脑瘤患者脑脊液肿瘤相关蛋白的蛋白质组学初步研究

通过比较脑肿瘤患者和正常人脑脊液的二维凝胶电泳图谱（two-dimensional electrophoresis,2DE）,并对差异蛋白进行质谱鉴定,以寻找肿瘤特异脑脊液蛋白。以脑肿瘤患者和正常人的脑脊液为研究对象,采用固相pH梯度（immobilized pH gradient,IPG）2DE分离总蛋白质,凝胶经银染显后,用ImageMaster2D图像分析软件进行比较分析、识别差异表达的蛋白质。结果得到肿瘤患者脑脊液蛋白点924个,正常脑组织蛋白点507个,匹配512个,匹配率分别为55．4％和84．3％,去冗余后发现,有35个蛋白点只在脑肿瘤患者脑脊液图谱中出现。肿瘤患者脑脊液和正常对照脑脊液双向电泳图谱有明显差别,但是本实验尚未对这些差异蛋白进行质谱鉴定,所以未找出肿瘤特异蛋白。     

HCV患者外周血单个核细胞蛋白质表达质谱分析

目的:应用比较蛋白质组学的方法分析丙型肝炎患者外周血单个核细胞蛋白质表达模式的变化及寻找丙型肝炎相关生物标记分子,进一步识别鉴定其差异表达蛋白质,分析其对丙型肝炎慢性化机制的意义.方法:应用固相化pH梯度双向凝胶电泳(2-DE)分离健康者(10)及HCV患者(28)PBMC的总蛋白质,凝胶银染显色后,PDQuest图像分析软件进行比较分析、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)获得差异蛋白点的肽质指纹图谱,通过SWISS-PROT数据库鉴定蛋白质.结果:得到两张2-DE图谱,HCV患者及健康者PBMC凝胶的蛋白质点数分别为625及614;初步筛选出HCV患者与健康者存在明显差异的12个蛋白点,经质谱分析,初步鉴定了10种蛋白质.这些差异蛋白质包括病毒蛋白、蛋白质合成与分解、三大代谢相关酶类、细胞结构相关蛋白质以及信号转导相关蛋白质.结论:应用2-DE及MALDI-TOF-MS方法建立了HCV患者PBMC双向凝胶电泳图谱,分离并初步鉴定了10种与HCV感染相关的差异表达的蛋白质,为研究HCV慢性化相关机制提供新的线索.     

肝细胞癌中差异表达N-连接糖蛋白的分析鉴定

目的分析鉴定与肝细胞癌(HCC)发生发展相关的差异表达的N-连接糖蛋白。方法应用刀豆蛋白凝集素(ConA)、晶状体凝集素(LCH)和雪花凝集素(GNA)组成的亲合层析柱富集10对HCC和癌旁非癌组织中N-连接糖蛋白、二维电泳(2DE)比较分析差异表达的蛋白质点、串联质谱鉴定差异表达蛋白,Western blotting验证人羧酸酯酶1(hCE1)、触珠蛋白(HP)及组织蛋白酶D(CD)的差异表达;体外侵袭实验检测CD表达沉默后对HCC的侵袭力影响。结果质谱、生物信息学等技术鉴定了28个与HCC相关的差异表达蛋白(HCC中高表达和低表达的蛋白均为14个),Western blotting验证hCE1、HP在HCC中明显低表达,组织蛋白酶D前体(pCD)在HCC中高表达;而ConA亲和层析柱富集的ConA-CD在HCC中显著高表达。CD-siRNA介导的CD表达沉默能够显著降低肝癌细胞系SNU449、SNU473的体外侵袭能力。结论 HP、hCE1蛋白表达变化和CD N-糖链变异可能参与了HCC发生发展过程。     

蛋白质磷酸酶2A癌性抑制因子在非小细胞肺癌组织中的表达及其临床意义

目的分析蛋白质磷酸酶2A癌性抑制因子在非小细胞肺癌组织中的表达及其临床意义。方法对220例非小细胞肺癌患者的癌变组织以及癌旁组织均实施免疫化学法检验,分析比较癌变组织以及癌旁组织中蛋白质磷酸酶2A癌性抑制因子(CIP2A)的表达情况。结果癌变组织的CIP2A阳性表达率(76.36%)显著高于癌旁组织(5.00%),P0.05。结论蛋白质磷酸酶2A癌性抑制因子可在非小细胞肺癌组织中高度表达,具有较为显著的临床意义。     

应用蛋白质组学分析结直肠癌促泌素和糖调节蛋白78蛋白变化及其意义

目的分析结直肠癌、配对正常黏膜组织差异表达蛋白质,寻找与结直肠癌发生、发展相关的分子标记物。方法固相pH梯度双向凝胶电泳分离结直肠癌患者配对癌及正常黏膜组织的总蛋白,液相色谱串联质谱鉴定差异表达蛋白质点。半定量逆转录聚合酶链反应（RT—PCR）、Western blot及免疫组织化学（EnVision法）方法检测促泌素（SCGN）及糖调节蛋白78（GRP78）两个差异表达蛋白质及组织定位,并检测54例神经内分泌肿瘤SCGN的表达。结果比较结直肠癌、配对正常黏膜组织蛋白质表达图谱,共获得5倍以上差异蛋白质点35个,癌组织中表达上调15个、下调20个。质谱分析鉴定14个蛋白质。RT-PCR及Western blot证实结直肠癌组织SCGN低表达,免疫组织化学染色发现正常结直肠黏膜神经内分泌细胞及98％（53／54）的神经内分泌肿瘤SCGN阳性。GRP78蛋白在结直肠癌组织中的表达明显高于正常黏膜组织,但在mRNA水平上癌组织和正常组织之间差异无统计学意义。结论双向凝胶电泳结合质谱分析是筛选肿瘤异常表达蛋白质的有效手段,所获得的35个差异表达候选蛋白质可能与结直肠癌的发生、发展有关。SCGN是一个候选神经内分泌标记物,GRP78蛋白表达增高可能涉及翻译后修饰。     

慢性间歇性缺氧大鼠肾脏组织蛋白质组的变化

目的:比较慢性间歇性缺氧大鼠与正常大鼠肾脏组织蛋白质双向电泳图谱,寻找和鉴定慢性间歇性缺氧大鼠肾脏组织中的差异蛋白表达谱。方法:以固相pH梯度等电聚焦为第一向和垂直SDS-PAGE为第二向,分别对正常对照大鼠和慢性间歇性缺氧大鼠的肾脏组织蛋白质样品进行二维分离,2-DE图谱经ImageMaster 2D Platinum V5.0软件分析,选取4个差异蛋白点用基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)进行鉴定。结果:通过对2-DE图谱蛋白斑点的匹配及对比分析,与慢性间歇性缺氧相关的差异表达蛋白斑点为112个;经质谱鉴定的4个差异表达的蛋白斑点,慢性间歇性缺氧组量下调的差异点1个,即线粒体ATP合成酶δ亚基;上调的差异点3个,分别为己糖激酶、儿茶酚-O-甲基转移酶、脱嘌呤/脱嘧啶核酸内切酶/氧化还原因子-1。结论:大鼠肾脏组织慢性间歇性缺氧损伤相关的差异蛋白表达变化可能通过多种途经影响肾脏功能。     

肝细胞癌患者血清蛋白质组成分的双向凝胶电泳-飞行时间质谱分析

目的　分析肝细胞癌患者与正常人血清双向凝胶电泳的差异蛋白质 ,寻找肝癌早期血清学诊断的可能指标。方法　固相 pH梯度双向凝胶电泳分离肝细胞癌患者及正常人血清总蛋白。银染显色 ,PDQuest 2DE软件分析 ,对差异蛋白质点用基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)测定其胶内酶解后的肽质指纹图谱 ,用PepIdent软件查询SWISS PROT数据库。 结果　获得了重复性较好的双向电泳银染图谱。图像分析检测 3块胶平均匹配的点数占平均蛋白点数的匹配率是 70 2 %。等电聚焦方向的平均偏差为 (1 0 2± 0 2 2 )mm ,十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)方向的平均偏差为 (0 97± 0 14 )mm。将 2 3个差异点进行胶内原位酶解 质谱指纹图分析 ,获得 15张肽质指纹图 ,查询数据库进行了初步鉴定。结论　固相 pH梯度双向凝胶电泳分离人血清总蛋白获得重复性较好的结果 ,但仍需进一步探索如何去除高丰度已知蛋白及脂类等物质的方法 ,以便得到分辨率更高的双向电泳银染图谱。MALDI TOF MS肽质指纹图分析鉴定的结果为人肝细胞癌血清学诊断指标的筛选提供了依据     

胃癌不同转移能力细胞系的2-DE图谱及差异分析

目的建立胃癌低转移能力细胞系RF1和高转移能力细胞系RF48的2DE图谱,并分离胃癌转移相关蛋白质。方法利用固相pH梯度技术(immobilizedpHgradient,IPG)双向凝胶电泳(twodimensioanlpolyacrylamidegelelectrophoresis,2DE)建立胃癌低转移能力细胞系RF1和高转移能力细胞系RF48二维电泳图谱;采用图像分析软件进行比较分析,识别差异表达的蛋白质,分离胃癌转移相关蛋白质。再利用基质辅助激光解析电离飞行时间质谱(MALDITOFMS)及数据库查询,对部分差异表达蛋白质点进行鉴定。结果建立胃癌低转移能力细胞系RF1和胃癌高转移能力细胞系RF48双向凝胶电泳图谱;三块凝胶的平均蛋白质点数分别为960±92和978±83,平均匹配的点数分别为780±69和769±53,匹配率达87%和86.3%;对25个差异蛋白质点进行肽质指纹图分析,鉴定出与瘤基因、细胞周期调控和信号转导有关的蛋白质。结论建立分辨率高且重复性较好的人胃癌低转移能力细胞系RF1和胃癌高转移能力细胞系RF48的双向凝胶电泳图谱,可以识别和鉴定出一些与胃癌转移相关的差异表达蛋白质。     

应用RNA-seq技术检测肺癌与癌旁组织差异表达基因分析

目的应用RNA-Seq技术分析肺癌与癌旁组织的差异基因表达,寻找与肺癌发病相关的基因,探讨肺癌的发病机制。方法对5例ⅡA期肺癌及癌旁组织进行转录组测序,获得差异表达基因集,采用qRT-PCR法检测10例肺癌(对照组)及癌旁组织中的6个关键基因(TFPI2、DKK1、CX3CL1、PSCA、DUOXA2、VSIG1)进行验证。结果ⅡA期肺癌与癌旁组织共有123个差异表达基因,其中上调基因35个(28.5%),下调基因88个(71.5%)。qRT-PCR结果表明,与细胞增殖、凋亡和黏附相关基因TFPI2、DKK1、CX3CL1表达上调,PSCA、DUOXA2、VSIG1表达下调,该检测与RNASeq检测结果一致。结论ⅡA期肺癌与癌旁组织中共有123个差异表达基因,细胞增殖、凋亡、黏附和代谢相关基因参与肺癌的发生、发展,为肺癌的发病机制提供参考。     

增生性瘢痕与正常皮肤的蛋白质组学研究

目的 比较增生性瘢痕与正常皮肤组织的蛋白质组表达差异,筛选出增生性瘢痕的特异蛋白质.方法 以中山大学附属第一医院烧伤外科2010年1月至5月8例增生性瘢痕患者的增生性瘢痕组织和3例整形外科患者正常躯干皮肤为研究对象,运用蛋白质组学技术、双向凝胶电泳对比分析增生性瘢痕组织和正常皮肤组织中蛋白表达差异,选择差异蛋白点进行MALDI - TOF/TOF质谱分析和生物学信息分析.结果 获得重复性较好的双向电泳荧光染色图谱,增生性瘢痕和正常皮肤组织凝胶电泳图谱中平均蛋白点分别为2975和3053,对其中表达差异超过4倍的蛋白点进行质谱分析和数据库检索,共鉴定出24种蛋白质,其中上调蛋白有11种,下调蛋白有13种.结论 成功地建立增生性瘢痕的双向电泳荧光染色图谱,鉴定出增生性瘢痕组织与正常皮肤组织间的差异蛋白质表达.     

以双向凝胶电泳为基础的食管癌组织蛋白质组学分析新策略

目的：探讨采用双向电泳为基础的蛋白质组学的研究手段在食管癌组织与食管癌旁组织差异分析中的应用。方法：双向凝胶电泳分离食管癌及其癌旁组织的蛋白质，图像分析软件ImageMaster4．01分析所获得的凝胶图谱并寻找差异点，用质谱仪鉴定差异点蛋白质。结果：提出了一套可行的癌组织差异分析策略，并发现在分子量50 000-60 000，等电点5—6的区域，食管癌组织和食管癌旁组织存在2个差异表达的蛋白，质谱鉴定为丙酮酸激酶M2/M1。结论：在合理的策略指导下，以双向凝胶电泳为基础的蛋白质组学技术是研究肿瘤的一种重要手段，我们得到的丙酮酸激酶M2/M1在食管癌与其癌旁组织中存在差异表达，可能对食管癌的诊断、治疗具有重要意义。     

Differential proteomic analysis of nuclear matrix in muscle-invasive bladder cancer: potential to improve diagnosis and prognosis.

INTRODUCTION: Although several molecular markers for bladder cancer have been identified, at present little information on prognostic biomarkers is available in the literature. Prognostication of this tumor is largely based on clinicopathological characteristics. Our aim was to identify nuclear matrix (NM) proteins that might serve to better characterize the phenotype of the invasive bladder cancer and to investigate their diagnostic and prognostic roles. METHODS: NM proteins expressed in normal (n=3) or non-tumoral (n=9) tissue specimens and muscle-invasive bladder cancer (n=21) specimens were analyzed by two dimensional (2D) gel electrophoresis. PDQuest image analysis software was used to generate a comparative NM proteome analysis. Selected spots were characterized by liquid chromatography coupled to tandem mass spectrometry and Western blot. RESULTS: We detected over 800 protein spots in each 2D map and 43 spots were identified. 30 proteins were differentially expressed by bladder tumor cells; among these, 19 proteins were detected in bladder tumoral tissues but not in normal and non-tumoral tissues and seven proteins correlated with tumor stage. One protein (p54nrb) was strongly correlated with vascular invasions and appeared to be also significantly (P<0.0001) associated with a decreased probability of survival. CONCLUSION: Important alterations in NM proteins occur in muscle-invasive bladder cancer. The differentially expressed proteins include biomarkers potentially useful for disease diagnosis, progression and prognosis. Our findings beyond improving the understanding of the biology of bladder cancer, could help to stratify patients into different prognostic subgroups and to select those who might be better candidate to multimodal therapeutic approaches.     

Elevated expression of complement C3 protein in chemically induced hepatotumorogenesis in Wistar rats: A correlative proteomics and histopathological study

Liver cancer remains the leading cause of cancer-related mortality worldwide. Early detection of liver cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The present study was designed to determine the differently expressed proteins at early stage in the serum of animals with liver cancer vis-à-vis controls and figure out the function of the proteins. One-dimensional electrophoresis (1D), two-dimensional electrophoresis (2DE) and liquid chromatography mass spectrometry (LC–MS/MS) were used to screen the serum proteins of liver cancer induced in animals by diethyl nitrosamine (DEN) + 2-acetyl amino fluorine (2-AAF). From optimized 2DE image and computer assisted PD Quest analysis were found to be differentially expressed spots when the serum from normal and treated animals were compared. Among these, one spot was selected whose expression level was higher in DEN + 2-AAF treated animal sera than in adjacent normal animal sera. The target spot was excised from the 2D gel of liver cancer sera and the peptide mass fingerprinting as obtained LC–MS/MS analysis after digesting the chosen protein spot. This was identified to be complement C3 protein. The changes in complement C3 expression level were validated by Western blot analysis. We reported that the changes in complement C3 concentration start at very early stage of tumorogenesis. The fully grown tumors were developed at 120 days and hepatotumorogenesis was confirmed by histopathological examination. This protein may therefore represent a powerful tool in search for candidate biomarkers for HCC.     

H22肝癌小鼠血清差异蛋白质的质谱鉴定

目的 分析正常小鼠和H22肝癌小鼠血清之间差异表达的蛋白并对部分差异蛋白进行鉴定分析,筛选可能与肝癌发生密切相关的蛋白质.方法 利用双向凝胶电泳技术（2-DE）与基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）,分析正常组和肿瘤组筛选并鉴定部分差异表达的蛋白,再进行生物学分析.结果 通过2-DE图谱研究发现,与正常组相比,肿瘤组存在明显差异的蛋白质点.选择其中可能为关键功能蛋白的蛋白点进行质谱分析,获得肽指纹图谱（PMF）,运用Mascot软件查询NCBI数据库分析并鉴定得出其中2个蛋白：假尿苷合成酶和线粒体核糖体大亚基蛋白.结论 肝癌的发生机制与假尿苷合成酶和线粒体核糖体大亚基蛋白2个差异表达蛋白密切相关,其参与肝癌的发生发展.     

First proteomic analysis of Legionella pneumophila based on its developing genome sequence

The first proteomic analysis of the respiratory pathogen Legionella pneumophila ATCC 33152 is presented in this report. Two-dimensional gel electrophoresis of total cell extracts was carried out. In total, 130 protein spots were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MS) or by quadruple time-of-flight tandem MS, including proteins correlated with virulence. For the first time, proteins of L. pneumophila were identified using mass spectrometric methods and mapped on a two-dimensional gel; this will be of considerable use for comparison of protein expression profiles of L. pneumophila wild-type and knock-out mutant strains and of L. pneumophila grown under different conditions.     

Matrix-assisted laser desorption/ionization mass spectrometry reveals decreased calcylcin expression in small cell lung cancer

To date, most of the proteomic analyses on lung cancer tissue samples have been performed using surgical specimens, which are obtained after a diagnosis is made. To determine if a proteomic signature obtained from bronchoscopic biopsy samples could be found to assist with diagnosis, 50 lung cancer bronchoscopic biopsy samples and 13 adjacent normal lung tissue samples were analyzed using histology-directed, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Lung tissue samples were cryosectioned, and sinapinic acid was robotically deposited on areas of each tissue section enriched in epithelial cells, either tumor or normal. Mass spectra were acquired using a MALDI-time of flight instrument. Small cell lung cancers (SCLCs) demonstrated clearly different protein profiles from normal lung tissue and from non-small cell lung cancers (NSCLCs). Calcyclin (m/z= 10,094.7) was identified to be underexpressed in small cell lung cancers, as compared with non-small cell lung cancers and normal lung tissue. An immunohistochemistry study using 152 NSCLCs and 21 SCLCs confirmed significantly reduced calcyclin stain in SCLCs. Thus, protein profiles obtained from bronchoscopic biopsy samples via MALDI MS distinguish cancerous epithelium from normal lung tissue and between NSCLCs and SCLCs.