肠道病毒71型感染前后宿主细胞蛋白质组的二维液相色谱分离和比较

以肠道病毒71型及其宿主细胞为研究主体,建立了一种二维液相色谱分离和分析比较病毒感染前后细胞蛋白表达谱的方法。该方法以高效液相色谱(HPLC)为技术平台,对细胞裂解物先后进行一维色谱聚焦分离和二维反相色谱分离。利用ProteoVue软件将二维色谱数据转换成模拟胶图,再利用DeltaVue软件对感染前后的宿主蛋白表达谱进行比较和分析,找出差异蛋白。二维液相色谱分离法能够根据蛋白的等电点和疏水性建立精确的细胞蛋白表达图谱,每0．2个pH为一个收集区段,在pH8．5～3．9的范围内可见蛋白条带约1200条。该方法良好的重现性、自动化以及结果分析的简易化,使之在细胞表达谱差异显示中的应用潜力巨大,并且为研究病毒与宿主相互作用提供了新的方法和思路。     

二维液相色谱法在羊草地上部总蛋白分离中的应用

取8周龄羊草的地上部分,用三氯乙酸-丙酮法沉淀总蛋白,沉淀裂解后将缓冲液置换为起始缓冲液,进行第一维色谱聚焦分离。将第一维分离收集的pH值为8.5至4.0之间的组分分别进行第二维无孔硅胶反相高效液相色谱分离,利用ProteoVue软件获得羊草植株总蛋白pI/UV图谱,即羊草植株总蛋白质表达谱。文中对二维液相色谱法分离羊草蛋白质进行了方法学的研究,在第二维分离中尝试用3种不同的洗脱梯度条件进行分离,优化二维液相色谱分离条件并与传统凝胶双向电泳进行了比较,另外还对二维液相色谱的重现性和准确性进行了检验。实验建立了利用二维液相色谱分离羊草总蛋白的技术方法。     

结核分枝杆菌菌体蛋白双向电泳技术的优化

目的：提取结核分枝杆菌菌体蛋白并建立一种利用双向电泳分离结核分枝杆菌蛋白质组的方法。方法：分离提取结核分枝杆菌菌体蛋白。样品采用不同pH梯度的鹏胶条进行第一向等电聚焦,12％SDS—PAGE凝胶进行二向电泳。银染后双向电泳图谱用Molecular Image Fx激光图像扫描仪扫描,PDQuest6．0软件完成配比分析。结果：优化了结核分枝杆菌菌体蛋白的提取方法,用裂解液8mol／L尿素结合2mol／L硫脲,140mmol／LDTT,0．5％biolyte,4％CHAPs,400mg／m1lOG处理,成功提取了蛋白,并通过结核分枝杆菌双向电泳技术体系的优化,建立了结核分枝杆菌菌体蛋白的分解图谱。pH4—7及pH7—10两胶面上共1387个点,占所检测到的蛋白总数的86％,绝大部分（1194个）蛋白位于pH4—7范围内。结论：为进一步开展结核分枝杆菌的比较蛋白质组学研究提供了方法学参考。     

基于等电聚焦-反相HPLC的虎纹捕鸟蛛毒素组学的初步研究

虎纹捕鸟蛛(Ornithoctonus huwena)是中国最毒的蜘蛛之一.已有研究表明,其粗毒中含有丰富的低分子量(<10 kD)多肽活性成分.为分析这些成分, 利用目标蛋白快速分离系统(ProteomeLab PF 2D)建立了一种新的二维液相色谱分离方法.该方法包括一维的基于蛋白质等电点(pI)的色谱聚焦分离和二维利用无孔硅胶反相柱的基于疏水性的高效液相色谱分离.得到的反相图谱通过仪器配套的ProteoVue软件转换成与凝胶电泳图像相似的pI/UV图,以更直观地显示多肽成分的数量、分布规律及相对丰度等.洗脱的多肽自动收集后用基质辅助激光解吸电离-飞行时间质谱进行分析.从一维分离的11个馏分(pH 4.53-8.59)中共检测到大约600个多肽条带.通过质谱分析测得130个多肽的精确分子量,同时通过De novo测序得到26种多肽(其中包括12种已知多肽)的部分序列信息,并利用这些序列信息对未知多肽进行了生物信息学分析.     

多维液相色谱分离技术在复杂蛋白质组学样品鉴定中的应用

目的：随着蛋白组学技术的发展,液相色谱-串联质谱的联用技术（液质联用）逐渐成为蛋白组学的主流技术。方法：通过结合各种不同原理的色谱分离类型,多维液相色谱分离技术能够极大的提高分离系统的峰容量,达到有效分离复杂程度很高的蛋白质组学样品的目的。结果：最广泛使用的多维液相色谱分离系统是离子交换色谱（IEX）和反相色谱（RP）的二维结合,近年来又发展出了分离能力更强的三维液相色谱分离系统,并且已经在蛋白质组学研究中得到了应用。结论：本文综述了多种多维液相色谱分离方法,在这些方法中,不同的分离原理的色谱类型被用于肽段或蛋白混合物的预分离中,有效促进了样品的充分分离,极大地提高了复杂样品的蛋白组学鉴定能力。     

二维制备高效液相色谱分离塞隆骨强极性多肽北大核心CSCD

本实验建立了一种新型的利用高正交性的二维制备型高效液相色谱系统分离强极性动物药多肽的方法。本文以塞隆骨水提取物为研究对象,以亲水性C18AQ制备型高效色谱柱为第一维分离柱,首先在一维分离中将目标混合物分成若干组份;然后以C18MP制备型高效液相色谱柱为第二维色谱分离柱,将第一维分离后得到的组份纯化为单体化合物。本研究最终得到5个塞隆骨单体化合物,化合物纯度均超过98%。经Nano-LCESI-MS/MS鉴定和搜库分析,这些多肽的序列分别为:KTAILVKE、RGAPQDQE、LVGPGAPGR、GFAGD和KPQWHP。此研究方法速度快、效率高且重复性好,可以对类似的研究提供借鉴。     

吗啡成瘾及戒断大鼠前额叶皮质蛋白质组变化的初步研究

目的：探讨与大鼠吗啡成瘾和戒断相关的前额叶皮质（PFC）蛋白。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,分别对吗啡成瘾和自然戒断大鼠及正常大鼠的PFC蛋白质样品进行二维分离,2-DE图谱经ImageMaster 2D Plat-inum 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,与吗啡成瘾和自然戒断相关的差异表达蛋白斑点为79个;经质谱鉴定出2个有意义的差异表达的蛋白斑点：Snap25亚型-βSnap25突触相关蛋白25,β-肌动蛋白。结论：PFC的某些蛋白可能与吗啡成瘾和戒断相关,其中尤其是神经毒性相关的蛋白可能与吗啡成瘾机制相关。     

蛋白质组分析中蛋白质分步提取方法的建立

利用细胞裂解液充分溶解细胞蛋白质是成功进行蛋白质组分析的先决条件.尝试利用三步提取法,即以三种溶解性能不同的裂解液分步提取细胞中的蛋白质组,并分别进行二维聚丙烯酰胺凝胶电泳（two-dimensional polyacrylamide gel electrophoresis, 2-D PAGE）分离.通过对2-D PAGE蛋白质图谱的比较,发现其与常规方法相比,具有蛋白质提取率高、双向电泳(two-dimensional electrophoresis,2-DE)分辨率高等优点.     

桶形芋螺毒管蛋白质组双向电泳条件优化

目的：优化双向电泳的条件,建立适用于桶形芋螺毒管蛋白质组分析的双向电泳方法。方法：对毒管蛋白的提取、上样量及SDS-PAGE凝胶浓度等影响因素进行优化。结果：乙酸提取法适宜于毒管蛋白的提取,对于pH3～10、17cm的IPG胶条,当上样量为0.75mg,聚焦70000Vhr,SDS-PAGE凝胶浓度为15%时,可提高双向电泳的分离效果,所得蛋白点清晰、数目达到1003个。结论：采用优化的条件进行双向电泳,能得到分辨率高、重现性好、完整的双向电泳图谱,为后续桶形芋螺毒管蛋白质组学研究打下基础。     

油松雌性不育系球果蛋白质双向电泳技术的建立

本文建立了油松雌性不育系雌球果蛋白质组研究中的双向电泳技术.第一向采用固定pH值梯度(IPG)胶条在IPGphorTM等电聚焦仪上进行等电聚焦,第二向在恒功率且恒温条件下于Ettan-DALTTMⅡ高通量电泳仪上进行SDS-PAGE电泳,以银染和考马斯亮蓝两种方法染色.通过对全蛋白的提取、胶条pH值和胶条肿胀等技术环节的优化和比较,得到了重复性很高,分离效果良好的蛋白质双向图谱.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

DNA endonuclease activities associated with melanoma cell chromatin

Chromatin-associated DNA endonucleases, extracted from Cloudman mouse melanoma cell nuclei, were separated on isoelectric focusing into seven fractions in two widely separated groups pH 3.4–5.4 and 7.5–9.3, each active on calf thymus DNA. All fractions in the former group, pI's 3.4, 4.4 and 5.4, produced at least one single-strand scission per molecule on circular duplex phage PM2 DNA, and transformed circular single-stranded phage fd DNA into linear strands of uniform length. In the second group there was no detectable activity against PM2 DNA, but two fractions pI's 7.5 and 8.0 were active on fd DNA as above, whereas the other two, pI's 8.5 and 9.0 transformed fd DNA into a number of different sized, discrete segments. These results indicate that, even allowing for possible enzymatic identity of some of the isoelectrically separated forms, at least three different DNA endonucleases are associated with mouse melanoma cell chromatin.     

Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis.

Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ductus deferens were 2.2, 2.3, 2.2, and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput = corpus greater than cauda and corpus greater than caput greater than cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine-borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 microM and 2.19 nmoles/min/microliters luminal fluid at pH 8.5 compared to 0.49 microM and 0.49 nmoles/min/microliters luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 microM) and higher Vmax (1.13 nmoles/min/microliters luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X-114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteome profile of human urine with two-dimensional liquid phase fractionation

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.     

Establishment of a PF2D-MS/MS platform for rapid profiling and semiquantitative analysis of membrane protein biomarkers

Current proteome profiling techniques have identified relatively few mammalian membrane proteins despite their numerous important functions. To establish a standard throughput-potential profiling platform for membrane proteins, Triton X-100-solubilized rat liver microsomal proteins were separated on a 2-D separation system (2-D liquid phase fractionation (PF2D)) in two different pH ranges (4.0-8.5 and 7.0-10.5). This system produced 182 proteins with more than two transmembrane domain (TMD), including 16 TMDs with high confidence. Comparative 2-D liquid maps with high resolution and reproducibility have been constructed for liver microsome from the phenobarbital (PB) treated rats. PF2D was also found to be useful for the semiquantification of some representative cytochrome P450 family proteins (e.g., cytochrome P450 2B2) that were induced by PB treatment compared with untreated controls. Thus, the combination of both high-detection capacity and rapid preliminary semiquantification in a PF2D platform could become a standard system for the routine analysis of membrane proteins.     

过氧化物酶6在大鼠附睾中的表达及分布

通过双向电泳结合质谱技术分离鉴定正常成年大鼠附睾头段与尾段管腔液中的蛋白组成,从附睾头段及尾段管腔液的22个差异蛋白点中鉴定出12个蛋白质.其中11个蛋白质在不同种属哺乳动物的附睾组织中已有鉴定报道,而过氧化物酶6(peroxiredoxin 6,Prdx6)为新发现的存在于附睾头段及尾段管腔液中的体液蛋白.采用RT-PCR、Western印迹及免疫组化技术,对该蛋白在大鼠附睾中的表达及分布进行了分析.实验表明,Prdx6与精子的成熟、贮存及保护有一定关系,其具体机制值得进一步深入研究.     

Reproducibility,sensitivity and compatibility of the ProteoExtract® subcellular fractionation kit with saturation labeling of laser microdissected tissues

Laser microdissection (LMD), a method of isolating specific microscopic regions of interest from a tissue that has been sectioned, is increasingly being applied to study proteomics. LMD generally requires tissues to be fixed and histologically stained, which can interfere with protein recovery and subsequent analysis. We evaluated the compatibility and reproducibility of protein extractions from laser microdissected human colon mucosa using a subcellular fractionation kit (ProteoExtract^®, Calbiochem). Four protein fractions corresponding to cytosol (fraction 1), membrane/organelle (fraction 2), nucleus (fraction 3) and cytoskeleton (fraction 4) were extracted, saturation labeled with Cy5 and 5 μg separated by both acidic (pH 4–7) and basic (pH 6–11) 2‐DE. The histological stains and fixation required for LMD did not interfere with the accurate subcellular fractionation of proteins into their predicted fraction. The combination of subcellular fractionation and saturation CyDye labeling produced very well resolved, distinct protein spot maps by 2‐DE for each of the subcellular fractions, and the total number of protein spots consistently resolved between three independent extractions for each fraction was 893, 1128, 1245 and 1577 for fractions 1, 2, 3 and 4, respectively. Although significant carryover of protein did occur between fractions, this carryover was consistent between experiments, and very low inter‐experimental variation was observed. In summary, subcellular fractionation kits are very compatible with saturation labeling DIGE of LMD tissues and provide greater coverage of proteins from very small amounts of microdissected material.