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Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis

ANALYTICAL BIOCHEMISTRY 2001年第1期297卷 25-31页

作者： Smolka, MB Zhou, HL Purkayastha, S Aebersold, R Inst Syst Biol Seattle WA 98105 USA Univ Estadual Campinas Inst Biol Dept Bioquim Campinas SP Brazil Appl Biosyst Inc Framingham MA 01701 USA

The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate. (C) 2001 Academic Press.

关键词：亲和力标记物/分析亲和力标记物/化学合成亲和力标记物/代谢半胱氨酸/分析电泳凝胶双向电泳聚丙烯酰氨凝胶同位素标记/方法乳清蛋白/化学乳清蛋白/代谢质谱分析法/方法分子量肽碎片/化学肽碎片/代谢蛋白质组/分析蛋白质组/化学蛋白质组/代谢酵母菌酿酒/化学酿酒酵母蛋白质类/化学十二烷硫酸钠/代谢时间因素胰蛋白酶/代谢尿素/代谢

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Synthesis and characterization of novel spin-labeled photoaffinity nonnucleoside analogues of ATP as structural and EPR probes for myosin

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BIOCONJUGATE CHEMISTRY 2000年第5期11卷 725-733页

作者： Chen, XR Grammer, J Cooke, R Pate, E Yount, RG Washington State Univ Dept Chem Dept Pure & Appl Math Sch Mol Biosci Pullman WA 99164 USA Univ Calif San Francisco Dept Biochem & Biophys San Francisco CA 94143 USA Univ Calif San Francisco CVRI San Francisco CA 94143 USA

Two new spin-labeled photoreactive nonnucleoside ATP analogues, 1-(4-azido-2-nitrophenyl)amino-3-( 1-oxyl-2,2,5,5-tetramethylpyrrolidinyl-3-carbamido)-2-propyl triphosphate (SL-NANTP) and 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2 2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate (SSL-NANTP), were synthesized and characterized. This study aims to develop a second generation of NANTP-based analogues containing immobile spin labels that can be used to monitor conformational changes in myosin during the contractile cycle of muscle. Previous studies have shown that both a photoaffinity nonnucleoside ATP analogue, 2-[(4-azido-2-nitrophenyl)amino] ethyl triphosphate (NANTP) [Nakamaye et al. (1985) Biochemistry 24, 5226-5235], and a photoaffinity ATP analogue, 3'(2')-O-4-[4-oxo-(4-amino-2,2,6,6-tetramethyl-piperidino-1-oxyl)-4-benzoyl] benzoyl adenosine 5'-triphosphate (SL-Bz(2)ATP) [Wang et al. (1999) J. Muscle Res. Cell MotiL. 20, 749-753], behave like ATP in their interactions with myosin. Remarkably, photolabeled myosin recovers all of its normal enzymatic properties after treatment with actin in the presence of MgATP [Luo et al. (1995) Biochemistry 34, 1978-1987]. For SL-NANTP, the spin label moiety is attached to NANTP via an aminomethyl side chain. In SSL-NANTP, attachment is via a restricted spiro ring. The two new probes interact with myosin subfragment-1 (S1) in a manner analogous to ATP, and after photoincorporation, labeled S1 recovers full activity after treatment with actin and MgATP. The electron paramagnetic resonance (EPR) spectrum resulting from S1 photolabeled with SL-NANTP shows a very high degree of probe mobility. However, the EPR spectrum of S1 photolabeled with SSL-NANTP shows that the probe is highly immobilized with respect to S1, constrained to move within a cone of angle 52 degrees (full-width, half-max). Unlike the parent, NANTP, which photolabels on the 23 kDa tryptic fragment of S1, SSL-NANTP photolabels on the 20 kDa f

关键词：肌动蛋白类/化学腺苷三磷酸/类似物和衍生物腺苷三磷酸/化学腺苷三磷酸/代谢亲和力标记物/化学合成亲和力标记物/化学叠氮化合物/化学合成叠氮化合物/化学电子自旋共振谱学/方法电泳聚丙烯酰氨凝胶指示剂和试剂肌球蛋白亚碎片/分析肌球蛋白亚碎片/化学肌球蛋白亚碎片/代谢肌球蛋白类/分析肌球蛋白类/化学肌球蛋白类/代谢自旋标记物钒酸盐类

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Development of an affinity-driven cross-linker: Isolation of a vitamin D receptor associated factor

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BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 2000年第4期10卷 361-364页

作者： Swamy, N Addo, JK Ray, R Boston Univ Sch Med Dept Med Vitamin D Lab Boston MA 02118 USA

A vitamin D analogue containing an affinity and a photoaffinity probe (affinity-driven cross-linker, Double Label) was synthesized. An unknown factor, associated with vitamin D receptor (VDR), was isolated from rat liver nuclear extract using a GST-VDR-ligand-binding domain fusion protein (GST-VDR-LBD), affinity labeled with Double Label, and protein-protein crosslinking by photolysis. (C) 2000 Elsevier Science Ltd. All rights reserved.

关键词：亲和力标记物/化学合成细胞核/化学交联试剂/化学合成 DNA结合蛋白质类/分离和提纯 DNA结合蛋白质类/代谢免疫印迹法肝/超微结构光亲和标记/化学合成蛋白质结合受体骨化三醇/分离和提纯受体骨化三醇/代谢重组融合蛋白质类/化学合成重组融合蛋白质类/代谢转录因子/分离和提纯转录因子/代谢维生素D/类似物和衍生物维生素D/化学合成维生素D/代谢动物大鼠

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A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes

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BIOORGANIC & MEDICINAL CHEMISTRY 2000年第3期8卷 507-513页

作者： Deussen, HJ Danielsen, S Breinholt, J Borchert, TV Novo Nordisk AS Prot Discovery DK-2880 Bagsvaerd Denmark

A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the E-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase(R) (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed. (C) 2000 Elsevier Science Ltd. All rights reserved.

关键词：亲和力标记物/化学合成生物素化作用定向分子进化酶抑制剂/化学合成酶联免疫吸附测定免疫磁化分离动力学脂肪酶/拮抗剂和抑制剂脂解作用硝基酚类/化学合成硝基酚类/化学有机磷化合物/化学合成有机磷化合物/化学丝氨酸蛋白酶抑制剂/化学合成构效关系

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Synthesis and preliminary analysis of a P-glycoprotein-specific [³H]-benzophenone photoaffinity label based on (-)-stipiamide

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BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 2000年第20期10卷 2275-2278页

作者： Andrus, MB Turner, TM Sauna, ZE Ambudkar, SV Brigham Young Univ Dept Chem & Biochem Provo UT 84602 USA NCI NIH Cell Biol Lab Div Basic Sci Bethesda MD 20878 USA

A benzophenone photoaffinity label 9 based on the polyene natural product (-)-stipiamide has been constructed using a diaminoethane spacer and the radioactive agent [H-3]-BZDC (N-succinimidyl p-benzoyl-(2,3-H-3)-dehydrocinnamate). Photoaffinity experiments show specific binding to human P-glycoprotein (Pgp) in the presence of cis-flupentixol but not with cyclosporin A. (C) 2000 Elsevier Science Ltd. All rights reserved.

关键词：亲和力标记物/化学合成亲和力标记物/化学亲和力标记物/药代动力学二苯甲酮类/化学合成二苯甲酮类/化学二苯甲酮类/药代动力学药物设计模型分子分子构象聚烯类/化学聚烯类/药代动力学琥珀酰亚胺类氚人类

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Calpain inhibitors based on the quiescent affinity label concept:: High rates of calpain inactivation with leaving groups derived from N-hydroxy peptide coupling reagents

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BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 2000年第20期10卷 2315-2319页

作者： Tripathy, R Ator, MA Mallamo, JP Cephalon Inc W Chester PA 19380 USA

A series of irreversible inhibitors of recombinant calpain has been synthesized and their rates of inactivation have been evaluated against calpain and cathepsin B, respectively. The design of the inhibitors was based on the quiescent affinity label concept. By choosing the appropriate affinity group and by employing leaving groups derived from N-hydroxy coupling reagents, good inhibitors of calpain with high rates of inactivation have been identified. However, poor aqueous stability limits their therapeutic utility. (C) 2000 Elsevier Science Ltd. All rights reserved.

关键词：亲和力标记物/化学合成亲和力标记物/化学亲和力标记物/药理学卡配因/拮抗剂和抑制剂组织蛋白酶B/拮抗剂和抑制剂半胱氨酸蛋白酶抑制剂/化学合成半胱氨酸蛋白酶抑制剂/化学半胱氨酸蛋白酶抑制剂/药理学二肽类/化学合成二肽类/化学二肽类/药理学药物设计指示剂和试剂寡肽类/化学合成寡肽类/化学寡肽类/药理学重组蛋白质类/拮抗剂和抑制剂构效关系人类

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Covalent labeling of adenylyl cyclase cytosolic domains with γ-methylimidazole-2′,5′-dideoxy-[γ-³²P]3′-ATP and the mechanism for P-site-mediated inhibition

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第49期274卷 34745-34750页

作者： Doronin, S Murray, L Dessauer, CW Johnson, RA SUNY Stony Brook Hlth Sci Ctr Dept Physiol & Biophys Stony Brook NY 11794 USA Univ Texas SW Med Ctr Dept Pharmacol Dallas TX 75235 USA

A truncated first cytosolic domain of type V adenylyl cyclase (VC1) and a truncated second cytosolic domain of type II adenylyl cyclase (IIC2) were used alone and in the readily reversible complex (***2) to evaluate interactions with each other and with reversible and irreversible P-site ligands, Enzyme activity was used to assess formation and dissolution of ***2. The data suggest that binding of 2',5'-dideoxy-3'-ATP to VC1 and IIC2 prevented formation of ***2 and that 2',5'-dideoxy-3'-ATP dissociation occurred slowly. To enable configuration specific cross-linking to the catalytic site, 2',5'-dideoxyadenosine 3'-[gamma-(1-methylimidazole)-triphosphate] (gamma-MetIm-2', 5'-dd-3'-ATP) and 2',5'-dd-adenosine 3'-(gamma-azidoanilido)-triphosphate (gamma-azidoanilido-2',5'-dd-3'-ATP) were synthesized, the former also as its gamma-P-32-labeled analog. gamma-Azidoanilido-2',5'-dd-3'-ATP exhibited an inhibitory potency comparable with that of 2',5'-dd-3'-ATP, gamma-MetIm-2',5'-dd-[gamma-P-32]3'-ATP labeled the individual VC1 and IIC2 domains comparably and covalently to similar to 20% within 1 h. Formation of ***2 resulted in reduced labeling of VC1 but enhanced labeling of IIC2. The data imply that formation of the catalytically active ***2 complex affects the interaction of each domain with the 2',5'-dd-3'-ATP, the binding of which also affects the interaction between the two cytosolic domains, leading to a pseudo irreversible inhibition.

关键词：腺苷三磷酸/类似物和衍生物腺苷三磷酸/化学合成腺苷三磷酸/代谢腺苷三磷酸/药理学亲和力标记物/化学合成亲和力标记物/代谢叠氮化合物/化学合成叠氮化合物/代谢交联试剂/代谢胞质溶胶/酶学双脱氧核苷酸类剂量效应关系药物大肠杆菌/代谢咪唑类/化学合成咪唑类/代谢同工酶类动力学时间因素

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A general method for producing bioaffinity MALDI probes

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ANALYTICAL CHEMISTRY 1999年第10期71卷 2014-2020页

作者： Wang, H Tseng, K Lebrilla, CB Univ Calif Davis Dept Chem Davis CA 95616 USA

A bioaffinity probe based on the idea of immobilizing avidin on the probe surface to extract biotinylated oligosaccharide is described. The probe is produced by taking advantage of the natural affinity of proteins for hydrophobic polymer films. The avidin is immobilized by simply drying the solution on a polymer film surface. This produces a bioaffinity probe that shows enhanced activity for biotin-labeled oligosaccharides. The probe is produced in a matter of minutes but is highly effective for concentrating biotinylated oligosaccharide on the surface. The best matrix for the analysis is DHB, and the best him for the probe is a polyester material commonly used for transparency film. The efficacy of the probe is illustrated with neutral and anionic oligosaccharides. Oligosaccharides derivatized with biotin are retained while those that are unlabeled are washed away. No trace of the unlabeled oligosaccharide is observed in the mass spectrum.

关键词：亲和力标记物/化学合成生物素/化学合成碳水化合物序列分子序列数据寡糖类/化学合成光谱法质量基质辅助激光解吸电离/方法

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Structure-based design of nonnatural ligands for the HLA-B27 protein

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JOURNAL OF RECEPTOR AND SIGNAL TRANSDUCTION RESEARCH 1999年第1-4期19卷 645-657页

作者： Dédier, S Krebs, S Lamas, JR Poenaru, S Folkers, G de Castro, JAL Seebach, D Rognan, D Swiss Fed Inst Technol Dept Pharm CH-8057 Zurich Switzerland Swiss Fed Inst Technol Organ Chem Lab CH-8092 Zurich Switzerland Univ Autonoma Madrid Ctr Biol Mol Severo Ochoa Fac Ciencias E-28049 Madrid Spain

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove, acid thus decrease more or less the affinity of the altered epitopes to HLA-B*2705. Bifunctional spacers ((R)-3-hydroxybutyrate and beta-homoalanine combinations) not only bridges the two distant anchoring amino acids but also strongly interact with the binding cleft and lead to an increase in binding to the MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.

关键词：亲和力标记物/化学合成亲和力标记物/化学亲和力标记物/代谢氨基酸序列圆二色性药物设计 HLA-B27抗原/化学 HLA-B27抗原/代谢配体寡肽类/化学合成寡肽类/化学寡肽类/代谢蛋白质变性重组融合蛋白质类/化学重组融合蛋白质类/代谢构效关系人类

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Reactive affinity probes for the mapping of the glycine-binding site of the NMDA receptor NR1 subunit

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JOURNAL OF RECEPTOR AND SIGNAL TRANSDUCTION RESEARCH 1999年第1-4期19卷 547-557页

作者： Kreimeyer, A Laube, B Sturgess, M Goeldner, M Foucaud, B ULP Fac Pharm Chim Bioorgan Lab CNRS UMR 7514 F-67401 Illkirch Graffenstaden France Max Planck Inst Brain Res D-60528 Frankfurt Germany Bearsden Bio Inc Aston PA 19014 USA

The glycine co-agonist binding site of the NMDA receptor is a target for the prevention and treatment of neurotoxic and neurodegenerative conditions. Until now, the interactions taking place at this site, and its structure, have been investigated by ligand structure-activity relationships and by site-directed mutagenesis. On the basis of a structural model which is currently proposed for this site, we have designed and synthesized six affinity markers by substituting electrophilic reactive groups in the 4, the 7 and the 3' positions of L 701,324, a high-affinity glycine site antagonist. These compounds compete with H-3-DCKA binding to rat brain membranes at equilibrium with nanomolar to low-micromolar affinities, and antagonize glycine-evoked currents in oocytes transfected with wild-type NR1-NR2B. However, they do not induce a time-shift in binding equilibria, and do not inactivate irreversibly the glycine evoked currents. Since they react only with cysteine at physiological pH, we conclude that there is no such residue in the site, in agreement with the model. Our affinity markers therefore represent potential topological probes for NMDA receptors with sequence positions related to the glycine-binding site mutated into cysteine.

关键词：亲和力标记物/化学合成亲和力标记物/化学亲和力标记物/代谢结合部位脑/代谢兴奋性氨基酸拮抗剂/化学合成兴奋性氨基酸拮抗剂/化学兴奋性氨基酸拮抗剂/代谢甘氨酸/化学动力学配体分子结构蛋白质构象喹诺酮类/化学喹诺酮类/代谢喹诺酮类/药理学动物大鼠

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