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Affinity Probes (affinity + probe)
Selected AbstractsA Targeted Releasable Affinity Probe (TRAP) for In Vivo PhotocrosslinkingCHEMBIOCHEM, Issue 9 2009Ping Yan Dr. Abstract A protein TRAP: The in vivo photocrosslinking of TRAP after its intracellular targeting to a binding sequence on the bait protein stabilizes protein interactions. Because the crosslinker is releasable, simple mass spectrometry can be used to identify the protein binding sites after purification. Protein crosslinking, especially coupled to mass-spectrometric identification, is increasingly used to determine protein binding partners and protein,protein interfaces for isolated protein complexes. The modification of crosslinkers to permit their targeted use in living cells is of considerable importance for studying protein-interaction networks, which are commonly modulated through weak interactions that are formed transiently to permit rapid cellular response to environmental changes. We have therefore synthesized a targeted and releasable affinity probe (TRAP) consisting of a biarsenical fluorescein linked to benzophenone that binds to a tetracysteine sequence in a protein engineered for specific labeling. Here, the utility of TRAP for capturing protein binding partners upon photoactivation of the benzophenone moiety has been demonstrated in living bacteria and mammalian cells. In addition, ligand exchange of the arsenic,sulfur bonds between TRAP and the tetracysteine sequence to added dithiols results in fluorophore transfer to the crosslinked binding partner. In isolated protein complexes, this release from the original binding site permits the identification of the proximal binding interface through mass spectrometric fragmentation and computational sequence identification. [source] High-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresisELECTROPHORESIS, Issue 12 2008Yilin Li Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source] A Targeted Releasable Affinity Probe (TRAP) for In Vivo PhotocrosslinkingCHEMBIOCHEM, Issue 9 2009Ping Yan Dr. Abstract A protein TRAP: The in vivo photocrosslinking of TRAP after its intracellular targeting to a binding sequence on the bait protein stabilizes protein interactions. Because the crosslinker is releasable, simple mass spectrometry can be used to identify the protein binding sites after purification. Protein crosslinking, especially coupled to mass-spectrometric identification, is increasingly used to determine protein binding partners and protein,protein interfaces for isolated protein complexes. The modification of crosslinkers to permit their targeted use in living cells is of considerable importance for studying protein-interaction networks, which are commonly modulated through weak interactions that are formed transiently to permit rapid cellular response to environmental changes. We have therefore synthesized a targeted and releasable affinity probe (TRAP) consisting of a biarsenical fluorescein linked to benzophenone that binds to a tetracysteine sequence in a protein engineered for specific labeling. Here, the utility of TRAP for capturing protein binding partners upon photoactivation of the benzophenone moiety has been demonstrated in living bacteria and mammalian cells. In addition, ligand exchange of the arsenic,sulfur bonds between TRAP and the tetracysteine sequence to added dithiols results in fluorophore transfer to the crosslinked binding partner. In isolated protein complexes, this release from the original binding site permits the identification of the proximal binding interface through mass spectrometric fragmentation and computational sequence identification. [source] Carbohydrate-Encapsulated Gold Nanoparticles for Rapid Target-Protein Identification and Binding-Epitope MappingCHEMBIOCHEM, Issue 7 2005Yu-Ju Chen Dr. Carbohydrate,lectin recognition plays important roles in cell,cell communication, proliferation, and differentiation. We report here a new approach of using a carbohydrate-encapsulated gold nanoparticle (shown in purple) as an affinity probe for the efficient separation and enrichment of target proteins, and then protein identification and epitope mapping by MALDI-TOF MS. [source] Properties of Human Mitochondrial RibosomesIUBMB LIFE, Issue 9 2003Thomas W. O'Brien Abstract Mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. Typical of mammalian mitochondrial ribosomes, the bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Human mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system. IUBMB Life, 55: 505-513, 2003 [source] Cysteine-capped ZnSe quantum dots as affinity and accelerating probes for microwave enzymatic digestion of proteins via direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2009Lokesh A. Shastri Fluorescent semiconductor quantum dots (QDs) exhibit great potential and capability for many biological and biochemical applications. We report a simple strategy for the synthesis of aqueous stable ZnSe QDs by using cysteine as the capping agent (ZnSe-Cys QDs). The ZnSe QDs can act as affinity probes to enrich peptides and proteins via direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. This nanoprobe could significantly enhance protein signals (insulin, ubiquitin, cytochrome c, myoglobin and lysozyme) in MALDI-TOFMS by 2.5,12 times compared with the traditional method. Additionally, the ZnSe-Cys QDs can be applied as heat absorbers (as accelerating probes) to speed up microwave-assisted enzymatic digestion reactions and also as affinity probes to enrich lysozyme-digested products in MALDI-TOFMS. Furthermore, after the enrichment experiments, the solutions of ZnSe-Cys QDs mixed with proteins can be directly deposited onto the MALDI plates for rapid analysis. This approach shows a simple, rapid, efficient and straightforward method for direct analysis of proteins or peptides by MALDI-TOFMS without the requirement for further time-consuming separation processes, tedious washing steps or laborious purification procedures. The present study has demonstrated that ZnSe-Cys QDs are reliable and potential materials for rapid, selective separation and enrichment of proteins as well as accelerating probes for microwave-digested reactions for proteins than the regular MALDI-MS tools. Additionally, we also believe that this work may also inspire investigations for applications of QDs in the field of MALDI-MS for proteomics. Copyright © 2009 John Wiley & Sons, Ltd. [source] Applications of silver nanoparticles capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight and atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry for rapid analysis of sulfur drugs and biothiols in human urineRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2008Kamlesh Shrivas A strategy is presented for the analysis of sulfur drugs and biothiols using silver nanoparticles (AgNPs) capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) and atmospheric pressure-matrix assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-ITMS). Biothiols adsorbed on the surface of AgNPs through covalent bonding were subjected to ultraviolet (UV) radiation that enabled desorption and ionization due to the excellent photochemical property of NPs. The proposed method has been successfully applied for the determination of cysteine and homocysteine in human urine samples using an internal standard. The limit of detection (LOD) and limit of quantification (LOQ) for cysteine and homocysteine in urine sample are 7 and 22,nM, respectively, with a relative standard deviation (RSD) of <10%. The advantages of the present method compared with the methods reported in the literature for biothiol analysis are simplicity, rapidity and sensitivity without the need for time-consuming separation and tedious preconcentration processes. Additionally, we also found that the bare AgNPs can be directly used as the matrix in MALDI-TOF MS for the analysis of sulfur drugs without the addition of an extra proton source. Copyright © 2008 John Wiley & Sons, Ltd. [source] | |||||||||||||