快速老化模型小鼠海马蛋白质组学初步研究

应用双向凝胶电泳结合质谱鉴定, 分析比较6月龄和12月龄快速老化模型小鼠(Senescence-accele-rated mouse, SAM)的快速老化亚系SAM-prone/8(SAMP8)及抗快速老化亚系SAM-resistance/1(SAMR1)海马蛋白表达的差异, 从蛋白质水平初步探讨与老化相关的学习记忆功能障碍的发生机制. 结果表明, 与同龄SAMR1比较, 6月龄SAMP8海马中有15个蛋白点表达显著上调, 5个蛋白点表达显著下调; 12月龄SAMP8海马中有12个蛋白点表达显著上调, 2个蛋白点表达显著下调, 2个蛋白点只在SAMP8中有表达. 应用质谱分析结合数据库检索, 共鉴定了22种蛋白质. 6月龄和12月龄SAMP8与SAMR1海马中表达有明显变化的蛋白按功能可分为如下4类: (1) 能量代谢相关蛋白; (2) 线粒体功能相关蛋白; (3) 信号转导相关蛋白; (4) 其它蛋白. 研究结果表明, SAMP8和SAMR1海马蛋白表达存在明显差异, 其中一些蛋白与SAMP8随龄出现的学习记忆功能减退相关, 并可能为研究或发现促智药物作用的新蛋白靶标提供线索.     

维拉帕米作用下急性心梗大鼠比较蛋白质组学研究

以急性心梗大鼠为研究对象, 应用双向凝胶电泳法(2-DE)分析比较了维拉帕米作用下急性心梗大鼠心肌蛋白表达的差异, 从蛋白质水平探讨了维拉帕米心肌保护作用的发生机制. 结果表明, 与假手术组及模型组相比, 维拉帕米给药组心肌组织中有8个蛋白点表达显著上调, 7个蛋白点表达显著下调. 采用质谱(MALDI-TOF-MS)分析结合数据库检索, 共鉴定了其中的15种蛋白质, 可按功能分为如下4类: (1) 能量代谢及线粒体功能相关蛋白; (2) 氧化应激相关蛋白; (3) 细胞骨架蛋白; (4) 其它蛋白. 研究结果表明, 维拉帕米的心肌保护作用与恢复心肌损伤过程中的能量供应及对抗氧化应激等作用有关.     

恐惧记忆相关蛋白的蛋白质组学研究

应用双向凝胶电泳结合质谱鉴定和数据库检索, 分析比较了CD1和C57BL/6J小鼠经条件性恐惧实验后海马蛋白表达的差异, 探讨了与恐惧记忆相关的蛋白质. CD1和C57BL/6J小鼠经条件性恐惧实验后, 海马蛋白表达存在明显差异, 29种蛋白(31个蛋白点)与恐惧记忆的形成显著相关. 其中24个蛋白点表达显著上调, 7个蛋白点显著下调. 与恐惧记忆相关的蛋白按功能可分为如下6类: (1) 能量代谢或线粒体功能相关蛋白; (2) 神经发育相关蛋白; (3) 信号转导相关蛋白; (4) 细胞骨架相关蛋白; (5) 氨基酸代谢和蛋白分解相关蛋白; (6) 伴侣蛋白. 这些恐惧记忆形成的相关蛋白深化了对恐惧记忆脑机制的认识, 为研究和治疗认知相关疾病提供了新靶标.     

蛋白质组学技术筛选与鉴定癫痫疾病的差异蛋白质

应用蛋白质组学双向凝胶电泳(Two-dimensional gel electrophoresis, 2DE)和质谱技术, 定量分析和鉴定了癫痫组(n=3)和正常组(n=3)脑组织的差异表达蛋白, 以从蛋白质水平上揭示癫痫病的发机制. 结果表明, 凝胶图谱可辨识2500~3000个蛋白点, 对21个显著差异表达蛋白点进行质谱鉴定和SwissProt数据库检索, 得到17个癫痫差异蛋白, 其中2个蛋白在癫痫组织中表达上调, 15个蛋白表达下调. 部分蛋白与癫痫的关系属首次报道. 这些蛋白与癫痫的发生发展相关, 可能成为癫痫的分子标志物和药物治疗的靶向蛋白.     

鼠肝癌淋巴道转移细胞模型的蛋白质组学研究

对2株来源于同一亲本细胞但淋巴道转移力显著不同的小鼠肝癌腹水型细胞株Hca-F(淋巴结转移率75%)和Hca-P(淋巴结转移率25%), 采用荧光差异双向凝胶电泳(2D DIGE)和DeCyder定量分析软件及HPLC-nESI-MS/MS技术, 定量分析和鉴定了小鼠肝癌细胞Hca-F和Hca-P的差异表达蛋白. 结果显示, 有116个蛋白质点表达水平存在明显差异(p<0.05), 在Hca-F中表达上调蛋白质点62个, 下调蛋白质点54个. 对所有116个蛋白质点进行了电喷雾串联质谱鉴定, 共鉴定出109种单一(Unique)蛋白. 其中部分蛋白已被报道与不同类型肿瘤的发生、浸润和转移相关, 多数蛋白质被首次报道与肝癌的淋巴道转移过程直接相关.     

拟南芥和甜菜夜蛾相互作用的差异蛋白分析

以甜菜夜蛾(Spodoptera exigua)和模式植物拟南芥(Arabidopsis thanliana)作为研究体系, 应用蛋白质双向凝胶电泳(Two-dimensional gel electrophoresis, 2-DE)分析了在甜菜夜蛾取食诱导条件下拟南芥蛋白表达的差异, 从蛋白质水平揭示昆虫取食诱导条件下植物的化学防御机制. 结果发现, 在昆虫取食诱导条件下, 有28个蛋白发生显著变化, 其中17个蛋白点上调表达, 11个蛋白点下调表达. 利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对差异蛋白进行了鉴定, 结果发现转酮酶、S-腺苷甲硫氨酸合成酶、二氢硫辛酰胺脱氢酶和脂肪酸合成酶在植物诱导化学防御中具有重要的作用, 其中脂肪酸合成酶与茉莉酸代谢通路相关.     

血管外膜肌成纤维细胞分化相关蛋白研究

为寻找涉及血管紧张素II (AngII)和转化生长因子β1 (TGF-β1)诱导的血管肌成纤维细胞(MF)分化的蛋白, 本研究采用双向电泳和质谱从整体水平检测了MF分化前后蛋白表达谱的变化, 共找到41个差异表达的蛋白点, 表达水平和/或蛋白位置在Ang II和TGF-β1刺激后都发生明显变化的蛋白点14个, 4个蛋白上调, 6个蛋白下调, 2个蛋白位置发生明显变化, 2个蛋白表达上调，位置也发生变化; 只在Ang II诱导的MF中表达发生变化的蛋白20个, 只在TGF-β1诱导的MF中表达发生变化的蛋白7个. 选取Ang II和TGF-β1共同调节的14个蛋白进行质谱鉴定, 结果除骨架蛋白外, 首次发现MF分化同泛素蛋白酶体系统和嘌呤合成有关; septin 2的下调可能是成纤维细胞分化的标志. 本研究运用蛋白质组学技术发现了新的参与MF分化的蛋白质, 为进一步研究和干预细胞表型转化提供了新的思路和靶点.     

质谱法分析2型糖尿病大鼠神经视网膜差异表达蛋白

采用二维电泳(2DE)分离了正常SD大鼠和2型糖尿病模型大鼠神经视网膜组织总蛋白, 并用Image Master 5.0软件分析比较了正常组和糖尿病组2DE图像, 正常组检测到 3122±37(n=3)个蛋白质点; 糖尿病组检测到2702±21(n=3)个蛋白质点. 约150个蛋白质点的表达水平在两组之间存在明显差异(P<0.05). 在糖尿病组中表达上调的点68个, 下调的点82个. 选择20个差异表达蛋白质点进行肽质量指纹谱(PMF)或串联质谱鉴定, 其中7个蛋白已有报道与糖尿病视网膜病变(DR)相关, 10个蛋白尚未见有报道.     

梅花鹿茸快速生长及骨化期相关差异表达蛋白分析

采用同位素标记相对定量(iTRAQ)技术、超高效液相色谱-质谱联用技术和生物信息学分析方法对东北梅花鹿快速生长期(60 d)与骨化期鹿茸(90 d)进行比较蛋白质组学研究,共鉴定出127种差异蛋白。与骨化期鹿茸比较,快速生长期鹿茸中显著上调的差异蛋白80种,显著下调差异蛋白47种,这些差异表达蛋白主要分布在细胞外基质、核小体、珠蛋白-血红蛋白复合物、肌动蛋白丝、内质网-高尔基中隔室、内质网官腔及核内膜等部位。快速生长期鹿茸显著上调的差异蛋白主要是参与血氧运输、神经生长和再生、软骨和骨组织发育及ATP合成的功能蛋白,而显著下调的差异蛋白主要为参与软骨内骨化过程的功能蛋白,这些蛋白在鹿茸不同生长时期表达量的变化与鹿茸的快速生长和骨化进程密切相关。本研究为系统揭示鹿茸快速生长及骨化的分子机制提供了基础数据,同时对研究鹿茸的药效物质基础及临床应用具有重要的指导意义。     

温敏核不育系水稻株1S及其矮秆突变SV14茎的蛋白质组学比较

采用双向凝胶电泳对温敏核不育水稻株1S和其矮秆突变体SV14的茎(穗颈下第1节和第2节)蛋白进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱.选取了26个蛋白质点采用MALDI-TOF-MS进行肽质谱指纹图分析,最终有12个蛋白质点得到了可靠鉴定.其中在SV14中相对于株1S上调的仅有OSJNBa0039C07.13 蛋白,其它蛋白均表现为下调.这些差异蛋白按照功能可分为4类: (1) 能量代谢相关蛋白;(2) 次生代谢相关蛋白;(3) 调控蛋白;(4) 未知蛋白.对光合系统Ⅱ氧延伸复合物蛋白质前体2,果糖二磷酸醛缩酶,UDP-葡糖醛酸脱羧酶对应的基因进行了半定量RT-PCR分析,发现这几个基因与蛋白质的表达不一致,可能是RNA发生了翻译后修饰而减少了蛋白表达量的结果.这些差异蛋白很可能与水稻矮化有关,为水稻矮秆基因的寻找提供了另一个有效途径.     

用于相对和绝对定量的等量异位标签-二维液相色谱-串联质谱法分析雌二醇和血清剥夺对乳腺癌细胞内蛋白质含量的影响

雌激素和血清中的激素及各种生长因子在乳腺癌的发生、发展过程中发挥着重要的作用。研究雌激素和血清对乳腺癌细胞中蛋白质组成和含量的影响对于阐明雌激素和血清对乳腺癌细胞影响的分子机理具有重要的意义。利用四重用于相对和绝对定量的等量异位标签(isobaric tags for relative and absolute quantification, iTRAQ)标记结合二维液相色谱-串联质谱(two-dimensional liquid chromatography-tandem mass spectrometry, 2D-LC-MS/MS)对雌二醇(17β-oestradiol, E2)和血清各自以及共同作用对MCF7乳腺癌细胞内蛋白质表达的影响进行了比较,共鉴定到置信度在95%以上的蛋白质576种,其中各组相对于正常培养组的细胞共找到26种差异在1倍以上的蛋白质,其中10种上调,16种下调。研究发现E2和血清可显著影响细胞内与蛋白质合成相关的蛋白质的水平。实验结果表明: iTRAQ-LC-MS/MS是进行多个样品差异蛋白组比较的一种有效方法。     

Proteome analysis of differential protein expression in infarcted rat heart after verapamil treatment

To explore the protein-level mechanism of action verapamil in acute myocardial infarcted rats, the myocardial proteome was analyzed by two-dimensional electrophoresis (2-DE). Compared with the sham-operated group and the infarcted group, the result shows that 8 protein expressions in the verapamil treated group were up-regulated, and 7 protein expressions in this group were down-regulated significantly. Using MALDI-TOF-MS, 15 proteins with significant changes were identified through a database search. These proteins can be divided into 4 groups by their biological function: (1) Energy metabolism and mitochondrial function related proteins; (2) oxidative stress-induced proteins; (3) cytoskeletal Proteins; (4) other proteins. The findings show that the myocardial protective effects of verapamil in the myocardial damage process are related to the recovery of energy supply as well as anti-oxidative stress property. __________ Translated from Chemical Journal of Chinese Universities, 2008, 29 (3) (in Chinese)     

温敏核不育系水稻株1S及其矮秆突变体SV14茎的蛋白质组学比较

采用双向凝胶电泳对温敏核不育水稻株1S和其矮秆突变体SV14的茎(穗颈下第1节和第2节)蛋白进行了分离, 通过银染显色, 获得了分辨率和重复性较好的双向电泳图谱. 选取了26个蛋白质点采用MALDI-TOF-MS进行肽质谱指纹图分析, 最终有12个蛋白质点得到了可靠鉴定. 其中在SV14中相对于株1S上调的仅有OSJNBa0039C07.13 蛋白, 其它蛋白均表现为下调. 这些差异蛋白按照功能可分为4类: (1) 能量代谢相关蛋白; (2) 次生代谢相关蛋白; (3) 调控蛋白; (4) 未知蛋白. 对光合系统Ⅱ氧延伸复合物蛋白质前体2, 果糖二磷酸醛缩酶, UDP-葡糖醛酸脱羧酶对应的基因进行了半定量RT-PCR分析, 发现这几个基因与蛋白质的表达不一致, 可能是RNA发生了翻译后修饰而减少了蛋白表达量的结果. 这些差异蛋白很可能与水稻矮化有关, 为水稻矮秆基因的寻找提供了另一个有效途径.     

凝胶电泳-毛细管液相色谱-质谱联用技术分析大肠癌肿瘤与癌旁组织的差异蛋白质组

通过凝胶电泳将大肠癌患者的手术标本组织(肿瘤与癌旁)中的总蛋白按照分子量分成不同的组分,经胶内水解后,利用毛细管液相色谱-质谱联用技术对各个组分的多肽混合物进行蛋白质组学分析,共鉴定出29种差异表达的蛋白质,其中肿瘤组织中表达上调的蛋白质19种,如Fibrinogen beta chain,Vimentin,Fi-brinogen gamma chain,Histone H2A type 1-B/E,Isoform 1 of Periostin,Fibrinogen alpha chain,Histone H3.1,Al-pha-enolase,Protein disulfide-isomerase A3等;在肿瘤组织中表达下调的有10种蛋白质,如Sarcomeric tropomy-osin kappa,Transgelin,Heat shock protein beta-1,Talin-1,Isoform 2 of Vinculin,Isoform 1 of Filamin-C等.通过蛋白质印迹实验对其中的踝蛋白1(Talin-1)和烯醇化酶(Alpha-enolase)的蛋白质组学鉴定结果进行了验证.生物信息学分析表明在大肠癌组织中表达上调的蛋白多具有胞外分泌的属性,提示这些蛋白很有可能会渗透到机体内的循环系统中,从而在患者血液或者腹水中能够被检测到;而在大肠癌组织中表达下调的蛋白则多为细胞骨架、胞外基质纤维等的组成成分,这些蛋白的下调常与肿瘤细胞向周围组织的侵袭和转移有密切关系.本研究为大肠癌的早期诊断、大肠癌初筛等方面的研究等提供了基础数据,对进一步深入研究大肠癌发病机制具有重要意义.     

Analysis of expression and comparative profile of normal placental tissue proteins and those in preeclampsia patients using proteomic approaches

Preeclampsia (PE) is a complex and serious condition of pregnancy. Trophoblasts in human placenta can be separated and collected by laser capture micro-dissection (LCM). Protein in trophoblasts have been extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), finally 962 unique proteins are identified by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Comparison of differential expressed proteins in normal and those in PE are investigated. Two-dimensional electrophoresis (2DE) and MS were used to identify differential expressed proteins. 13 differential expressed proteins include signal transduction protein, molecular chaperone, cell skeleton proteins are identified, in which 3 proteins are down-regulated and 10 proteins are up-regulated. They might be correlated with the cause of PE.     

Exploring the Proteomic Alterations from Untreated and Cryoablation and Irradiation Treated Giant Cell Tumors of Bone Using Liquid-Chromatography Tandem Mass Spectrometry

Giant cell tumors of bone (GCT) are benign tumors that show a locally aggressive nature and affect bones’ architecture. Recently, cryoablation and irradiation treatments have shown promising results in GCT patients with faster recovery and less recurrence and metastasis. Therefore, it became a gold standard surgical treatment for patients. Hence, we have compared GCT-untreated, cryoablation, and irradiation-treated samples to identify protein alterations using high-frequency liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Our label-free quantification analysis revealed a total of 107 proteins (p < 0.01) with 26 up-regulated (<2-folds to 5-fold), and 81 down-regulated (>0.1 to 0.5 folds) proteins were identified from GCT-untreated and treated groups. Based on pathway analysis, most of the identified up-regulated proteins involved in critical metabolic functions associated with tumor proliferation, angiogenesis, and metastasis. On the other hand, the down-regulated proteins involved in glycolysis, tumor microenvironment, and apoptosis. The observed higher expressions of matrix metalloproteinase 9 (MMP9) and TGF-beta in the GCT-untreated group associated with bones’ osteolytic process. Interestingly, both the proteins showed reduced expressions after cryoablation treatment, and contrast expressions identified in the irradiation treated group. Therefore, these expressions were confirmed by immunoblot analysis. In addition to these, several glycolytic enzymes, immune markers, extracellular matrix (ECM), and heat shock proteins showed adverse expressions in the GCT-untreated group were identified with favorable regulations after treatment. Therefore, the identified expression profiles will provide a better picture of treatment efficacy and effect on the molecular environment of GCT.     

Identification of human hepatocellular carcinoma-related proteins by proteomic approaches

Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. Analysis of human serum from HCC patients using two-dimensional gel electrophoresis (2DE) combined with nano-high-performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC–ESI-MS/MS) identified fourteen different proteins differentially expressed between HCC patients and the control group. Twelve proteins were up-regulated and two down-regulated. By using nano-HPLC–MS/MS system to analyze proteome in human serum, 317 proteins were identified, twenty-nine of which to high confidence levels (protein matched at last two unique peptide sequences). Of these twenty-nine proteins, six were present only in HCC patients and may serve as biomarkers for HCC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of apolipoprotein and apolipoprotein precursors in pancreatic cancer serum samples via two-dimensional liquid chromatography and mass spectrometry

Major advances in cancer control depend upon early detection, early diagnosis and efficacious treatment modalities. Current existing markers of pancreatic ductal adenocarcinoma, generally incurable by available treatment modalities, are inadequate for early diagnosis or for distinguishing between pancreatic cancer and chronic pancreatitis. We have used a proteomic approach to identify proteins that are differentially expressed in sera from pancreatic cancer patients, as compared to control. Normal, chronic pancreatitis and pancreatic cancer serum samples were depleted of high molecular weight proteins by acetonitrile precipitation. Each sample was separated by chromatofocusing, and then further resolved by reversed-phase (RP)-HPLC. Effluent from the RP-HPLC column was split into two streams with one directly interfaced to an electrospray time-of-flight (ESI-TOF) mass spectrometer for molecular weight (MW) determination of the intact proteins. The remainder went through a UV detector with the corresponding peaks collected with a fraction collector, subsequently used for MS/MS analysis. The ion intensities of proteins with the same MW obtained from ESI-TOF-MS analysis were compared, with the differentially expressed proteins determined. An 8915 Da protein was found to be up-regulated while a 9422 Da protein was down-regulated in the pancreatic cancer sera. Both proteins were identified by MS and MS/MS as proapolipoprotein C-II and apolipoprotein C-III(1), respectively. The MS/MS data of proapolipoprotein C-II was searched using "semi-trypsin" as the search enzyme, thus confirming that the protein at 8915 Da was proapolipoprotein C-II. In order to confirm the identity of the protein at 9422 Da, we initially identified a protein of 8765 Da with a similar mass spectral pattern. Based on MS and MS/MS, its intact molecular weight and "semi-trypsin" database search, the protein at 8765 Da was identified as apolipoprotein C-III(0). The MS and MS/MS data of the proteins at 8765 Da and 942 Da were similar, thus confirming the protein at 9422 Da as being apolipoprotein C-III(1). The detection of differentially expressed proapolipoprotein C-II and apolipoprotein C-III(1) in the sera of pancreatic cancer patients may have utility for detection of this deadly malignancy.