RESUMO
Oxabornyl polyenes represent a unique group of polyketides characterized by a central polyene core flanked by a conserved oxabornyl moiety and a structurally diverse oxygen heterocyclic ring. They are widely distributed in fungi and possess a variety of biological activities. Due to the significant spatial separation between the two stereogenic ring systems, it is difficult to establish their overall relative configurations. Here, we isolated three oxabornyl polyenes, prugosenes A1-A3 (1-3), from Talaromyces sp. JNU18266-01. Although these compounds were first reported from Penicillium rugulosum, their overall relative and absolute configurations remained unassigned. By employing ozonolysis in combination with ECD calculations, we were able to establish their absolute configurations, and additionally obtained seven new chemical derivatives (4-10). Notably, through NMR data analysis and quantum chemical calculations, we achieved the structural revision of prugosene A2. Furthermore, prugosenes A1-A3 exhibited potent antiviral activity against the respiratory syncytial virus, with compound 1 displaying an IC50 value of 6.3 µM. Our study thus provides a valuable reference for absolute configuration assignment of oxabornyl polyene compounds.
Assuntos
Polienos , Polienos/química , Polienos/farmacologia , Estrutura Molecular , Talaromyces/química , Antivirais/farmacologia , Antivirais/química , Vírus Sinciciais Respiratórios/efeitos dos fármacos , HumanosRESUMO
In light of the worthy design flexibility and the good signal amplification capacity, the recently developed DNA motor (especially the DNA walker)-based fluorescent biosensors can offer an admirable choice for realizing bioimaging. However, this attractive biosensing strategy not only has the disadvantage of uncontrollable initiation but also usually demands the supplement of exogenous driving forces. To handle the above obstacles, some rewarding solutions are proposed here. First, on the surface of an 808 nm near-infrared light-excited low-heat upconversion nanoparticle, a special ultraviolet upconversion luminescence-initiated three-dimensional (3D) walking behavior is performed by embedding a photocleavage linker into the sensing elements, and such light-controlled target recognition can perfectly overcome the pre-triggering of the biosensor during the biological delivery to significantly boost the sensing precision. After that, a peculiar self-driven walking pattern is constructed by employing MnO2 nanosheets as an additional nanovector to physically absorb the sensing frame, for which the reduction of the widespread glutathione in the biological medium can bring about sufficient self-supplied Mn2+ to guarantee the walking efficiency. By selecting an underlying next-generation broad-spectrum cancer biomarker (survivin messenger RNA) as the model target, we obtain that the newly formed autonomous 3D DNA motor shows a commendable sensitivity (where the limit of detection is down to 0.51 pM) and even an outstanding specificity for distinguishing single-base mismatching. Beyond this sound assay performance, our sensing approach is capable of working as a powerful imaging platform for accurately operating in various living specimens such as cells and bodies, showing a favorable diagnostic ability for cancer care.
Assuntos
Técnicas Biossensoriais , Nanopartículas , DNA/genética , Glutationa , Luminescência , Compostos de Manganês , ÓxidosRESUMO
The further development of high-performance fluorescent biosensors to image intracellular microRNAs is beneficial to cancer medicine. By virtue of the need for enzymes and hairpin DNA probes, the entropy-driven reaction-assisted signal amplification strategy has shown an enormous potential to accomplish this task. Nevertheless, this good option still meets with poor biostability, low cell uptake efficiency, and unsatisfactory accuracy. On the basis of these challenges, we put forward here a battery of solving pathways. First, the straight DNA probes are anchored onto the vertexes of dual DNA tetrahedrons, and thus the enzyme resistance of the whole sensing system is observably enhanced. A metal-organic framework (ZIF-8 nanoparticle), which can be effectively dissociated into a weakly acidic environment, then is employed as an additional delivery vehicle to encapsulate such a DNA tetrahedron sustained biosensor and finally bring about a more efficient endocytosis. Last, a kind of photocleavage-linker triggered photoresponsive manner is incorporated to achieve an exceptional precise target identification, by which the biosensor can only be initiated under the irradiation of an externally mild 365 nm ultraviolet light source. In accordance with the above efforts, worthy assay performance toward microRNA-196a has given rise to this newly constructed biosensor, whose sensitivity is down to 2.7 pM and also able to distinguish single-base variation. Beyond that, the amplifier can work as a powerful imaging toolbox to accurately determine the targets in living cells, providing a promising intracellular sensing platform.
Assuntos
Técnicas Biossensoriais , Estruturas Metalorgânicas , MicroRNAs , DNA , Entropia , MicroRNAs/genéticaRESUMO
Despite that the currently discovered CRISPR-Cas12a system is beneficial for improving the detection accuracy and design flexibility of luminescent biosensors, there are still challenges to extend target species and strengthen adaptability in complicated biological media. To conquer these obstacles, we present here some useful strategies. For the former, the limitation to nucleic acids assay is broken through by introducing a simple functional DNA regulation pathway to activate the unique trans-cleavage effect of this CRISPR system, under which the expected biosensors are capable of effectively transducing a protein (employing dual aptamers) and a metal ion (employing DNAzyme). For the latter, a time-gated luminescence resonance energy transfer imaging manner using a long-persistent nanophosphor as the energy donor is performed to completely eliminate the background interference and a nature-inspired biomimetic periodic chip constructed by photonic crystals is further combined to enhance the persistent luminescence. In line with the above efforts, the improved CRISPR-Cas12a luminescent biosensor not only exhibits a sound analysis performance toward the model targets (carcinoembryonic antigen and Na+) but also owns a strong anti-interference feature to actualize accurate sensing in human plasma samples, offering a new and applicative analytical tool for laboratory medicine.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Biomimética , Sistemas CRISPR-Cas/genética , DNA/genética , Humanos , LuminescênciaRESUMO
Herein, the biological effects of heavy rare earth ion Er(III) on rice mitochondria were comprehensively investigated mainly by spectroscopic methods. The experimental results demonstrated that Er(III) could lead to the swelling of rice mitochondria, collapse of mitochondrial transmembrane potential, decrease of membrane fluidity, promotion of H(+) permeability and suppression of K(+) permeability. These further indicated that Er(III) could induce the mitochondrial permeability transition (MPT) and the dysfunction of rice mitochondria. The ultra-structure change of mitochondria observed by transmission electron microscopy (TEM) also proved that Er(III) induced MPT. Moreover, the testing results of the protective effect of four different agents on mitochondrial swelling implied that the thiol chelation on the mitochondrial inner membrane was the main reason that caused the MPT.
Assuntos
Érbio/farmacologia , Mitocôndrias/metabolismo , Oryza/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Oryza/efeitos dos fármacosRESUMO
Ytterbium (Yb), a widely used rare earth element, is treated as highly toxic to human being and adverseness to plant. Mitochondria play a significant role in plant growth and development, and are proposed as a potential target for ytterbium toxicity. In this paper, the biological effect of Yb(3+) on isolated rice mitochondria was investigated. We found that Yb(3+) with high concentrations (200 ~ 600 µM) not only induced mitochondrial membrane permeability transition (mtMPT), but also disturbed the mitochondrial ultrastructure. Moreover, Yb(3+) caused the respiratory chain damage, ROS formation, membrane potential decrease, and mitochondrial complex II activity reverse. The results above suggested that Yb(3+) with high concentrations could induce mitochondrial membrane dysfunction. These findings will support some valuable information to the safe application of Yb-based agents.
Assuntos
Membranas Mitocondriais/metabolismo , Oryza/efeitos dos fármacos , Oryza/metabolismo , Itérbio/toxicidade , Transporte de Elétrons/efeitos dos fármacos , Complexo II de Transporte de Elétrons/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Ácido Succínico/metabolismoRESUMO
Lycibarbarspermidines are unusual phenolamide glycosides characterized by a dicaffeoylspermidine core with multiple glycosyl substitutions, and serve as a major class of bioactive ingredients in the wolfberry. So far, little is known about the enzymatic basis of the glycosylation of phenolamides including dicaffeoylspermidine. Here, we identify five lycibarbarspermidine glycosyltransferases, LbUGT1-5, which are the first phenolamide-type glycosyltransferases and catalyze regioselective glycosylation of dicaffeoylspermidines to form structurally diverse lycibarbarspermidines in wolfberry. Notably, LbUGT3 acts as a distinctive enzyme that catalyzes a tandem sugar transfer to the ortho-dihydroxy group on the caffeoyl moiety to form the unusual ortho-diglucosylated product, while LbUGT1 accurately discriminates caffeoyl and dihydrocaffeoyl groups to catalyze a site-selective sugar transfer. Crystal structure analysis of the complexes of LbUGT1 and LbUGT3 with UDP, combined with molecular dynamics simulations, revealed the structural basis of the difference in glycosylation selectivity between LbUGT1 and LbUGT3. Site-directed mutagenesis illuminates a conserved tyrosine residue (Y389 in LbUGT1 and Y390 in LbUGT3) in PSPG box that plays a crucial role in regulating the regioselectivity of LbUGT1 and LbUGT3. Our study thus sheds light on the enzymatic underpinnings of the chemical diversity of lycibarbarspermidines in wolfberry, and expands the repertoire of glycosyltransferases in nature.
Assuntos
Glicosiltransferases , Lycium , Glicosiltransferases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosilação , Lycium/enzimologia , Lycium/metabolismo , Lycium/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Glicosídeos/metabolismo , Glicosídeos/química , Cristalografia por Raios X , Piperidinas/metabolismo , Piperidinas/química , Especificidade por SubstratoRESUMO
As an essential micronutrient for humans, the metabolism of copper is fine-tuned by evolutionarily conserved homeostatic mechanisms. Copper toxicity occurs when its concentration exceeds a certain threshold, which has been exploited in the development of copper ionophores, such as elesclomol, for anticancer treatment. Elesclomol has garnered recognition as a potent anticancer drug and has been evaluated in numerous clinical trials. However, the mechanisms underlying elesclomol-induced cell death remain obscure. The discovery of cuproptosis, a novel form of cell death triggered by the targeted accumulation of copper in mitochondria, redefines the significance of elesclomol in cancer therapy. Here, we provide an overview of copper homeostasis and its associated pathological disorders, especially copper metabolism in carcinogenesis. We summarize our current knowledge of the tumor suppressive mechanisms of elesclomol, with emphasis on cuproptosis. Finally, we discuss the strategies that may contribute to better application of elesclomol in cancer therapy.
Assuntos
Cobre , Neoplasias , Humanos , Espécies Reativas de Oxigênio/metabolismo , Cobre/metabolismo , Hidrazinas , Neoplasias/tratamento farmacológicoRESUMO
To enhance the biological degradation of volatile organic sulfur compounds, a microbial fuel cell (MFC) system with superior activity is developed for dimethyl disulfide (DMDS) degradation. The MFC achieves a removal efficiency near 100% within 6 h (initial concentration: 90 mg L-1) and a maximum biodegradation rate constant of 0.743 mM h-1. The DMDS removal load attains 2.684 mmol h-1 L-1, which is 6.18-2440 times the loads of conventional biodegradation processes reported. Meanwhile, the maximum power density output and corresponding current density output are 5.40 W m-3 and 40.6 A m-3, respectively. The main mechanism of extracellular electron transfer is classified as mediated electron transfer, supplemented by direct transfer. Furthermore, the mass balance analysis indicates that methanethiol, S0, S2-, SO42-, HCHO, and CO2 are the main intermediate and end products involved in the hybrid metabolism pathway of DMDS. Overall, these findings may offer basic information for bioelectrochemical degradation of DMDS and facilitate the application of MFC in waste gas treatment. ENVIRONMENTAL IMPLICATION: Dimethyl disulfide (DMDS), which features poor solubility, odorous smell, and refractory property, is a typical pollutant emitted from the petrochemical industry. For the first time, we develop an MFC system for DMDS degradation. The superior DMDS removal load per unit reactor volume is 6.18-2440 times those of conventional biodegradation processes in literature. Both the electron transfer route and the hybrid metabolism pathway of DMDS are cleared in this work. Overall, these findings give an in-depth understanding of the bioelectrochemical DMDS degradation mechanism and provide an efficient alternative for DMDS removal.
Assuntos
Fontes de Energia Bioelétrica , Elétrons , Dissulfetos , Transporte de ElétronsRESUMO
Microbial electrolysis cell (MEC) with a biocathode could provide extra reaction driving force for gaseous chlorobenzene (CB) removal. In this work, external potentials (-0.1 to -0.7â¯V vs. SHE) were applied to regulate the biocathodic activity. Results showed -0.3â¯V was the optimum potential, while the removal efficiency, dechlorination efficiency and Coulombic efficiency achieved 94%, 65%, and 89%, respectively. Electrochemical stimulation enriched dechlorination microorganisms (Achromobacter and Gordonia), and significantly improved CB mineralization efficiency, which was twice higher than that without additional potential at 300â¯mgâ¯m-3 inlet concentration. Furthermore, electron transfer between biocathode and microorganisms was mainly through direct electron transfer (DET). A new integrated redox pathway for CB anaerobic degradation was proposed, in which CB was sequentially converted into 2-chlorophenol and 3-chlorocatechol, then dechlorinated to catechol, and finally mineralized into CO2. Overall, this work provided an insight into gaseous CB bioelectrochemical degradation through the potential regulation.
Assuntos
Eletrólise , Gases , Clorobenzenos , Eletrodos , OxirreduçãoRESUMO
Taking advantage of outstanding precision in target recognition and trans-cleavage ability, the recently discovered CRISPR/Cas12a system provides an alternative opportunity for designing fluorescence biosensors. To fully exploit the analytical potential, we introduce here some meaningful concepts. First, the collateral cleavage of CRISPR/Cas12a is efficiently activated in a functional DNA regulation manner and the bottleneck which largely applicable to nucleic acids detection is broken. After selection of a representative aptamer and DNAzyme as the transduction pathways, the sensing coverage is extended to a small organic compound (ATP) and a metal ion (Na+). The assay sensitivity is significantly improved by utilizing a bead-supported enrichment strategy wherein emerging holographic optical tweezers are used to enhance imaging stability and simultaneously achieve multiflux analysis. Last, a sandwich-structured energy-concentrating upconversion nanoparticle triggered boosting luminescent resonance energy transfer mode is comined to face with complicated biological samples by skillfully confining the emitters into a very limited inner shell. Following the above attempts, the developed CRISPR/Cas12a biosensors not only present an ultrasensitive assay behavior toward these model non-nucleic acid analytes but also can serve as a formidable toolbox for determining real samples including single cell lysates and human plasma, proving a good practical application capacity.
Assuntos
Técnicas Biossensoriais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas , DNA , Humanos , Pinças ÓpticasRESUMO
Benefiting from the outstanding signal amplification effect and the admirable construction flexibility, the currently proposed DNA motors (particularly DNA walkers) based biosensing concepts have provided a forceful fluorescence imaging tool for intracellular detection. Even so, this promising sensing means is not only subject to poor controllability and prone to produce false signals but also requires exogenous powering forces owing to the common employment of DNAzyme. In response to these challenges, we are herein motivated to present some meaningful solving strategies. For one thing, the surfaces of gold nanoparticles are conducted with a photo-gated walking behavior by introducing a photocleave mode, under which the light-switchable DNA walkers are capable of being selectively activated via an external ultraviolet source to faultlessly prevent the sensing frame from being pre-initiated during cellular uptake and intracellular delivery. For another, the intracellular biothiols are consumed by MnO2 nanosheets to effectively avoid the competitions to Au-S bonds to eliminate potential false outputs and also self-supply sufficient cofactors (Mn2+) to actualize a self-powered operation pattern as well as facilitate the endocytosis process. Following these breakthroughs, a favorable analysis performance towards a model tumor biomarker (survivin mRNA) is endowed with the newly raised biosensor, whose sensitivity is low to pM level with a sound specificity for identifying single base mismatching. Moreover, the significantly improved autonomous three-dimensional DNA walkers can be used to determine and dynamically trace the targets in live cancer cells with an exceptional precise and efficient manner, commendably impelling the sensing ability of DNA motors in biological specimens.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Nanopartículas Metálicas , MicroRNAs , DNA/genética , Ouro , Compostos de Manganês , Óxidos , RNA Mensageiro/genética , Survivina/genéticaRESUMO
Owing to their favorable design flexibility and eminent signal amplification ability, DNA nanomachine-supported biosensors have provided an attractive avenue for intracellular fluorescence imaging, especially for DNA walkers. However, this promising option not only suffers from poor controllability but also needs to be supplied with additional driving forces on account of the frequent employment of metal ion-dependent DNAzymes. Aiming at overcoming these obstacles, we introduce some fruitful solutions. On one hand, innovative light-activated walking behavior induced by a photocleavage mode is established on the surfaces of gold nanoparticles, and such a photoselective sensing system can be perfectly prevented from pre-activating during the intracellular delivery process and made to achieve target identification only under irradiation using a moderate ultraviolet light source. On the other hand, this light-switchable sensing frame is encapsulated within a dissociable metal-organic framework (ZIF-8) to facilitate endocytosis and ensure sufficient internal cofactors (Zn2+) to realize a self-driven pattern in the acidic environment of the cell lysosome. Based on the abovementioned efforts, the newly constructed autonomous three-dimensional DNA walkers present satisfactory sensitivity (a limit of detection of down to 19.4 pM) and specificity (even distinguishing single-base changes) toward a model biomarker (microRNA-21). More importantly, the sensing method allows determination of the variations in targets in living cancer cells with exceptional precision and efficiency, offering a powerful assay platform for intracellular imaging.
Assuntos
DNA/química , DNA/metabolismo , Luz , MicroRNAs/metabolismo , Nanoestruturas/química , Nanotecnologia/métodos , Imagem Óptica/métodos , Sobrevivência Celular , Células HeLa , Humanos , Células MCF-7 , Estruturas Metalorgânicas/químicaRESUMO
Apart from gene editing capacity, the newly discovered CRISPR/Cas systems offer an exciting option for biosensing field because of their excellent target recognition accuracy. However, the currently constructed sensors are not only limited to nucleic acid analysis but also suffer from poor adaptability in complex samples and unsatisfying sensitivity. We herein introduce some advanced concepts to break through these bottlenecks. First, the sensing targets are extended by skillfully designing a functional DNA such as aptamer (for protein) and DNAzyme (for metal ion) to regulate the transduction of non-nucleic acid species and further activate the trans cleavage of CRISPR/Cas12a. Second, a boosting upconversion luminescent resonance energy is triggered by using a peculiar energy-confining notion, whereby the luminescence domain is intensively restricted in a very narrow space (~2.44 nm) and up to 92.9% of the green emission can be quenched by the approaching BHQ-1 modified reporters. Third, a bio-inspired periodic arrangement biomimetic chip (photonic crystal) is employed to selectively reflect the upconversion luminescence to achieve noteworthy signal enhancement (~35-fold). By utilizing very simple detection devices (a 980 nm portable laser and a smartphone), the CRISPR/Cas12a biosensor shows commendable sensitivity and specificity toward model targets (ATP and Na+, limits of detection are ~ 18 nM and ~0.37 µM, respectively). More importantly, the analysis of real complex samples demonstrate that the as-proposed platform can work as a powerful toolbox for monitoring the ATP fluctuation in single cell and point-of-care testing Na+ in human plasma, enabling a broad application prospect.
Assuntos
Técnicas Biossensoriais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Biomimética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , HumanosRESUMO
Mitochondrial dysfunction is considered as a crucial mechanism of nanomaterial toxicity. Herein, we investigated the effects of polyhydroxylated fullerene (C60(OH)44, fullerenol), a model carbon-based nanomaterial with high water solubility, on isolated mitochondria. Our study demonstrated that fullerenol enhanced the permeabilization of mitochondrial inner membrane to H(+) and K(+) and induced mitochondrial permeability transition (MPT). The fullerenol-induced swelling was dose-dependent and could be effectively inhibited by MPT inhibitors such as cyclosporin A (CsA), adenosine diphosphate (ADP), ruthenium red (RR) and ethylenediaminetetraacetic acid (EDTA). After treating the mitochondria with fullerenol, the mitochondrial membrane potential (MMP) was found collapsed in a concentration-independent manner. The fluorescence anisotropy of hematoporphyrin (HP) changed significantly with the addition of fullerenol, while that of 1,6-diphenyl-hexatriene (DPH) changed slightly. Moreover, a decrease of respiration state 3 and increase of respiration state 4 were observed when mitochondria were energized with complex II substrate succinate. The results of transmission electron microscopy (TEM) provided direct evidence that fullerenol damaged the mitochondrial ultrastructure. The investigations can provide comprehensive information to elucidate the possible toxic mechanism of fullerenols at subcellular level.
Assuntos
Fulerenos/toxicidade , Mitocôndrias Hepáticas/efeitos dos fármacos , Animais , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Mitocôndrias Hepáticas/fisiologia , Mitocôndrias Hepáticas/ultraestrutura , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Membranas Mitocondriais/ultraestrutura , Ratos WistarRESUMO
The effects of lanthanum and calcium on heat production of mitochondria isolated from Carassius auratus liver were investigated by microcalorimetry, and their effects on mitochondrial swelling and membrane potential (Δψ) were determined by spectroscopic methods. La(3+) showed only inhibitory action on mitochondrial energy turnover with inhibition concentration of 50 % (IC50) being 71.2 µmol L(-1). Similarly, Ca(2+) restrained the heat production of mitochondria, and the IC50 of Ca(2+) was much higher than that of La(3+). In the spectroscopic experiments, La(3+) and Ca(2+) induced fish liver mitochondrial swelling and decreased membrane potential (Δψ), and the induction ability of La(3+) was stronger than that of Ca(2+). It is concluded that the effects of La(3+) and Ca(2+) on fish liver mitochondria differ, and La represents toxic action rather than Ca analogy.
Assuntos
Cálcio/farmacologia , Carpa Dourada/metabolismo , Lantânio/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Animais , PermeabilidadeRESUMO
The effects of lanthanum on heat production of mitochondria isolated from Wistar rat liver were investigated with microcalorimetry; simultaneously, the effects on mitochondrial swelling and membrane potential (Δψ) were determined by spectroscopic methods. La(3+) showed only inhibitory action on mitochondrial energy turnover with IC50 being 55.8 µmol L(-1). In the spectroscopic experiments, La(3+), like Ca(2+), induced rat liver mitochondrial swelling and decreased membrane potential (Δψ), which was inhibited by the specific permeability transition inhibitor, cyclosporine A (CsA). The induction ability of La(3+) was stronger than that of Ca(2+). These results demonstrated that La(3+) had some biotoxicity effect on mitochondria; the effects of La(3+) and Ca(2+) on rat liver mitochondrial membrane permeability transition (MPT) are different, and La represents toxic action rather than Ca analogy.
Assuntos
Lantânio/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/farmacologia , Calorimetria/métodos , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Imunossupressores/farmacologia , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Ratos Wistar , EspectrofotometriaRESUMO
Here we describe an algorithm for identifying peptides/ proteins of known sequence and unknown peptides from partial spectra generated by an in-source decay (ISD) technique coupled with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. The identification of protein fragments is processed with a software program called CMATCH, which generates candidate subsequences for both known peptides/proteins and unknown peptides for the major product ions in the spectral range m/z 400-5000 and then matches these to known protein sequences contained in a reference database for the known peptides/proteins. CMATCH, which is compiled for MSDOS or WINDOWS95/NT, has two main advantages: first, the candidate subsequences are generated automatically without the need for supplementary information concerning the distribution of either N-terminal or C-terminal ions in the spectra for both known peptides/proteins and unknown peptides; second, the highest coordinated homologous sequences are picked up automatically from the reference database as the best matches with known peptides/proteins. Examples from the ISD spectra of several test proteins demonstrate the efficacy of this protein identification software.