大疣蛛毒素-Ⅵ的分离纯化及部分生物学活性鉴定

从雷氏大疣蛛(Macrothele raveni)粗毒中,结合阳离子交换和反相高效液相色谱分离到一种多肽神经毒素,命名为大疣蛛毒素-VI(Raventoxin-VI).经MALDI-TOF质谱分析,它的分子量为(5 371.6±0.5) Da.利用Edman降解气相蛋白质测序仪测得其氨基酸序列是NH2-NIIKGRVVKLCGGCAQKCCDREPRCDPCRTCVENVGT-GGGYLSSNKKCNGS-COOH,其中8个Cys形成4对二硫键.Raventoxin-Ⅵ能阻断小鼠膈神经膈肌标本神经肌肉接头传递,脑室注射能使小鼠瘫软.从一级结构上没有发现与该毒素有较高相似性的其他蜘蛛毒素.     

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。     

大鼠附睾蛋白质组的提取和二维液相色谱分离

背景与目的：提取犬鼠附睾液蛋白并建立一种利用二维液相色谱法分离附睾蛋白组的方法。方法：分离提取犬鼠附睾液蛋白。样品利用起始缓冲液置换后,进行一维色谱聚焦分离,然后收集pH8．5—4．0之间的组份进行二维反相离压液相色谱分离,最后将获得的二维UV图通过ProteoVue软件转换成PI／UV图谱。结果：成功提取了附睾液蛋白,并通过二维液相色谱成功建立了大鼠头体尾部附睾液蛋白的二维PI／UV图谱,收集了一维色谱聚焦分离的pH8．5—4．0区间的20个组份,并将每个组份进行二维色谱分离后转换为PI/UV图谱。结论：为进一步全面研究附睾蛋白功能和体液差异蛋白质组研究打下了基础。     

二维制备高效液相色谱分离塞隆骨强极性多肽

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。     

大疣蛛毒素-VI的分离纯化及部分生物学活性鉴定

从雷氏大疣蛛(Macrotheleraveni)粗毒中,结合阳离子交换和反相高效液相色谱分离到一种多肽神经毒素,命名为大疣蛛毒素 VI(Raventoxin VI).经MALDI TOF质谱分析,它的分子量为(5371.6±0.5)Da.利用Edman降解气相蛋白质测序仪测得其氨基酸序列是NH2 NIIKGRVVKLCGGCAQKCCDREPRCDPCRTCVENVGT GGGYLSSNKKCNGS COOH,其中8个Cys形成4对二硫键.Raventoxin Ⅵ能阻断小鼠膈神经膈肌标本神经肌肉接头传递,脑室注射能使小鼠瘫软.从一级结构上没有发现与该毒素有较高相似性的其他蜘蛛毒素.     

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。     

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。     

牦牛骨抗氧化肽的分离纯化及鉴定

为了分离纯化及鉴定牦牛骨胶原多肽,以得到具有DPPH自由基清除率的抗氧化肽。合成抗氧化肽,验证其对DPPH自由基清除率。牦牛骨胶原多肽经陶瓷羟基磷灰石柱层析和C18反相高效液相色谱层析后得到组分F11。当其质量浓度为1 mg/mL时,对DPPH自由基清除率为34.45%。用反相高效液相色谱-质谱联用对F11组分进行序列测定,得到氨基酸序列为GPAGPPGPIGNVGAPGPK和GKSGDRGETGPAGP AGPIGPVGAR的抗氧化肽,分子量分别为1 570.810 3 Da和2 160.104 Da。合成抗氧化多肽GPAGPPGPIGN VGAPGPK和GKSGDRGETGPAGPAGPIGPVGAR,当其质量浓度为1 mg/mL时,对DPPH自由基清除率分别为16.59%和35.17%。本研究分离鉴定出牦牛骨抗氧化肽,牦牛骨可能成为一种潜在的抗氧化剂。     

一种羊肚菌源多肽MIP-16的分离纯化及其神经保护活性

分离纯化人工栽培的梯棱羊肚菌子实体多肽(MIP-16),对其结构和神经保护活性进行研究。采用磷酸盐缓冲液提取,分子排阻色谱分离,反相高效液相色谱纯化获得梯棱羊肚菌子实体多肽,通过液相色谱-质谱连用技术完成氨基酸序列鉴定。构建6-羟基多巴胺处理后引起凋亡的PC12细胞模型,验证MIP-16对PC12细胞的保护作用。结果显示:MIP-16由16个氨基酸组成,相对分子质量为1 762 Da,氨基酸组成序列为Thr-Ile-Thr-Leu-Glu-Val-Glu-Ser-Ser-Asn-Ile-Thr-Asn-Asp-Val-Lys。细胞实验发现,MIP-16能够抑制PC12细胞丙二醛(MDA)产生,降低活性氧(ROS)水平,调节Bcl-2和Bax的比例,并降低Caspase蛋白的表达。MIP-16通过细胞线粒体途径抑制PC12细胞凋亡,具有神经保护活性,可作为一种辅助治疗帕金森综合症的药物开发。     

黄连中α-葡萄糖苷酶抑制剂的筛选分离及质谱分析

为筛选黄连中α-葡萄糖苷酶抑制剂,本研究采用高效液相色谱-电喷雾质谱联用技术(HPLC-DAD-MS)对黄连提取物中的化学成分进行分析鉴定,并采用高速逆流色谱分离其中的活性成分。选用反相C18色谱柱,以0.02%醋酸溶液(A)和甲醇(B)为流动相,进行梯度洗脱;利用电喷雾质谱(ESI-MS)正离子模式在线检测化学成分;以α-葡萄糖苷酶作为生物靶分子,以超滤质谱技术筛选酶抑制剂。再经高速逆流色谱分离纯化,以乙酸乙酯-正丁醇-乙醇-水(3.0∶1.7∶0.5∶6.0,v/v/v/v)为两相溶剂系统,所得分离收集液经高效液相色谱法检测。实验通过HPLC-DAD-MS共鉴定出5个化学成分,分别为药根碱、表小檗碱、黄连碱、巴马亭和小檗碱。通过HSCCC分离得到两种α-葡萄糖苷酶抑制剂巴马亭和小檗碱。利用液相色谱-超滤-质谱-高速逆流色谱联用技术可以快速分离鉴定黄连中的化合物。此方法对于筛选有效成分具有快速和灵敏等优势。     

Classifications of ovarian cancer tissues by proteomic patterns

Ovarian cancer is a morphologically and biologically heterogeneous disease. The identification of type-specific protein markers for ovarian cancer would provide the basis for more tailored treatments, as well as clues for understanding the molecular mechanisms governing cancer progression. In the present study, we used a novel approach to classify 24 ovarian cancer tissue samples based on the proteomic pattern of each sample. The method involved fractionation according to pI using chromatofocusing with analytical columns in the first dimension followed by separation of the proteins in each pI fraction using nonporous RP HPLC, which was coupled to an ESI-TOF mass analyzer for molecular weight (MW) analysis. A 2-D mass map of the protein content of each type of ovarian cancer tissue samples based upon pI versus intact protein MW was generated. Using this method, the clear cell and serous ovarian carcinoma samples were histologically distinguished by principal component analysis and clustering analysis based on their protein expression profiles and subtype-specific biomarker candidates of ovarian cancers were identified, which could be further investigated for future clinical study.     

Isolation and certain properties of the thermostable endoglucanase from E. coli C600 (pKNE-102) coded by a Clostridium thermocellum gene

A previously unknown endoglucanase encoded by the C. thermocellum gene was isolated from the recombinant strain of E. coli (pKNE-102). The purification procedure included ammonium sulfate precipitation, heat treatment, chromatography on a polyvinyl matrix (Toyopearl HW-50F) and chromatofocusing on a high performance Mono P HR 5/20 column. Sodium dodecyl sulfate electrophoresis analysis of the Toyopearl HW-50F effluent revealed two protein bands with Mr of 41 kDa and 42 kDa. These protein components differed also by their pI values (4.45, and 4.40, respectively) and could be separated by chromatofocusing. Both components were found to be active and exhibited similar enzymatic properties as well as high thermal stability.     

Anionic and neutral glutathione S-transferase isoenzymes in the freshwater worm Tubifex tubifex (O.F.M.)

The anionic and the neutral glutathione S-transferase (GST) isoenzymes from the freshwater worm Tubifex tubifex (O.F.M.) were separated in one step by chromatofocusing on a Polybuffer exchanger 94 column, eluted with Polybuffer 74. Their pI values ranged between 4.12 and 6.98, and their molecular weight between 30 000 and 38 400. The apparent Km values towards glutathione and 1-chloro-2,4-dinitrobenzene were also determined. The high number of non-cationic GST isoenzymes is unusual. Tubifex worms seems well equipped for attacking environmental pollutants.     

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.     

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.     

Cytoplasmic 3H-arginine polypeptides of Ehrlich ascites tumour cells

Summary Low molecular weight ninhydrin positive peptide fractions of the Ehrlich tumour cell cytoplasm were isolated and characterized. After preliminary gel filtration of the cytoplasm on Sephadex G-25 column, the peptide mixture was fractionated on cationic exchanger SP-Sephadex C-25 column and eluted with increasing pH gradient. Five peaks were obtained. Only the first peak contained sugar component. All five peptides were studied with respect to molecular weight, isoelectric point and electrophoretic homogeneity. The cytoplasm of Ehrlich tumour cells contains one peptide of acidic (pI - 5.0), two slightly basic (pI - 7.7 and pI - 7.7) and two strongly basic nature (pI - 8.7 and pI - 8.9). Molecular weights varied from 8 500 to 18 500 daltons. The origin of these peptides is briefly discussed.     

Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate

A comprehensive on-line sample clean-up with an integrated two-dimensional HPLC system was developed for the analysis of natural peptides. Samples comprised of endogenous peptides with molecular weights up to 20 kDa were generated from human hemofiltrate (HF) obtained from patients with chronic renal failure. The (poly-)peptides were separated using novel silica-based restricted access materials with strong cation-exchange functionalities (SCX-RAM). The size-selective sample fractionation step is followed by cation-exchange chromatography as the first dimension. The subsequent second dimension of separation is based on hydrophobic interaction using four parallel short reversed-phase (RP) columns implemented via a fully automated column switching technique. More than 1000 peaks were resolved within the total analysis time of 96 min. Substances of selected peaks were sampled to analyse their molecular weights by off-line MALDI-TOF mass spectrometry and to determine their amino acid sequence by Edman degradation. The potential for comprehensive peptide mapping and identification is demonstrated.     

Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes

Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics--molecular mass, basic isoelectric point, and dye column binding--were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.