Proteomic analysis on metastasis-associated proteins of human hepatocellular carcinoma tissues

Purpose: A comparative proteomic approach was used to identify and analyze proteins related to metastasis of hepatocellular carcinoma (HCC). Methods: Proteins extracted from 12 HCC tissue specimens (six with metastases and six without) were separated by two-dimensional gel electrophoresis (2-DE). The protein spots exhibiting statistical alternations between the two groups through computerized image analysis were then identified by mass spectrometry. In addition immunohistochemistry (IHC), Western blotting and RT-PCR were performed to verify the expression of certain candidate proteins. Results: 16 proteins including HSP27, S100A11, CK18 were annotated by mass spectrometry, relevant to chaperone function, cell mobility, cytoskeletal architecture, respectively. Most were previously unconnected with metastasis of HCC. Of these HSP27 was found overexpressed consistently in 2-DE patterns of all metastatic HCC tissues compared with nonmetastatic ones. IHC and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. Conclusion: There are various proteins joined together in HCC metastasis. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets unique to the metastatic phenotype of HCC.     

Differential proteomic analysis of human hepatocellular carcinoma cell line metastasis-associated proteins

PURPOSE: The comparative study of differentially expression of protein profiles of hepatocellular carcinoma cell lines with various metastasic potential and screening key molecules related to hepatocellular carcinoma metastasis and recurrence. METHODS: Using two-dimensional electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), we analyzed differentially displayed proteomics of human hepatocellular carcinoma cell lines Hep3B, MHCC97L, MHCC97H with different metastasic potential. RESULTS: Approximate 1,000 protein spots were detected on silver-stained gel by ImageMaster (977+/-113 spots in Hep3B, 1092+/-40 in MHCC97L, and 889+/-14 in MHCC97H). Fifty distinct different protein spots were analyzed with online LC-ESI-MS/MS. Only 26 protein spots had a positive result, including annexin1, S100A4, and so on. In comparison with nonmetastasis Hep3B cell lines, there were 16 proteins overexpressed in MHCC97H and MHCC97L, 10 proteins underexpressed in MHCC97H and MHCC97L. Applying cell immunohistochemistry and RT-PCR, we further validated two interesting and different proteins, annexin1 and S100A4. CONCLUSION: The protein profile of metastatic hepatocellular carcinoma cell lines displayed obvious differences compared with non-metastatic liver cancer cell lines. The results imply that various different proteins may lead to HCC metastasis together.     

Comparative proteomics analysis of human gastric cancer

AIM： To isolate and identify differentially expressed proteins between cancer and normal tissues of gastric cancer by two-dimensional electrophoresis （2-DE） and matrix-assisted laser desorption/ionization time-of- flight mass spectrometry （MALDI-TOF-MS）. METHODS： Soluble fraction proteins of gastric cancer tissues and paired normal tissues were separated by 2-DE. The differentially expressed proteins were selected and identified by MALDI-TOF-MS and database search. RESULTS： 2-DE profiles with high resolution and reproducibility were obtained. Twenty-three protein spots were excised from sliver staining gel and digested in gel by trypsin, in which fifteen protein spots were identified successfully. Among the identified proteins, there were ten over-expressed and five under-expressed proteins in stomach cancer tissues compared with normal tissues. CONCLUSION： In this study, the well-resolved, reproducible 2-DE patterns of human gastric cancer tissue and paired normal tissue were established and optimized and certain differentially-expressed proteins were identified. The combined use of 2-DE and MS provides an effective approach to screen for potential tumor markers.     

Proteomic analysis of gastric cancer and immunoblot validation of potential biomarkers

AIM: To search for and validate differentially expressed proteins in patients with gastric adenocarcinoma.METHODS: We used two-dimensional gel electrophoresis and mass spectrometry to search for differentially expressed proteins in patients with gastric adenocarcinoma. A set of proteins was validated with immunoblotting.RESULTS: We identified 30 different proteins involved in various biological processes: metabolism, development, death, response to stress, cell cycle, cell communication, transport, and cell motility. Eight proteins were chosen for further validation by immunoblotting. Our results show that gastrokine-1, 39S ribosomal protein L12 (mitochondrial precursor), plasma cell-induced resident endoplasmic reticulum protein, and glutathione S-transferase mu 3 were significantly underexpressed in gastric adenocarcinoma relative to adjacent non-tumor tissue samples. On the other hand, septin-2, ubiquitin-conjugating enzyme E2 N, and transaldolase were significantly overexpressed. Translationally controlled tumor protein was shown to be differentially expressed only in patients with cancer of the gastric cardia/esophageal border.CONCLUSION: This work presents a set of possible diagnostic biomarkers, validated for the first time. It might contribute to the efforts of understanding gastric cancer carcinogenesis.     

Proteome Analysis of Human Pancreatic Ductal Adenocarcinoma Tissue Using Two-Dimensional Gel Electrophoresis and Tandem Mass Spectrometry for Identification of Disease-Related Proteins

A comparative proteomic approach has been used to identify and analyze proteins related to pancreatic cancer. Proteomes of eight pairs of clinical pancreatic ductal adenocarcinoma (PDAC) tissue samples and samples of normal adjacent tissue were obtained by two-dimensional gel electrophoresis (2DE). Comprehensive analysis of proteins was focused on total protein spots for which there were statistical differences between the two groups. Proteins were identified by peptide mass fingerprinting with tandem mass spectrometry (MS–MS). Western blotting and immunohistochemistry (IHC) were also performed to verify the expression of some candidate proteins. Thirty protein spots were identified, including proteases, antioxidant proteins, signal-transduction proteins, calcium-binding proteins, structural proteins, chaperones, and others. Western blotting and IHC confirmed up-regulated expression of two candidate proteins, nucleotide diphosphatase kinase (NDPK) and annexin II, in tumorous tissues. These results suggest that combination of 2DE with MS is an effective strategy for discovery of differently expressed proteins in PDAC which may be molecular markers for diagnosis or therapeutic targets. Rui Tian and Li-Ming Wei contributed equally to this work     

Identification of proteins of human colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

AIM: To conduct the proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOFMS). METHODS: The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis of the tryptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were used to search NCBI, SWISS-PROT and MSDB databases by using Mascot software. RESULTS: PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified. CONCLUSION: The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established. Protein expression profile of SW480 has been obtained. It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis.     

Identification of differential proteins in colorectal cancer cells treated with caffeic acid phenethyl ester

AIM: To investigate the molecular mechanisms of the anti-cancer activity of caffeic acid phenethyl ester (CAPE).METHODS: Protein profiles of human colorectal cancer SW480 cells treated with or without CAPE were analysed using a two-dimensional (2D) electrophoresis gel-based proteomics approach. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250. Digital images were taken with a GS-800 Calibrated Densitometer, and image analysis was performed using PDQuest 2-D Analysis software. The altered proteins following CAPE treatment were further identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry following a database search. The identified proteins were validated by Western blot and immunofluorescence assay.RESULTS: CAPE induced human colorectal cancer cell apoptosis. Four up-regulated proteins and seven down-regulated proteins in colorectal cancer cells treated with CAPE were found. The identified down-regulated proteins in CAPE-treated colorectal cancer cells were Triosephosphate Isomerase (Tim), Proteasome subunit alpha 4 (PSMA4) protein, Guanine nucleotide binding protein beta, Phosphoserine aminotransferase 1 (PSAT1), PSMA1, Myosin XVIIIB and Tryptophanyl-tRNA synthetase. Notably, CAPE treatment led to the down-regulation of PSAT1 and PSMA1, two proteins that have been implicated in tumorigenesis. The identified up-regulated proteins were Annexin A4, glyceraldehyde-3-phosphate dehydrogenase, Glucosamine-6-phosphate deaminase 1 (GNPDA1), and Glutathione peroxidase (GPX-1). Based on high match scores and potential role in cell growth control, PSMA1, PSAT1, GNPDA1 and GPX-1 were further validated by Western blotting and immunofluorescence assay. PSMA1 and PSAT1 were down-regulated, while GNPDA1 and GPX-1 were up-regulated in CAPE-treated colorectal cancer cells.CONCLUSION: These differentiated proteins in colorectal cancer cells following CAPE treatment, may be potential molecular targets of CAPE and involved in the anti-cancer effect of CAPE.     

人肝癌细胞膜上HSP70的表达

用流式细胞仪和Cell-ELISA检测了 4株人肝癌细胞膜上HSP70的表达。结果表明正常情况下人肝癌细胞膜上HSP70的表达较低 ,经热处理后表达增加 (P <0 0 1)并显示出细胞之间的差异 (P <0 0 1)。推测其低表达可能与肝癌细胞逃逸机体免疫监视有关 ,对制定肝癌的防治对策有重要的指导意义。     

人肝细胞性肝癌组织转移相关分子的比较蛋白质组学研究

目的 应用比较蛋白质组学方法研究肝细胞性肝癌(HCC)转移相关的关键蛋白分子。方法 用双向凝胶电泳(2DE)对有转移的HCC组织和未发生转移的HCC组织总蛋白进行分离，两组间差异蛋白点用质谱和数据库搜索鉴定，并在蛋白质和mRNA水平上进一步检测验证。结果 鉴定出l6个差异表达蛋白，包括S100钙结合蛋白(S100)、热休克蛋白27(HSP27)、细胞角蛋白18(CK18)等。对在转移性HCC组织中表达显著增高的HSP27，应用western blot分析在蛋白水平上验证了2DE结果，RT-PCR检测出两组问mRNA水平也存在差异，但不显著，免疫组织化学检测显示HSP27主要定位于肝细胞胞浆内。结论 HCC转移与多种蛋白表达相关，其中HSP27高表达可能在转移中发挥作用，可能为潜在的判断HCC转移的分子标记或控制转移的治疗靶点。     

MALDI-TOF-MS分析新疆食管鳞状细胞癌的蛋白组学

目的:分析新疆食管鳞状细胞癌和食管正常上皮细胞的差异表达蛋白.方法:运用激光捕获显微切割技术(LCM)分别获取食管鳞状癌细胞和食管正常上皮细胞,应用二维凝胶电泳技术(2-DE)分离纯化细胞,Imagemaster 2D软件比较分析两者电泳图谱的差异,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定分析两者表达的差异蛋白.结果:建立了食管鳞状癌细胞和食管正常上皮细胞的2-DE图谱,获得43个差异蛋白点,通过质谱鉴定出17种蛋白,其中15种蛋白如Trangelin2、HSP27、S100A11、GSTP等在食管鳞状癌细胞中表达明显增高,2种蛋白如SCCA1,在食管鳞状癌细胞中表达明显降低.结论:提示17种差异蛋白可能与食管鳞状细胞癌的发生和发展有关,为筛选食管鳞状细胞癌的特异性分子标志物奠定基础.     

大肠癌潜在标志物-热休克蛋白27

目的:探讨大肠癌特异性标志物,为大肠癌的早期诊断、预后判断和治疗提供帮助,同时为理解大肠癌的发病机制提供线索.方法:收集6例大肠癌患者,应用高灵敏的二维凝胶电泳和MALDI-TOF-MS技术检测出肿瘤黏膜和邻近正常结肠黏膜之间差异表达的蛋白.对其中之一热休克蛋白27(HSP27)进行Western blot和免疫组织化学验证.结果:筛选出42个具有明显表达差异的蛋白质点,质谱鉴定出10个差异表达蛋白,包括HSP27、二硫异构酶、核不均一核糖核蛋白A2/B1、磷酸丙糖异构酶、丙酮酸激酶等.Western blot和免疫组织化学结果证实HSP27在大肠癌中有过度表达,提示其可能为重要的肿瘤标志物.结论:大肠癌肿瘤组织与大肠正常黏膜之间存在差异表达蛋白,HSP27在大肠癌中有异常表达,可能作为大肠癌发生、发展的候选生物标志物.     

Proteomic analysis of hippocampus in the rat

Objective To analyze the protein expression in the rat hippocampus by the proteomic approach. Methods Proteins from hippocampal tissue homogenates of the rat were separated by two-dimensional gel electrophoresis (2-DE), and stained with colloidal Coomassie blue to produce a high-resolution map of the rat hippocampus proteome. Selected proteins from this map were digested with trypsin, and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) The mass spectrometric data were used to identify the proteins through searches of the NCBI protein sequence database. Results 37 prominent proteins with various functional characteristics were identified. The identified brain protein classes covered metabolism enzymes, cytoskeleton proteins, heat shock proteins, antioxidant proteins,signalling proteins, proteasome-related proteins, neuron-specific proteins and glial-associated proteins. Furthermore,3 hypothetical proteins, unknown proteins so far only proposed from their nucleic acid structure, were identified.Conclusion This study provides the first unbiased characterization of proteins of the rat hippocampus and will be used for future studies of differential protein expression in rat models of neurological disorders.     

不同转移潜能肝癌细胞株的差异蛋白质组的二维液相色谱分析

目的:对不同转移潜能肝癌细胞株MHCC97- H(高转移)和MHCC97-L(低转移)差异表达的蛋白质进行二维液相色谱分离和MALDI-TOF质谱鉴定.方法:将肝癌细胞株MHCC97-H和MHCC97- L细胞裂解样品按蛋白质PI进行一维的色谱聚焦分离,然后每个PI组分再按疏水性经二维反相无孔硅胶HPLC分离,利用ProteoVue软件将UV光吸收图谱转换成PI对疏水性的胶图,再利用DeltaVue软件比较升高或降低的差异蛋白.收集差异蛋白峰进行胰酶酶解,然后进行MALDI-TOF质谱鉴定.结果:2D图谱显示共有72个差异蛋白条带,共鉴定出了9个差异蛋白.分别为M2型丙酮酸激酶、ATP合成酶α亚单位、热休克蛋白60、Toll样受体9、含黄素单加氧酶、钙网硬蛋白前体、锰超氧化物岐化酶、nm23-H1、G-蛋白偶连受体激酶5;其中4个蛋白在高转移细胞株MHCC97-H中表达升高,5个蛋白在低转移细胞株MHCC97-L表达升高.结论:这些差异蛋白可能在肝癌的转移中起关键作用.     

吲哚美辛抗人结肠癌细胞系HCT116的功能蛋白质组研究

目的　探讨吲哚美辛 (IN)对人结肠癌细胞系HCT116蛋白质表达谱的影响及其抗结肠癌的作用机制。方法　应用固相 pH梯度双向凝胶电泳 (2 DE)技术分离IN治疗组和对照组HCT116细胞的总蛋白 ,银染显色 ,采用ImageScan扫描获取电子图像 ,ImageMaster 2D图像分析软件分析 2组的2 DE图谱并识别差异表达的蛋白质。应用基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)得到相应的肽质量指纹图谱 ,分析并鉴定差异蛋白质。结果　获得了背景清晰、分辨率高、重复性好的HCT116细胞系 2 DE图谱 ,对照组蛋白质点数为 12 99± 5 5 ,其匹配率为 94 .2 % ;IN治疗组蛋白质点数为 12 0 8± 4 7,其匹配率为 91.0 %。两组共有 4 5个差异蛋白质点 ,其中 ,治疗组有 9个蛋白质点表达上调 ,34个蛋白质点表达下调 ,2个蛋白质点仅在对照组表达。对差异蛋白质点进行了肽质指纹图分析发现 ,IN可通过BfL 1蛋白诱导HCT116细胞凋亡 ,并鉴定出一些与细胞凋亡、细胞周期及信号转导相关的蛋白质。结论　IN可通过BfL 1等多种途径诱导HCT116细胞的凋亡并抑制其增殖。     

不同转移潜能人肝癌细胞系酪氨酸磷酸化蛋白质差异分析

目的研究不同转移潜能人肝癌细胞系中酪氨酸磷酸化蛋白质表达的差异并筛查与肝癌转移相关的酪氨酸磷酸化蛋白质分子。方法应用双电泳(2-D E)、免疫印迹法及基质辅助激光解析电离飞行时间质谱分析,对三种不同转移潜能人肝癌细胞系Hep 3B、MHCC97L和MHCC97H进行酪氨酸磷酸化蛋白质组分析。结果对照2-DE胶和免疫印迹发光胶片,Hep3B检测到10个点,MHCC 9 7L检测到19个点, MHCC97H检测到17个点。经质谱鉴定,得到膜连蛋白Ⅰ等19个差异点。结论细胞内酪氨酸磷酸化蛋白的表达差异与肝癌的侵袭转移有关。     

中国大陆日本血吸虫地理株间成虫蛋白质组分的差异

目的分离、鉴定中国大陆日本血吸虫不同地理株间成虫差异表达蛋白。方法分别收集、制备日本血吸虫不同地理株成虫可溶性蛋白,经固相pH梯度双向凝胶电泳分离,凝胶银染,并用PDQuest8.0凝胶图像分析软件进行比较分析,筛选出差异表达蛋白点并采用基质辅助激光解析离子飞行时间质谱仪进行鉴定。结果湖南株、江西株和江苏株雄虫分别检出698±10、650±19、629±23个蛋白点,雌虫分别检出670±12、682±22、625±28个蛋白点,约90%蛋白点分子量处于20～90kD范围内,蛋白等电点在5～8之间。不同地理株雌虫与雄虫分别两两比较,雄虫发现14个差异点,雌虫发现18个差异点。对其中7个雄虫差异表达蛋白及3个雌虫差异表达蛋白经MALDI-TOF-MS鉴定分析,获得其肽质量指纹图谱、等电点、分子量等相关信息。7个雄虫差异表达蛋白分别为:SJCHGC00821蛋白、SJCHGC00475蛋白、3-磷酸甘油醛脱氢酶、谷胱甘肽转移酶片段、谷胱甘肽-S-转移酶、D-核酮糖-5-磷酸-3-表异构酶、G蛋白α亚基AgGq1,3个雌虫表达蛋白分别为:预测蛋白、GAF型传感器信号转导组氨酸激酶、组织蛋白酶B样半胱氨酸蛋白酶前体。结论中国大陆日本血吸虫不同地理株虫体间蛋白质存在差异,部分蛋白表达差异与虫体对吡喹酮敏感性密切相关。     

Heat shock protein 70 chaperoned alpha-fetoprotein in human hepatocellular carcinoma cell line BEL-7402

AIM: To investigate the interaction between heat shock protein 70 (HSP70) and a-fetoprotein (AFP) in human hepatocellular carcinoma (HCC) cell line BEL-7402. METHODS: The expression and localization of HSP70 and AFP in human HCC cell line BEL-7402 were determined by immunocytochemistry and indirect immunofluorescence cytochemical staining. The interaction between HSP70 and AFP in HCC cells was analyzed by immunoprecipitation and Western blot. RESULTS: Immunocytochemical staining detection showed that HCC cell BEL-7402 expressed a high level of HSP70 and AFP synchronously. Both were stained in cell plasma. AFP existed in the immunoprecipitate of anti-HSP70 mAb, while there was HSP70 in the immunoprecipitate of anti-AFP mAb. CONCLUSION: HSP70 chaperones AFP in human HCC cell BEL-7402. The interaction between HSP70 and AFP in human HCC cell can be a new route to study the pathogenesis and immunotherapy of HCC.     

Comparative proteomic analysis of differentially expressed proteins in human pancreatic cancer tissue

BACKGROUND:Pancreatic cancer is one of the most common malignant tumors.Early diagnosis of pancreatic cancer is difficult because of the latent onset and lack of good biomarkers.This study aimed to look for and identify differentially expressed proteins in tissues of pancreatic cancer and adjacent noncancerous tissues by proteomic approaches so as to provide information about possible pancreatic cancer markers and therapeutic targets.METHODS:Proteins extracted from 3 paired adjacent noncancerous and cancero...