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1.
Mol Cell Biochem ; 479(3): 579-590, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37129769

RESUMO

Antibody drug conjugates (ADCs) with twelve FDA approved drugs, known as a novel category of anti-neoplastic treatment created to merge the monoclonal antibody specificity with cytotoxicity effect of chemotherapy. However, despite many undeniable advantages, ADCs face certain problems, including insufficient internalization after binding, complex structures and large size of full antibodies especially in targeting of solid tumors. Camelid single domain antibody fragments (Nanobody®) offer solutions to this challenge by providing nanoscale size, high solubility and excellent stability, recombinant expression in bacteria, in vivo enhanced tissue penetration, and conjugation advantages. Here, an anti-human CD22 Nanobody was expressed in E.coli cells and conjugated to Mertansine (DM1) as a cytotoxic payload. The anti-CD22 Nanobody was expressed and purified by Ni-NTA resin. DM1 conjugated anti-CD22 Nanobody was generated by conjugation of SMCC-DM1 to Nanobody lysine groups. The conjugates were characterized using SDS-PAGE and Capillary electrophoresis (CE-SDS), RP-HPLC, and MALDI-TOF mass spectrometry. Additionally, flow cytometry analysis and a competition ELISA were carried out for binding evaluation. Finally, cytotoxicity of conjugates on Raji and Jurkat cell lines was assessed. The drug-to-antibody ratio (DAR) of conjugates was calculated 2.04 using UV spectrometry. SDS-PAGE, CE-SDS, HPLC, and mass spectrometry confirmed conjugation of DM1 to the Nanobody. The obtained results showed the anti-CD22 Nanobody cytotoxicity was enhanced almost 80% by conjugation with DM1. The binding of conjugates was similar to the non-conjugated anti-CD22 Nanobody in flow cytometry experiments. Concludingly, this study successfully suggest that the DM1 conjugated anti-CD22 Nanobody can be used as a novel tumor specific drug delivery system.


Assuntos
Imunoconjugados , Maitansina , Neoplasias , Anticorpos de Domínio Único , Anticorpos Monoclonais/farmacologia , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Maitansina/química , Neoplasias/tratamento farmacológico , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Camelidae/imunologia
2.
Anal Biochem ; 681: 115319, 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-37716512

RESUMO

The study of antibody-antigen interactions, through epitope mapping, enhances our understanding of antibody neutralization and antigenic determinant recognition. Epitope mapping, employing monoclonal antibodies and mass spectrometry, has emerged as a rapid and precise method to investigate viral antigenic determinants. In this report, we propose an approach to improve the accuracy of epitopic peptide interaction rate recognition. To achieve this, we investigated the interaction between the nucleocapsid protein of fig mosaic virus (FMV-NP) and single-chain variable fragment antibodies (scFv-Ab). These scFv-Ab maintain high specificity similar to whole monoclonal antibodies, but they are smaller in size. We coupled this with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The experimental design involved using two different enzymes to digest FMV-NP separately. The resulting peptides were then incubated separately with the desired scFv-Ab at different incubation times and antibody concentrations. This allowed us to monitor the relative rate of epitopic peptide interaction with the antibody. The results demonstrated that, at a 1:1 ratio and after 2 h of interaction, the residues 122-136, 148-157, and 265-276 exhibited high-rate epitopic peptide binding, with reductions in peak intensity of 78%, 21%, and 22%, respectively. Conversely, the residues 250-264 showed low-rate binding, with a 15% reduction in peak intensity. This epitope mapping approach, utilizing scFv-Ab, two different enzymes, and various incubation times, offers a precise and dependable analysis for monitoring and recognizing the binding kinetics of antigenic determinants. Furthermore, this method can be applied to study any kind of antigens.

3.
J Proteome Res ; 21(11): 2566-2585, 2022 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-36173113

RESUMO

Safranal, as an aroma in saffron, is one of the cytotoxic compounds in saffron that causes cell death in triple-negative breast cancer cells. Our recent research reported the anti-cancer effects of safranal, which further demonstrated its impact on protein translation, mitochondrial dysfunction, and DNA fragmentation. To better understand the underlying mechanisms, we identified acetylated and phosphorylated peptides in safranal-treated cancer cells. We conducted a comprehensive phosphoproteomics and acetylomics analysis of safranal-treated MDA-MB-231 cells by using a combination of TMT labeling and enrichment methods including titanium dioxide and immunoprecipitation. We provide a wide range of phosphoproteome regulation in different signaling pathways that are disrupted by safranal treatment. Safranal influences the phosphorylation level on proteins involved in DNA replication and repair, translation, and EGFR activation/accumulation, which can lead the cells into apoptosis. Safranal causes DNA damage which is followed by the activation of cell cycle checkpoints for DNA repair. Over time, checkpoints and DNA repair are inhibited and cells are under a mitotic catastrophe. Moreover, safranal prevents repair by the hypo-acetylation of H4 and facilitates the transcription of proapoptotic genes by hyper-acetylation of H3, which push the cells to the brink of death.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Cicloexenos/farmacologia , Terpenos/farmacologia , Apoptose
4.
Rapid Commun Mass Spectrom ; 36(1): e9212, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34661948

RESUMO

RATIONALE: Surface functionalization is considered to be the foundation for developing nanomaterial applications in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. However, the surface properties of nanostructures can influence their interaction with the analyte and consequently the mass data. In the present study, functionalized nanoparticles (NPs) were used for MALDI-MS and laser desorption/ionization mass spectrometry (LDI-MS) experiments in order to evaluate the effect of the surface properties of NPs on tailoring the intensity of mass signals. METHODS: Regarding the LDI-MS analyses, the surface of superparamagnetic iron oxide nanoparticles (SPIONs) was coated with nitrosonium tetrafluoroborate, citric acid, nitrodopamine, and gallic acid. Additionally, the SPIONs were applied as a matrix to analyze three small peptides. In the MALDI-MS analyses, silica NPs were selected as co-matrix and functionalized with cysteine, sulfobetaine, and amine alkoxysilanes. Then, the silica NPs were utilized as additives in the MALDI-MS samples of four proteins in a mass range between ~2000 and 60,000 Da. RESULTS: The results of LDI-MS analyses demonstrated more than one order enhancement in the signal intensity of analytes based on the amount of electrostatic interaction and laser energy absorption by the surface ligands. However, those of MALDI-MS experiments indicated a significant signal improvement when achieving the colloidal stability of silica NPs in the matrix solution. CONCLUSIONS: Based on the results, the surface properties of NPs affected the (MA)LDI-MS analyses indispensably. Finally, the functionalization of SPIONs represented a new model for the future development of NPs with both affinity and enhanced ionization abilities in mass spectrometry.


Assuntos
Nanopartículas Magnéticas de Óxido de Ferro/química , Peptídeos/química , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Dióxido de Silício/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação
5.
Chirality ; 34(10): 1371-1382, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35778873

RESUMO

Noscapine is an isolated compound from the opium poppy, with distinctive chiral structure and chemistry, interacts with other compounds due to having multiple π-acceptors, hydrogen bond acceptors, and ionic sites. Therefore, it has promising applicability for the enantioselective separation of a wide range of polar, acidic, basic, and neutral compounds. A new noscapine derivative chiral stationary phase (ND-CSP) has been synthesized by consecutive N-demethylation, reduction, and N-propargylation of noscapine followed by attachment of a solid epoxy-functionalized silica bed through the 1,3-dipolar Huisgen cycloaddition. The noscapine derivative-based stationary phase provides a considerable surface coverage, which is greater than some commercial CSPs and can validate better enantioresolution performance. The major advantages inherent to this chiral selector are stability, reproducibility after more than 200 tests, and substantial loading capacity. The characterization by Fourier transform infrared (FTIR) spectroscopy and elemental analysis indicated successful functionalization of the silica surface. Chromatographic method conditions like flow rate and mobile phase composition for enantioseparation of various compounds such as warfarin, propranolol, mandelic acid, and a sulfanilamide derivative were optimized. Comparing the experimental results with docking data revealed a clear correlation between the calculated binding energy of ND-CSP and each enantiomer with the resolution of enantiomer peaks.


Assuntos
Noscapina , Cromatografia Líquida de Alta Pressão/métodos , Reprodutibilidade dos Testes , Dióxido de Silício/química , Estereoisomerismo
6.
Metab Brain Dis ; 37(1): 229-241, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34302583

RESUMO

The hydrogen/deuterium exchange (HDX) is a reliable method to survey the dynamic behavior of proteins and epitope mapping. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a quantifying tool to assay for HDX in the protein of interest. We combined HDX-MALDI-TOF MS and molecular docking/MD simulation to identify accessible amino acids and analyze their contribution into the structural changes of profilin-1 (PFN-1). The molecular docking/MD simulations are computational tools for enabling the analysis of the type of amino acids that may be involved via HDX identified under the lowest binding energy condition. Glycine to valine amino acid (G117V) substitution mutation is linked to amyotrophic lateral sclerosis (ALS). This mutation is found to be in the actin-binding site of PFN-1 and prevents the dimerization/polymerization of actin and invokes a pathologic toxicity that leads to ALS. In this study, we sought to understand the PFN-1 protein dynamic behavior using purified wild type and mutant PFN-1 proteins. The data obtained from HDX-MALDI-TOF MS for PFN-1WT and PFN-1G117V at various time intervals, from seconds to hours, revealed multiple peaks corresponding to molecular weights from monomers to multimers. PFN-1/Benzaldehyde complexes identified 20 accessible amino acids to HDX that participate in the docking simulation in the surface of WT and mutant PFN-1. Consistent results from HDX-MALDI-TOF MS and docking simulation predict candidate amino acid(s) involved in the dimerization/polymerization of PFNG117V. This information may shed critical light on the structural and conformational changes with details of amino acid epitopes for mutant PFN-1s' dimerization, oligomerization, and aggregation.


Assuntos
Esclerose Lateral Amiotrófica , Medição da Troca de Deutério , Profilinas , Esclerose Lateral Amiotrófica/genética , Biologia Computacional , Deutério , Humanos , Simulação de Acoplamento Molecular , Profilinas/química , Profilinas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
J Proteome Res ; 20(1): 895-908, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33225711

RESUMO

Saw-scaled or carpet vipers (genus Echis) are considered to cause a higher global snakebite mortality than any other snake. Echis carinatus sochureki (ECS) is a widely distributed snake species, also found across the thirteen provinces of Iran, where it is assumed to be responsible for the most snakebite envenomings. Here, we collected the Iranian specimens of ECS from three different geographically distinct populations, investigated food habits, and performed toxicity assessment and venom proteome profiling to better understand saw-scaled viper life. Our results show that the prey items most commonly found in all populations were arthropods, with scorpions from the family Buthidae particularly well represented. LD50 (median lethal dose) values of the crude venom demonstrate highly comparable venom toxicities in mammals. Consistent with this finding, venom characterization via top-down and bottom-up proteomics, applied to both crude venoms and size-exclusion chromatographic fractions, revealed highly comparable venom compositions among the different populations. By combining all proteomics data, we identified 22 protein families from 102 liquid chromatography and tandem mass spectrometry (LC-MS/MS) raw files, including the most abundant snake venom metalloproteinases (SVMPs, 29-34%); phospholipase A2 (PLA2s, 26-31%); snake venom serine proteinases (SVSPs, 11-12%); l-amino acid oxidases (LAOs, 8-11%), C-type lectins/lectin-like (CTLs, 7-9%) protein families, and many newly detected ones, e.g., renin-like aspartic proteases (RLAPs), fibroblast growth factors (FGFs), peptidyl-prolyl cis-trans isomerases (PPIs), and venom vasodilator peptides (VVPs). Furthermore, we identified and characterized methylated, acetylated, and oxidized proteoforms relating to the PLA2 and disintegrin toxin families and the site of their modifications. It thus seems that post-translational modifications (PTMs) of toxins, particularly target lysine residues, may play an essential role in the structural and functional properties of venom proteins and might be able to influence the therapeutic response of antivenoms, to be investigated in future studies.


Assuntos
Proteômica , Venenos de Víboras , Animais , Cromatografia Líquida , Irã (Geográfico) , Espectrometria de Massas em Tandem , Venenos de Víboras/toxicidade
8.
Rapid Commun Mass Spectrom ; 35(2): e8964, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33002226

RESUMO

RATIONALE: Plasma-assisted ionization is widely used in mass spectrometry; in this study, a low-pressure glow discharge is introduced as a new method to improve the detection of large proteins, and bovine serum albumin (BSA) is used as a protein model. The treatment of analyte, matrix, and the matrix/analyte mixture is evaluated under optimal conditions. METHODS: Low-pressure radio-frequency capacitively coupled plasma (RF-CCP) treatment is utilized in the sample preparation step of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to enhance the protein MALDI ion signal. Plasma treatment can be an effective tool for enhancing the non-covalent binding of the analyte with the matrix, incorporation of the analyte into the matrix, production of matrix/analyte crystals, and analyte protonation through plasma activation, resulting in an improved MALDI ion signal. RESULTS: Fourier-transform infrared (FTIR) spectroscopy allows us to distinguish between the functional groups of plasma-treated and control samples. In addition, optical emission spectroscopy (OES) determines the plasma species, and zeta potential analysis characterizes the potential difference between plasma-treated and control samples before MALDI-TOF MS analysis. Plasma-treated BSA can provide a five-times enhancement of ion intensity. The combination of the plasma-treated analyte with the plasma-treated matrix leads to an increase in the ion intensity by a factor of 14. CONCLUSIONS: Low-pressure glow discharge plasma treatment greatly enhances MALDI ion signals, with a noticeable increase in incorporation, co-crystallization, protonation, and the concentration of the sample functional groups.


Assuntos
Gases em Plasma/química , Proteínas/análise , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Íons , Soroalbumina Bovina , Espectroscopia de Infravermelho com Transformada de Fourier
9.
Anal Chem ; 92(15): 10460-10469, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32484340

RESUMO

The ability of mass spectrometry for discrimination between protein and peptide masses which are unique to specific pathogens provides an accurate and fast method for the detection of different types of pathogens, especially viruses. Capsid proteins are specific to each virus and can be used as a biomarker for detection of this pathogen. On the other hand, single-chain variable fragment (scFv) antibodies have been recently used to enhance the accuracy of immunoassay techniques. So conjugation of mass spectrometry and scFv antibody provides a very accurate and fast method for the detection of viruses. In this report, for the first time, we have immobilized scFv antibody of fig mosaic virus (FMV) on the magnetic nanoparticles (MNPs) to extract the virus capsid protein from complex biological media and subsequently identified this protein through both its intact molecular mass and peptide mass fingerprint (PMF) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).


Assuntos
Compostos Férricos/química , Nanopartículas Metálicas/química , Vírus de Plantas/isolamento & purificação , Anticorpos de Cadeia Única/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fenômenos Magnéticos , Mapeamento de Peptídeos , Sensibilidade e Especificidade
10.
Anal Biochem ; 609: 113909, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-32827466

RESUMO

Protein A, a major cell wall component of Staphylococcus aureus, is one of the first immunoglobulin-binding proteins that is discovered about 80 years ago. However, a great deal of development in both purification methods and application of antibodies in treatment have been done. There are many publications based on the untargeted (size exclusion, ion exchange and hydrophobic interactions) and targeted (affinity) methods by scientists in academic/industry groups. In this review, we have focused on the study of both native and engineered Protein A to understand its mechanism in the purification of antibodies. What domain of Protein A dose interact with antibody? Where are contact regions? What is the non-covalent interaction mechanism of Protein A and antibody? Does alkaline condition, in the washing step, influence on antibody structure and activity? On the other hand, the immobilization of Protein A on various sorbents such as agarose, silica, polysaccharide, polymers, and magnetic nanoparticles have investigated. Also, the application of Protein A as biosensor for detection of the antibody is discussed. We have tried to find interesting and stimulating answers to all these questions.


Assuntos
Anticorpos/metabolismo , Cromatografia de Afinidade/métodos , Proteína Estafilocócica A/metabolismo , Anticorpos/isolamento & purificação , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Nanopartículas/química , Engenharia de Proteínas , Estabilidade Proteica , Sefarose/química , Proteína Estafilocócica A/química , Proteína Estafilocócica A/genética , Staphylococcus aureus/metabolismo
11.
Chirality ; 32(11): 1289-1298, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32797693

RESUMO

So far, no detailed view has been expressed regarding the interactions between vancomycin and racemic compounds including mandelic acid. In the current study, a chiral stationary phase was prepared by using 3-aminopropyltriethoxysilane and succinic anhydride to graft carboxylated silica microspheres and subsequently by activating the carboxylic acid group for vancomycin immobilization. Characterization by elemental analysis, Fourier transform infrared spectroscopy, solid-state nuclear magnetic resonance, and thermogravimetric analysis demonstrated effective functionalization of the silica surface. R and S enantiomers of mandelic acid were separated by the synthetic vancomycin column. Finally, the interaction between vancomycin and R/S mandelic acid enantiomers was simulated by Auto-dock Vina. The binding energies of interactions between R and S enantiomers and vancomycin chiral stationary phase were different. In the most probable interaction, the difference in mandelic acid binding energy was approximately 0.2 kcal/mol. In addition, circular dichroism spectra of vancomycin interacting with R and S enantiomers showed different patterns. Therefore, R and S mandelic acid enantiomers may occupy various binding pockets and interact with different vancomycin functions. These observations emphasized the different retention of R and S mandelic acid enantiomers in vancomycin chiral column.


Assuntos
Ácidos Mandélicos/química , Ácidos Mandélicos/isolamento & purificação , Simulação de Acoplamento Molecular , Vancomicina/química , Conformação Molecular , Estereoisomerismo , Termodinâmica
12.
Rapid Commun Mass Spectrom ; 33 Suppl 1: 20-27, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30076652

RESUMO

The venom produced by snakes contains complex mixtures of pharmacologically active proteins and peptides which play a crucial role in the pathophysiology of snakebite diseases. The deep understanding of venom proteomes can help to improve the treatment of this "neglected tropical disease" (as expressed by the World Health Organization [WHO]) and to develop new drugs. The most widely used technique for venom analysis is liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based bottom-up (BU) proteomics. Considering the fact that multiple multi-locus gene families encode snake venom proteins, the major challenge for the BU proteomics is the limited sequence coverage and also the "protein inference problem" which result in a loss of information for the identification and characterization of toxin proteoforms (genetic variation, alternative mRNA splicing, single nucleotide polymorphism [SNP] and post-translational modifications [PTMs]). In contrast, intact protein measurements with top-down (TD) MS strategies cover almost complete protein sequences, and prove the ability to identify venom proteoforms and to localize their modifications and sequence variations.


Assuntos
Proteoma , Proteômica , Venenos de Serpentes , Animais , Cromatografia Líquida de Alta Pressão , Proteoma/análise , Proteoma/química , Análise de Sequência de Proteína , Venenos de Serpentes/análise , Venenos de Serpentes/química , Espectrometria de Massas em Tandem
13.
Anal Biochem ; 557: 123-130, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30030996

RESUMO

5-methylcytosine (5mC) is an epigenetic mark which has a profound effect on various fundamental processes in cells. In present study, at first Hydrophilic Interaction Liquid Chromatography (HILIC) was compared with Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) based on their selectivity (α), retention factor (k), and resolution (R) for cytosine (C) and 5mC nucleobases. We tried to justify the separation mechanism on the basis of mobile phase and solute polarity, structural characterization of solute and stationary phases, log Do/w, and pka under both modes. Then, these two modes were compared in order to select the best column for measurement of methylation level in two real samples with less analytical complexity (i.e. animal and bacteria) and a highly complex sample (i.e. plant), after chemical hydrolysis of DNA. In this favor, diol and cyano (CN) columns in HILIC mode as well as C8 and C18 in RP-HPLC were investigated. Optimum separation and the best validation parameters were obtained for CN column with Limit of Detection (LOD) of 1.4 pmol and Limit of Quantification (LOQ) of 4.8 pmol for 5mC. When the CN column was used in HILIC-UV procedure, separation of 5mC and C bases was achieved in all types of hydrolyzed DNA solutions.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Metilação de DNA , DNA/análise , Interações Hidrofóbicas e Hidrofílicas , Animais , Bovinos , DNA/metabolismo
14.
Cell Mol Biol (Noisy-le-grand) ; 64(9): 6-15, 2018 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-30030949

RESUMO

Resistance to antibiotics is an emerging and growing threat. To address this threat, attempts are being made by researchers to identify the Volatile Organic Compounds (VOCs) of bacteria. It is believed that unique combinations could be found among the VOCs produced by each microorganism. The current study aimed to identify and compare the VOCs of antibiotic-resistant and standard strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. A polymer of divinylbenzene /carboxen /polydimethylsiloxane was applied for absorption of volatile compounds in headspace bacterial samples in form of a solid phase micro-extraction fiber holder. Gas chromatography-mass spectrometry technique was used for identification of volatile compounds. The analysis of the VOCs indicated that some VOCs appeared only in standard strains while others were common only among resistant strains. Exclusive VOCs to a specific strain were also detected. This study demonstrated that resistant strains of bacteria produced VOCs that were different from those of the standard strains. In addition, VOCs released by bacteria after passing the logarithmic growth phase showed no significant differences. The identification of VOCs can be a precise way to differentiate bacterial species, also it can be said that the VOCs produced by different pathogenic microorganisms can be the suitable biomarkers for their detection.


Assuntos
Bactérias/química , Infecções Respiratórias/microbiologia , Compostos Orgânicos Voláteis/análise , Acinetobacter baumannii/química , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Farmacorresistência Bacteriana , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Klebsiella pneumoniae/química , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Infecções Respiratórias/patologia , Microextração em Fase Sólida , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Compostos Orgânicos Voláteis/isolamento & purificação
15.
Metab Brain Dis ; 33(6): 1975-1984, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30203378

RESUMO

Single amino acid mutations in profilin 1 (PFN1) have been found to cause amyotrophic lateral sclerosis (ALS). Recently, we developed a mouse model for ALS using a PFN1 mutation (glycine 118 to valine, G118V), and we are now interested in understanding how PFN1 becomes toxically lethal with only one amino acid substitution. Therefore, we studied mutation-related changes in the PFN1 protein and hypothesized that such changes significantly disturb its structure. Initially, we expressed and studied the purified PFN1WT and PFN1G118V proteins from bacterial culture. We found that the PFN1G118V protein has a different mean residue ellipticity, as measured by far-UV circular dichroism, accompanied by a spectral shift. The intrinsic fluorescence of PFN1G118V showed a small fluctuation in maximum fluorescence absorption and intensity. Moreover, we examined the time course of PFN1 aggregation using SDS-PAGE, western blotting, and MALDI-TOF/TOF and found that compared with PFN1WT, PFN1G118V had an increased tendency to form aggregates. Dynamic light scattering data confirmed this, showing a larger size distribution for PFN1G118V. Our data explain why PFN1G118V tends to aggregate, a phenotype that may be the basis for its neurotoxicity.


Assuntos
Esclerose Lateral Amiotrófica/genética , Mutação/genética , Profilinas/química , Profilinas/genética , Agregados Proteicos/genética , Humanos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
16.
Chirality ; 29(10): 579-588, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28749545

RESUMO

An interesting mode of chromatography for preparation of pure enantiomers from pure samples is the method of stacked injection as a pseudocontinuous procedure. Maximum throughput and minimal production costs can be achieved by the use of total chiral column length in this mode of chromatography. To maximize sample loading, often touching bands of the two enantiomers is automatically achieved. Conventional equations show direct correlation between touching-band loadability and the selectivity factor of two enantiomers. The important question for one who wants to obtain the highest throughput is "How to optimize different factors including selectivity, resolution, run time, and loading of the sample in order to save time without missing the touching-band resolution?" To answer this question, tramadol and propranolol were separated on cellulose 3,5-dimethyl phenyl carbamate, as two pure racemic mixtures with low and high solubilities in mobile phase, respectively. The mobile phase composition consisted of n-hexane solvent with alcohol modifier and diethylamine as the additive. A response surface methodology based on central composite design was used to optimize separation factors against the main responses. According to the stacked injection properties, two processes were investigated for maximizing throughput: one with a poorly soluble and another with a highly soluble racemic mixture. For each case, different optimization possibilities were inspected. It was revealed that resolution is a crucial response for separations of this kind. Peak area and run time are two critical parameters in optimization of stacked injection for binary mixtures which have low solubility in the mobile phase.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Injeções , Solubilidade , Solventes/química , Estereoisomerismo
17.
J Sep Sci ; 40(18): 3602-3611, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28714264

RESUMO

Hydrophilic interaction liquid chromatography on bare silica presents some benefits for analysis and purification of ionizable basic alkaloids. This mode was used to separate five major opium alkaloids: morphine, codeine, thebaine, papaverine, and noscapine. Central composite design based on response surface methodology was applied for experimental design, modeling, and optimization in a single-step gradient method. The main effects and their interactions (initial percentage of modifier, changing range of modifier in run time, pH of buffer, and its concentration) were investigated in 30 experiments. Multivariate curve resolution-alternating least squares, by resolving overlapped curves, helped in the accurate calculation of baseline resolution factors to be modeled and optimized more accurately. Then three crucial resolution factors besides elution time were modeled in quadratic and cubic equations and optimized. In addition to the four factors, five extra logarithmic, and nonlogarithmic factors extracted from the four factors to give nine factors overall were inspected on mechanism of retention. It was shown that a linear combination consist of four independence variables successfully describes morphinans retentivity in a single-step gradient method.


Assuntos
Alcaloides/isolamento & purificação , Cromatografia Líquida , Ópio/química , Codeína/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Morfina/isolamento & purificação , Noscapina/isolamento & purificação , Papaverina/isolamento & purificação , Tebaína/isolamento & purificação
18.
Biomed Chromatogr ; 31(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27461958

RESUMO

Profiling and monitoring concentrations of key hormones in body have long been critical aims in clinical therapy. As a crucial hormone, identification and quantification of ghrelin is a fundamental, often key, step in understanding human physiological mechanisms. Through the advances and improvements of different analytical techniques, ghrelin measurement is generally feasible, and the number of successful reports is progressively being increased with new aspects of selectivity, sensitivity and ease of use in various circumstances. Herein we discuss current chromatographic methods for sample collection, separation and a mass spectrometry method for detection and measurement of ghrelin and other proghrelin-derived peptides in biological metrics. We describe the most commonly applied analytical LC-MS procedures for determination of proghrelin-derived peptides and provide illustrative instances representing the state of the art. This review is intended for bioanalytical chemists or clinical researchers who are interested in this field of research.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Grelina/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Grelina/sangue , Humanos , Peptídeos/análise , Peptídeos/sangue
19.
Biomed Chromatogr ; 31(8)2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28058728

RESUMO

Male factor infertility is involved in almost half of all infertile couples. Lack of the ejaculated sperm owing to testicular malfunction has been reported in 6-10% of infertile men, a condition named nonobstructive azoospermia (NOA). In this study, we investigated untargeted metabolomic profiling of the seminal plasma in NOA men using gas chromatography-mass spectrometry and advance chemometrics. In this regard, the seminal plasma fluids of 11 NOA men with TESE-negative, nine NOA men with TESE-positive and 10 fertile healthy men (as a control group) were collected. Quadratic discriminate analysis (QDA) technique was implemented on total ion chromatograms (TICs) for identification of discriminatory retention times. We developed multivariate classification models using the QDA technique. Our results revealed that the developed QDA models could predict the classes of samples using their TIC data. The receiver operating characteristic curves for these models were >0.88. After recognition of discriminatory retention time's asymmetric penalized least square, evolving factor analysis, correlation optimized warping and alternating least squares strategies were applied for preprocessing and deconvolution of the overlapped chromatographic peaks. We could identify 36 discriminatory metabolites. These metabolites may be considered discriminatory biomarkers for different groups in NOA.


Assuntos
Azoospermia/metabolismo , Metaboloma , Sêmen/metabolismo , Espermatogênese , Azoospermia/diagnóstico , Azoospermia/fisiopatologia , Análise Discriminante , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Masculino , Metabolômica/métodos
20.
Anal Biochem ; 494: 31-6, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26582432

RESUMO

Diazinon insecticide is widely applied in rice (Oryza sativa L.) fields in Iran. However, concerns are now being raised about its potential adverse impacts on rice. In this study, a time-course metabolic change in rice plants was investigated after diazinon treatment using gas chromatography-mass spectrometry (GC-MS) and subsequently three different methods, MetaboAnalyst, MetaboNetwork, and analysis of reporter reactions, as a potential multivariate method were used to find the underlying changes in metabolism with stronger evidence in order to link differentially expressed metabolites to biological pathways. Results clearly showed the similarity of acetylcholinesterase (AChE) of rice plants to that of animals in terms of its inhibitability by diazinon and emphasized that subsequent accumulation of AChE mainly affects the metabolism of osmolites and tricarboxylic acid intermediates subsequent accumulation of ACh mainly affects the metabolism of osmolites and TCA intermediates.


Assuntos
Diazinon/análise , Cromatografia Gasosa-Espectrometria de Massas , Inseticidas/análise , Oryza/química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Redes e Vias Metabólicas , Metabolômica , Oryza/metabolismo
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