疗敏感性不同的结直肠癌中存在蛋白质表达的差异,这些差异表达蛋白可能与化疗敏感性有关,并可能用于化疗敏感性的预测.     

蛋白质组学研究及其在肿瘤诊断和治疗中的应用

蛋白质组学是研究蛋白质组成和动态变化的一门新兴学科，蛋白质组学研究技术的主要步骤包括样品制备、双向凝胶电泳(2-DE)、蛋白质的鉴定技术及相应的信息技术和数据库4大方面。目前，蛋白质组学研究已应用于膀胱癌、乳腺癌、结肠癌、食管癌、卵巢癌和白血病等恶性肿瘤的早期诊断及治疗疗效的评价。     

蛋白质组学研究及其在肿瘤诊断和治疗中的应用

蛋白质组学是研究蛋白质组成和动态变化的一门新兴学科，蛋白质组学研究技术的主要步骤包括样品制备、双向凝胶电泳（2－DE）、蛋白质的鉴定技术及相应的信息技术和数据库4大方面。目前，蛋白质组学研究已应用于膀胱癌、乳腺癌、结肠癌、食管癌、卵巢癌和白血病等恶性肿瘤的早期诊断及治疗疗效的评价。     

蛋白质组学及其在肿瘤研究中的应用

蛋白质组学技术如样品制备、双向电泳(2-DE)、凝胶染色、质谱分析、生物信息学、差异凝胶电泳(DIGE)、同位素亲和标签(ICAT)、蛋白质芯片等的研究取得较大进展,蛋白质组学在探讨肿瘤发病机制及诊断等方面得到广泛应用.现就蛋白质组学相关技术研究进展及其在肿瘤研究中的应用作一综述.     

蛋白质组技术在大肠癌生物标志物筛选中的应用

蛋白质组学是研究蛋白质的组成和动态变化的一门新兴学科,蛋白质组技术主要包括双向凝胶电泳(2-DE)、质谱(MS)、蛋白质芯片及蛋白质信息技术和数据库.目前,蛋白质组技术已成为研究肿瘤发生发展机制及寻找肿瘤标志物的重要工具.现就蛋白质组技术在分离、鉴定大肠癌生物标志物的应用进展作一介绍.     

蛋白质组学及其在肿瘤研究中的应用

蛋白质组学技术如样品制备、双向电泳(2-DE)、凝胶染色、质谱分析、生物信息学、差异凝胶电泳(DIGE)、同位素亲和标签(ICAT)、蛋白质芯片等的研究取得较大进展，蛋白质组学在探讨肿瘤发病机制及诊断等方面得到广泛应用。现就蛋白质组学相关技术研究进展及其在肿瘤研究中的应用作一综述。     

蛋白质组学在乳腺癌研究中的应用

蛋白质组学在肿瘤研究中的应用是近几年的研究热点,许多新方法也不断应用于蛋白质组学的研究中,包括组织芯片、蛋白质芯片、差异显示双相凝胶电泳以及最新的表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)蛋白质芯片技术,这些新方法在乳腺癌的早期诊断、预后评价以及信号转导等方面都取得一定进展。     

Heterogeneity of sialoglycoproteins purified from normal and malignant cells

We have identified previously a group of sialoglycoproteins with an apparent molecular weight of 110,000, which appear homogeneous by sodium dodecyl sulfate:polyacrylamide gel electrophoresis but exhibit isoelectric point heterogeneity by two-dimensional polyacrylamide gel electrophoresis. This technique demonstrated that there are multiple glycoproteins of similar molecular weight which differ between normal cells and transformants. We have now purified glycoproteins by concanavalin A:Sepharose chromatography and preparative polyacrylamide gel electrophoresis from both a nontumorigenic normal mouse fibroblast line, A31, and a highly malignant nonimmunogenic transformant, 3T12T. Differences in the isoelectric point distribution of the sialoglycoproteins which were observed between the normal and transformed two-dimensional gel patterns using crude membranes could also be demonstrated with the purified glycoproteins. Treatment of the isolated sialoglycoproteins with neuraminidase to remove sialic acid resulted in significant isoelectric point shifts but did not eliminate all of the heterogeneity. Even following neuraminidase treatment, the purified glycoprotein fraction upon isoelectric focusing showed differences in patterns between normal and transformed cells. Preliminary characterization of the alterations seen in the two cell lines are presented and show decreased fucose and increased sialic acid in transformed cell glycoprotein.     

Two-dimensional gel electrophoresis of cellular proteins reveals myeloid origin of blasts in two children with otherwise undifferentiated leukemia

In previous studies, two-dimensional polyacrylamide gel electrophoresis allowed the detection of 11 polypeptide markers that distinguished between subtypes of acute lymphoblastic leukemia and between acute lymphoblastic and acute myelocytic leukemia. This report describes the occurrence of polypeptide markers in two-dimensional gels that indicate a myeloid origin of blasts obtained from two children who presented with acute leukemia, the cells of origin of which could not be determined, at the time of diagnosis, by morphologic, cytochemical, or immune marker analysis. The authors conclude that two-dimensional electrophoresis provides a new tool for the delineation of the cell of origin in acute leukemia.     

Two-dimensional electrophoresis analysis of differential protein expression in squamous carcinoma of the cervix

Objective： To establish and optimize the two-dimensional gel electrophoresis （2-DE） maps of squamous carcinoma of the cervix and to study the protein difference between squamous carcinoma of the cervix （SCC） and normal cervical tissue. Methods： Using Two-dimensional gel electrophoresis followed by computer-assisted image analysis, the differential proteins between squamous carcinoma of the cervical tissue and normal cervical tissue were compared. Then using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the differential proteins were identified. Results： The well-resolved and reproducible two-dimensional gel electrophoresis patterns of squamous carcinoma of the cervix tissue and normal cervical tissue were obtained. After silver staining, the average matching ratio of squamous carcinoma of the cervix was 86.1%. There was a good reproducibility of spot position in 2-DE map, with average deviation in IEF direction of 0.95±0.13 mm, while in SDS-PAGE direction it was 1.20±0.18 mm. Ten protein spots were identified by mass spectrometry, some of which were involved in cell proliferation, cell apoptosis, intracellular enzymes, structural proteins, cycle regulation, and tumor occurrence. Conclusion： The differentially expressed proteins provide a fundamental basis for further study of human squamous carcinoma of the cervix and screening of its specific markers.     

Proteomic phenotyping: metastatic and invasive breast cancer

The adriamycin resistant breast cancer cell line (MCF-7/ADR) is a subject of ongoing debate concerning its origin and or source. Previous studies in our laboratory showed that MCF-7/ADR has a unique cytosolic protein expression pattern when compared to that of the parental MCF-7 cell line and other drug resistant MCF-7 cell lines. Protein expression patterns obtained using two-dimensional gel electrophoresis and mass spectrometry indicated that this MCF-7/ADR cell line shares some similarities with the metastatic breast cancer cell lines MDA-MB. Further comparisons with available two-dimensional gel electrophoresis maps in the literature indicate that MCF-7/ADR has a protein expression signature even closer to of the ductal infiltrating breast carcinoma cell line 8701. These observations suggest that MCF-7/ADR cells might have originated in a selection of ductal infiltrating carcinoma cells, which were present among the original MCF-7 cell population. These ductal infiltrating carcinoma cells may possess an intrinsic adriamycin resistance phenotype.     

Comprehensive mutational scanning of the p53 coding region by two- dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long- distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li- Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.      

Distinctive protein pattern in two-dimensional electrophoretograms of cancerous prostatic tissues

In this report, we describe methods used to analyze the protein composition of sectioned frozen prostatic tissues by two-dimensional gel electrophoresis. Our results show a high degree of homology in two-dimensional electrophoretograms of proteins extracted from frozen sections of malignant prostate glands. Such homology was not apparent in protein patterns of benign hypertrophic prostatic tissue sections. Typically, 600 discrete proteins were resolved on two-dimensional electrophoretograms and 9 proteins were present in all patterns of prostate adenocarcinomatous tissues. These nine proteins were not observed in any of the protein electrophoretograms developed from nonmalignant prostate tissue. Three proteins were found common to nonmalignant prostate glands but were not present in prostatic adenocarcinoma.     

A reference map of human nasopharyngeal squamous carcinoma proteome

In order to conduct a comparative proteomics study of human nasopharyngeal carcinoma (NPC) to understand the molecular mechanisms that participate in the formation of NPC, the two-dimensional gel electrophoresis (2-DE) reference map of human NPC tissue proteome was described. To provide a high level of reproducibility between gels and accurately array each protein expressed in NPC tissue proteome, the two-dimensional polyacrylamide gel electrophoresis system, modified colloidal Coomassie Brilliant Blue staining method and ImageMaster 2D Platinum image analysis software were used. The NPC 2-DE maps show that high quality and good reproducibility of the 2-DE gel pattern was attained. An average total of 1,100 protein spots were separated by 2-DE, visualized by a modified colloidal Coomassie Brilliant Blue staining method. A synthesized 2-DE reference gel was acquired after detailed analysis of the NPC 2-DE gel maps, and 216 medium to high abundant spots were identified as landmark spots of NPC 2-DE gel, which expressed on >75% of gels. To provide an unambiguous identification of the landmark spots in gels, MALDI-TOF, ESI-Q-TOF mass spectrometry and database search were used to identify the proteins expressed in NPC tissue proteome. Between the 216 landmark spots, all proteins were identified with MALDI-TOF at first, 41 of which were identified with both MALDI-TOF and ESI-Q-TOF. All identified proteins were classified in terms of their subcellular localization and physiological function with information from SWISS-PROT and NCBI websites. According to our knowledge this is the first 2-DE reference map of human NPC. This reference map will serve as a basis for further studies of human NPC and the reference map data will be used to expand the proteome database of human NPC, which can be accessed in our website (http://www.xyproteomics.org/).     

A novel role for human sulfiredoxin in the reversal of glutathionylation

Modification of protein cysteine residues by disulfide formation with glutathione (glutathionylation) is a reversible posttranslational modification of critical importance in controlling cell signaling events following oxidative and/or nitrosative stress. Here, we show that human sulfiredoxin, a small redox protein conserved in eukaryotes, can act as a novel regulator of the redox-activated thiol switch in cells by catalyzing deglutathionylation of a number of distinct proteins in response to oxidative and/or nitrosative stress. Actin and protein tyrosine phosphatase 1B were identified in vitro as targets of sulfiredoxin 1 (Srx1)-dependent deglutathionylation and confirmed in vivo by two-dimensional gel electrophoresis analysis. In addition, we show that Srx1-dependent deglutathionylation is functionally relevant through restoration of phosphatase activity. Human sulfiredoxin contains one cysteine residue (Cys(99)) that is conserved in all family members. Mutation of the cysteine residue inhibits deglutathionylation but did not affect its capacity to bind intracellular proteins. Furthermore, sulfiredoxin is not an acceptor molecule for the GS(-) moiety during the reaction process. Using two-dimensional gel electrophoresis, we identified multiple protein targets in vivo that are deglutathionylated by sulfiredoxin following oxidative and/or nitrosative stress. This novel deglutathionylation function of sulfiredoxin suggests it has a central role in redox control with potential implications in cell signaling.     

Stem cells and proteomics

With the accomplishment of the draft of human genome project and the sequencing of several decades of organisms and the adventure of post-genomics, proteomics, an important subject of post-genomics, has been applied in many fields such as biology, oncology and medicine, etc. People’s attention has gradually transferred from revealing the genetic information to discovering the functions of the genetic materials. In consideration of the limited number of genes and the relative stability of thei…     

Carboquone alters the protein synthesis of Chinese hamster V79 cell

The effect of carboquone (CQ) on protein synthesis in Chinese hamster V79 cells was determined. While the syntheses of higher molecular weight proteins decreased, the relative level of a 43-kilodalton (kd) protein increased following exposure to CQ at the concentration of 0.5 microgram/ml, in a time-related fashion, determined using one-dimensional gel electrophoresis. Using two-dimensional gel electrophoresis, the 43-kd protein may be actin protein, and the syntheses of another two proteins of 8.5/45 and 4.8/270 (designated isoelectric point/molecular weight, kd) were increased in the CQ-treated V79 cells. These changes of the proteins induced by CQ in V79 cells were noted in HeLa cells. Three CQ-induced proteins are candidates for the elucidation of the antineoplastic effect of CQ.