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1.
Emerg Microbes Infect ; 10(1): 810-821, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33847245

RESUMO

EK1 peptide is a membrane fusion inhibitor with broad-spectrum activity against human coronaviruses (CoVs). In the outbreak of COVID-19, we generated a lipopeptide EK1V1 by modifying EK1 with cholesterol, which exhibited significantly improved antiviral activity. In this study, we surprisingly found that EK1V1 also displayed potent cross-inhibitory activities against divergent HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. Consistently, the recently reported EK1 derivative EK1C4 and SARS-CoV-2 derived fusion inhibitor lipopeptides (IPB02 ∼ IPB09) also inhibited HIV-1 Env-mediated cell-cell fusion and infection efficiently. In the inhibition of a panel of HIV-1 mutants resistant to HIV-1 fusion inhibitors, EK1V1 and IPB02-based inhibitors exhibited significantly decreased or increased activities, suggesting the heptad repeat-1 region (HR1) of HIV-1 gp41 being their target. Furthermore, the sequence alignment and molecular docking analyses verified the target site and revealed the mechanism underlying the resistance. Combined, we conclude that this serendipitous discovery provides a proof-of-concept for a common mechanism of viral fusion and critical information for the development of broad-spectrum antivirals.


Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antivirais/isolamento & purificação , Relação Dose-Resposta a Droga , Inibidores da Fusão de HIV/isolamento & purificação , Inibidores da Fusão de HIV/farmacologia , Humanos , Lipopeptídeos/isolamento & purificação , Lipopeptídeos/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
2.
Molecules ; 26(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800800

RESUMO

Self-assembling peptides have gained attention because of their nanotechnological applications. Previous work demonstrated that the self-assembling peptide f1-8 (Pf1-8) that is generated from the tryptic hydrolysis of ß-lactoglobulin can form a hydrogel after several purification steps, including membrane filtration and consecutive washes. This study evaluates the impact of each processing step on peptide profile, purity, and gelation capacity of each fraction to understand the purification process of Pf1-8 and the peptide-peptide interactions involved. We showed that peptide-peptide interactions mainly occurred through electrostatic and hydrophobic interactions, influencing the fraction compositions. Indeed, the purity of Pf1-8 did not correlate with the number of wash steps. In addition to Pf1-8, two other hydrophobic peptides were identified, peptide f15-20, and peptide f41-60. The gelation observed could be induced either through peptide-peptide interactions or through self-assembling, both being driven by non-covalent bond and more specifically hydrophobic interactions.


Assuntos
Hidrogéis/química , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Multimerização Proteica
3.
Mar Drugs ; 19(4)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916201

RESUMO

More than 7000 red algae species have been classified. Although most of them are underused, they are a protein-rich marine resource. The hydrolysates of red algal proteins are good candidates for the inhibition of the angiotensin-I-converting enzyme (ACE). The ACE is one of the key factors for cardiovascular disease, and the inhibition of ACE activity is related to the prevention of high blood pressure. To better understand the relationship between the hydrolysates of red algal proteins and the inhibition of ACE activity, we attempted to identify novel ACE inhibitory peptides from Pyropia pseudolinearis. We prepared water soluble proteins (WSP) containing phycoerythrin, phycocyanin, allophycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase. In vitro analysis showed that the thermolysin hydrolysate of the WSP had high ACE inhibitory activity compared to that of WSP. We then identified 42 peptides in the hydrolysate by high-performance liquid chromatography and mass spectrometry. Among 42 peptides, 23 peptides were found in chloroplast proteins. We then synthesized the uncharacterized peptides ARY, YLR, and LRM and measured the ACE inhibitory activity. LRM showed a low IC50 value (0.15 µmol) compared to ARY and YLR (1.3 and 5.8 µmol). In silico analysis revealed that the LRM sequence was conserved in cpcA from Bangiales and Florideophyceae, indicating that the novel ACE inhibitory peptide LRM was highly conserved in red algae.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Proteínas de Plantas/farmacologia , Rodófitas/metabolismo , Inibidores da Enzima Conversora de Angiotensina/síntese química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Humanos , Hidrólise , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/isolamento & purificação , Peptidil Dipeptidase A/química , Proteínas de Plantas/isolamento & purificação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
4.
Molecules ; 26(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806637

RESUMO

Marine collagen peptides have high potential in promoting skin wound healing. This study aimed to investigate wound healing activity of collagen peptides derived from Sipunculus nudus (SNCP). The effects of SNCP on promoting healing were studied through a whole cortex wound model in mice. Results showed that SNCP consisted of peptides with a molecular weight less than 5 kDa accounted for 81.95%, rich in Gly and Arg. SNCP possessed outstanding capacity to induce human umbilical vein endothelial cells (HUVEC), human immortalized keratinocytes (HaCaT) and human skin fibroblasts (HSF) cells proliferation and migration in vitro. In vivo, SNCP could markedly improve the healing rate and shorten the scab removal time, possessing a scar-free healing effect. Compared with the negative control group, the expression level of tumor necrosis factor-α, interleukin-1ß and transforming growth factor-ß1 (TGF-ß1) in the SNCP group was significantly down-regulated at 7 days post-wounding (p < 0.01). Moreover, the mRNA level of mothers against decapentaplegic homolog 7 (Smad7) in SNCP group was up-regulated (p < 0.01); in contrast, type II TGF-ß receptors, collagen I and α-smooth muscle actin were significantly down-regulated at 28 days (p < 0.01). These results indicate that SNCP possessed excellent activity of accelerating wound healing and inhibiting scar formation, and its mechanism was closely related to reducing inflammation, improving collagen deposition and recombination and blockade of the TGF-ß/Smads signal pathway. Therefore, SNCP may have promising clinical applications in skin wound repair and scar inhibition.


Assuntos
Cicatriz/tratamento farmacológico , Colágeno/farmacologia , Queratinócitos/efeitos dos fármacos , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , Poliquetos/química , Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Cicatriz/metabolismo , Colágeno/química , Humanos , Queratinócitos/metabolismo , Masculino , Camundongos , Fragmentos de Peptídeos/química , Transdução de Sinais , Pele/metabolismo
5.
Protein Expr Purif ; 183: 105861, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33667651

RESUMO

Sensitive and specific serology tests are essential for epidemiological and public health studies of COVID-19 and for vaccine efficacy testing. The presence of antibodies to SARS-CoV-2 surface glycoprotein (Spike) and, specifically, its receptor-binding domain (RBD) correlates with inhibition of SARS-CoV-2 binding to the cellular receptor and viral entry into the cells. Serology tests that detect antibodies targeting RBD have high potential to predict COVID-19 immunity and to accurately determine the extent of the vaccine-induced immune response. Cost-effective methods of expression and purification of Spike and its fragments that preserve antigenic properties are essential for development of such tests. Here we describe a method of production of His6-tagged S319-640 fragment containing RBD in E. coli. It includes expression of the fragment, solubilization of inclusion bodies, and on-the-column refolding. The antigenic properties of the resulting product are similar, but not identical to the RBD-containing fragment expressed in human cells.


Assuntos
COVID-19/virologia , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Expressão Gênica , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Domínios Proteicos , Redobramento de Proteína , SARS-CoV-2/genética , Solubilidade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/isolamento & purificação
6.
Mar Drugs ; 19(2)2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33546257

RESUMO

Oxidative stress-induced endothelial dysfunction is strongly linked to the pathogenesis of cardiovascular diseases. A previous study revealed that seahorse hydrolysates ameliorated oxidative stress-mediated human umbilical vein endothelial cells (HUVECs) injury. However, the responsible compounds have not yet been identified. This study aimed to identify cytoprotective peptides and to investigate the molecular mechanism underlying the cytoprotective role in H2O2-induced HUVECs injury. After purification by gel filtration and HPLC, two peptides were sequenced by liquid chromatography-tandem mass spectrometry as HGSH (436.43 Da) and KGPSW (573.65 Da). The synthesized peptides and their combination (1:1 ratio) showed significant HUVECs protection effect at 100 µg/mL against H2O2-induced oxidative damage via significantly reducing intracellular reactive oxygen species (ROS). Two peptides and their combination treatment resulted in the increased heme oxygenase-1 (HO-1), a phase II detoxifying enzyme, through the activation of nuclear transcription factor-erythroid 2-related factor (Nrf2). Additionally, cell cycle and nuclear staining analysis revealed that two peptides and their combination significantly protected H2O2-induced cell death through antiapoptotic action. Two peptides and their combination treatment led to inhibit the expression of proapoptotic Bax, the release of cytochrome C into the cytosol, the activation of caspase 3 by H2O2 treatment in HUVECs, whereas antiapoptotic Bcl-2 expression was increased with concomitant downregulation of Bax/Bcl-2 ratio. Taken together, these results suggest that seahorse-derived peptides may be a promising agent for oxidative stress-related cardiovascular diseases.


Assuntos
Citoproteção/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Smegmamorpha , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citoproteção/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/isolamento & purificação
7.
J Food Sci ; 86(3): 1081-1088, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33565626

RESUMO

Hyperuricemia is related to plenty of diseases, seriously damaging human health. Current clinical drugs used to treat hyperuricemia have many adverse effects. In this study, kidney bean hydrolysate (KBH) was found to exert high xanthine oxidase inhibitory (XOI) activity. Compared to KBH (50.31 ± 2.73%), XOI activities of three fractions (Mw <5 kDa, Mw <3 kDa, Mw  < 1 kDa) by ultrafiltration were higher and increased to 58.58 ± 3.57%, 59.34 ± 1.78%, and 55.05 ± 5.00%, respectively (P < 0.05). A total of 69 peptides were identified by HPLC-ESI-MS/MS and analyzed binding affinities with XO with the help of molecular docking. AVDSLVPIGR, DWYDIK, LDNLLR, ISPIPVLK, ISSLEMTR showed well binding affinities with XO and DWYDIK presented the highest XOI activity (68.63 ± 5.07%) among five synthetic peptides (P < 0.05). Additionally, visual analysis results indicated that DWYDIK was pushed into the hydrophobic channel and formed hydrogen bonds with pivotal amino acids of xanthine oxidase. Overall, KBH could be a promising candidate as anti- hyperuricemia functional food. PRACTICAL APPLICATION: This research initially revealed that kidney bean peptides could significantly inhibit the activity of xanthine oxidase, indicating kidney bean peptides could be a treatment for hyperuricemia. Kidney bean peptides may have commercial potentials as a safer alternative with few side effects to drugs.


Assuntos
Simulação por Computador , Inibidores Enzimáticos/farmacologia , Fragmentos de Peptídeos/farmacologia , Phaseolus/química , Extratos Vegetais/farmacologia , Xantina Oxidase/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/isolamento & purificação , Humanos , Hiperuricemia/tratamento farmacológico , Hiperuricemia/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/isolamento & purificação , Extratos Vegetais/análise , Extratos Vegetais/isolamento & purificação , Espectrometria de Massas em Tandem , Ultrafiltração
8.
Food Chem ; 340: 128152, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33032150

RESUMO

Soy protein isolates were fermented by three commercial Lactobacillus helveticus strains for a maximum of seven days to investigate the resulting proteolysis. The proteolytic activity of the most active strain (LH88) was further analyzed (LC-MS/MS and GC-MS) and it was shown that the ß-conglycinin α subunit 1, ß-conglycinin α' subunit, glycinin G1, and 2S albumin were specifically degraded. Peptigram analysis and visualization of the crystal structure showed that the hydrolysis sites of ß-conglycinin α subunit, α' subunit, and the glycinin G1 were located on the surface of the molecule or at the mobile disordered region, hence being highly accessible for the proteinase of LH88. The proteins were partially further degraded to free amino acids, and subsequently catabolized to volatile compounds. However, most of the proteins remained native, even after seven days of fermentation, thus additional modification of protein structure or adjustment of fermentation conditions are required for effective generation of flavor compounds.


Assuntos
Lactobacillus helveticus/metabolismo , Proteínas de Soja/metabolismo , Aminoácidos/análise , Técnicas de Cultura Celular por Lotes , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Lactobacillus helveticus/crescimento & desenvolvimento , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Peptídeos/análise , Peptídeos/metabolismo , Proteólise , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/isolamento & purificação , Proteínas de Armazenamento de Sementes/metabolismo , Proteínas de Soja/química , Proteínas de Soja/isolamento & purificação , Espectrometria de Massas em Tandem , Compostos Orgânicos Voláteis/análise
9.
J Ethnopharmacol ; 264: 113382, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32918991

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Crassostrea gigas Thunberg and other oysters have been traditionally used in China as folk remedies to invigorate the kidney and as natural aphrodisiacs to combat male impotence. AIM OF THE STUDY: Erectile dysfunction (ED) has become a major health problem for the global ageing population. The aim of this study is therefore to evaluate the effect of peptide-rich preparations from C. gigas oysters on ED and related conditions as increasing evidence suggests that peptides are important bioactive components of marine remedies and seafood. MATERIALS AND METHODS: Crassostrea oyster peptide (COP) preparations COP1, COP2 and COP3 were obtained from C. gigas oysters by trypsin, papain or sequential trypsin-papain digestion, respectively. The contents of testosterone, cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) and the activity of nitric oxide synthase (NOS) in mice and/or cells were measured by enzyme-linked immunosorbent assays. Real-time PCR was used to assess the expression of genes associated with sex hormone secretion pathways. The model animal Caenorhabditis elegans was also used to analyze the gene expression of a conserved steroidogenic enzyme. In silico analysis of constituent peptides was performed using bioinformatic tools based on public databases. RESULTS: The peptide-rich preparation COP3, in which >95% peptides were <3000 Da, was found to increase the contents of male mouse serum testosterone and cAMP, both of which are known to play important roles in erectile function, and to increase the activity of mouse penile NOS, which is closely associated with ED. Further investigation using mouse Leydig-derived TM3 cells demonstrates that COP3 was able to stimulate the production of testosterone as well as NO, a pivotal mediator of penile erection. Real-time PCR analysis reveals that COP3 up-regulated the expression of Areg and Acvr2b, the genes known to promote sex hormone secretion, but not Fst, a gene involved in suppressing follicle-stimulating hormone release. Furthermore, COP3 was also shown to up-regulate the expression of let-767, a well-conserved C. elegans gene encoding a protein homologous to human 17-ß-hydroxysteroid dehydrogenases. Preliminary bioinformatic analysis using the peptide sequences in COP3 cryptome identified 19 prospective motifs, each of which occurred in more than 10 peptides. CONCLUSIONS: In this paper, Crassostrea oyster peptides were prepared by enzymatic hydrolysis and were found for the first time to increase ED-associated biochemical as well as molecular biology parameters. These results may help to explain the ethnopharmacological use of oysters and provide an important insight into the potentials of oyster peptides in overcoming ED-related health issues.


Assuntos
Fatores Biológicos/isolamento & purificação , Fatores Biológicos/farmacologia , Crassostrea/enzimologia , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , Testosterona/sangue , Animais , Caenorhabditis elegans , Células Cultivadas , Biologia Computacional/métodos , Relação Dose-Resposta a Droga , Ensaios Enzimáticos/métodos , Hidrólise , Masculino , Camundongos
10.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886672

RESUMO

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética
11.
Mol Genet Genomics ; 295(6): 1529-1535, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32894358

RESUMO

Lanthipeptides are a subgroup of ribosomally encoded and post-translationally modified peptides (RiPPs) which frequently possess potent biological activity. Here we provide the first comprehensive bioinformatic analysis of the lanthipeptide-producing capability of the Salinispora genus, a marine actinomycete. One hundred twenty-two Salinispora arenicola, tropica, and pacifica genomic sequences were analyzed for lanthipeptide gene clusters, and the resulting 182 clusters were divided into seven groups based on sequence similarities. Group boundaries were defined based on LanB and LanM sequences with greater than 80% similarity within groups. Of the seven groups, six are predicted to encode class I lanthipeptides while only one group is predicted to encode class II lanthipeptides. Leader and core peptides were predicted for each cluster along with the number of possible lanthionine bridges. Notably, all of the predicted products of these clusters would represent novel lanthipeptide scaffolds. Of the 122 Salinispora genomes analyzed in this study, 92% contained at least one lanthipeptide gene cluster suggesting that Salinispora is a rich, yet untapped, source of lanthipeptides.


Assuntos
Alanina/análogos & derivados , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Micromonosporaceae/metabolismo , Fragmentos de Peptídeos/metabolismo , Sulfetos/metabolismo , Alanina/isolamento & purificação , Alanina/metabolismo , Proteínas de Bactérias/genética , Genômica , Micromonosporaceae/genética , Micromonosporaceae/crescimento & desenvolvimento , Fragmentos de Peptídeos/isolamento & purificação , Sulfetos/isolamento & purificação
12.
PLoS One ; 15(7): e0236740, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722706

RESUMO

Tryptic digestion of proteins followed by liquid chromatography with tandem mass spectrometry analysis is an extensively used approach in proteomics research and biopharmaceutical product characterization, owing to the high level of cleavage fidelity produced with this technique. However, nonspecific trypsin cleavages have been frequently reported and shown to be related to a number of digestion conditions and predigestion sample treatments. In this work, we reveal that, for a number of commercial trypsins, reconstitution and storage conditions can have a significant impact on the occurrence of trypsin nonspecific cleavages. We analyzed the tryptic digestion of a variety of biotherapeutics, using trypsins reconstituted under different conditions. The results indicate that, for many commercial trypsins, commonly recommended reconstitution/storage conditions (mildly acidic, e.g., 50 mM acetic acid, 1 mM HCl) can actually promote nonspecific trypsin activities, which are time dependent and can be as high as 20% in total relative abundance. In contrast, using water for reconstitution and storage can effectively limit nonspecific cleavages to 1%. Interestingly, the performances of different commercial trypsins were found to be quite distinct in their levels of nonspecific cleavages and responses to the two reconstitution conditions. Our findings demonstrate the importance of choosing the appropriate trypsin for tryptic digestion and the necessity of assessing the impact of trypsin reconstitution and storage on nonspecific cleavages. We advocate for manufacturers of commercial trypsins to reevaluate manufacturing processes and reconstitution/storage conditions to provide good cleavage specificity.


Assuntos
Ácidos/química , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Proteólise , Proteômica/métodos , Tripsina/metabolismo , Humanos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/isolamento & purificação
13.
J Vis Exp ; (160)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32597857

RESUMO

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts must be purified for downstream applications. There are various chromatographic methods available for this purpose yet not all labs can afford high performance methods and isolation from complex biological samples can be difficult. Here we demonstrate that preparative liquid-phase isoelectric focusing (IEF) can separate molecules, including small molecules and peptides from complex plant extracts, based on their isoelectric points (pI). We have used the method for complex biological sample fractionation and characterization. As a proof of concept, we fractionated a Gymnema sylvestre plant extract, isolating a family of terpenoid saponin small molecules and a peptide. We also demonstrated effective microbial protein separation using the Candida albicans fungus as a model system.


Assuntos
Produtos Biológicos/isolamento & purificação , Candida albicans/metabolismo , Proteínas Fúngicas/isolamento & purificação , Focalização Isoelétrica/métodos , Fragmentos de Peptídeos/isolamento & purificação , Extratos Vegetais/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Produtos Biológicos/química , Proteínas Fúngicas/química , Gymnema sylvestre/química , Fragmentos de Peptídeos/química , Bibliotecas de Moléculas Pequenas/química
14.
Mikrochim Acta ; 187(2): 144, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31970520

RESUMO

Iron(III-immobilized magnetic nano-composites (MNCs) were first fabricated using one-step aqueous self-assembly of oligopeptides (Glu-Pro-Ala-Lys-Ala-Lys-Ala-Lys; EPAK-VI) for the highly selective capture of phosphopeptides from complex biological samples. Under physiological conditions, EPAK-VI can readily self-organize into a robust and complete coating layer mainly composed of ß-sheets and ß-turns on the surface of Fe3O4@GO and Fe3O4@C MNCs. Tailored by the cyclic structure of proline, the Glu-Pro motifs of EPAK-VI are vertically erected on the surface and thus serve as an effective linker to chelate Fe3+ through carboxyl (COO-) group in the glutamic acid (E) residues. The ionic hydrogen bonds between the ε-amino groups and the surface negative charges coupled with intermolecular hydrogen bonds render the EPAK-VI coating on the MNCs insusceptible to repeated extreme washing conditions. The Fe3+-EPAK-VI coated MNCs exhibit high enrichment efficiency for ß-casein tryptic digest (0.05 fmol µL-1), excellent selectivity from mixed digests (ß-casein/bovine serum albumin, mass ratio 1:500), and high recovery rate (over 80%). Graphical abstractSchematic representation of the fabrication of Fe3+-immobilized MNCs for phosphopeptide enrichment.


Assuntos
Nanopartículas de Magnetita/química , Nanocompostos/química , Oligopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Animais , Caseínas/sangue , Caseínas/química , Caseínas/isolamento & purificação , Bovinos , Grafite/química , Humanos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/isolamento & purificação , Fosfopeptídeos/sangue , Proteólise , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/química
15.
J Pharm Biomed Anal ; 181: 113095, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31962249

RESUMO

An analytical method for the degree of protein deamidation has been developed by using carboxy group derivatization and liquid chromatography-tandem mass spectrometry (LCMS/MS). The fragment peptides (LGEYGFQNALIVR and YNGVFQECCQAEDK) obtained by digesting bovine serum albumin (BSA) with trypsin and their asparagine deamidated peptides (LGEYGFQDALIVR and YDGVFQECCQAEDK) were selected as model peptides, and their carboxy groups were derivatized with ethylamine. This derivatization enabled a clear distinction between natural peptides and deamidated peptides by mass, allowing for facile distinction by LCMS/MS before and after deamidation. Good linearity was confirmed for four peptides used in this study via isotope dilution mass spectrometry, showing that protein deamidation can be evaluated by the present method. To confirm the validity of this method for the evaluation of deamidation, natural peptides and deamidated peptides were mixed in arbitrary ratios, and degree of deamidation in these solution was analyzed. This confirmed that accurate evaluation was possible at deamidation degree values of ca. 10 %, 5 %, 2.5 %, and 1 %. Additionally, an accelerated storage test of BSA demonstrated that the deamidation of asparagine at position 404 of BSA progressed by 4 % in 9 weeks at 40 °C and pH 8 in the dark, and that the deamidation process can be traced over time.


Assuntos
Asparagina/química , Glutamina/química , Fragmentos de Peptídeos/isolamento & purificação , Amidas/química , Ácidos Carboxílicos/química , Química Farmacêutica , Cromatografia Líquida de Alta Pressão/métodos , Armazenamento de Medicamentos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Proteólise , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificação , Espectrometria de Massas em Tandem/métodos
16.
Biosens Bioelectron ; 149: 111840, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31726274

RESUMO

ß-amyloid 1-40 oligomers (Aß40O) is considered to be one of the important biomarkers for the diagnosis and treatment of Alzheimer's disease (AD). To explore a method with excellent performance is favorable for measuring the low concentration of Aß40O in AD patients. Here, we developed a simple and fast method with a double stranded DNA (dsDNA)/graphene oxide (GO) based sensor, which was a fluorescent probe for a highly sensitive detection of Aß40O down to 0.1 nM with a linear detectable range from 0.1 nM to 40 nM. The proposed sensor effectively reduced non-specific adsorption and improved the specificity of detection because of the covalent conjugation of a binding DNA (bDNA) containing Aß40O-targeting aptamer (AptAß) onto GO surface, as well as the optimization of the number of mismatch base pairs of dsDNA. Moreover, AD patients and healthy persons were distinguished by this present method. All advantages of this method are exactly what the clinical detection of AD biomarkers need. This novel aptasensor might pave a way towards the early diagnosis of AD.


Assuntos
Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos beta-Amiloides/química , Pareamento Incorreto de Bases/genética , DNA/química , Grafite/química , Humanos , Limite de Detecção , Fragmentos de Peptídeos/química
17.
Anal Chim Acta ; 1093: 160-167, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31735210

RESUMO

In this study, poly(butyl methacrylate-co-ethyleneglycol dimethacrylate) polymeric monoliths were in situ developed within 0.75 mm i.d. poly(ethylene-co-tetrafluoroethylene) (ETFE) tubing by UV polymerization via three different free-radical initiators (α,α'-azobisisobutyronitrile (AIBN), 2,2-dimethoxy-2-phenylacetophenone (DMPA) and 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MTMPP). The influence of the nature of each photo-initiator and irradiation time on the morphological features of the polymer was investigated by scanning electron microscopy, and the chromatographic properties of the resulting microbore columns were evaluated using alkyl benzenes as test substances. The beds photo-initiated with MTMPP gave the best performance (minimum plate heights of 38 µm for alkyl benzenes) and exhibited a satisfactory reproducibility in the chromatographic parameters (RSD < 11%). These monolithic columns were also successfully applied to the separation of phenylurea herbicides, proteins and a tryptic digest of ß-casein.


Assuntos
Acetofenonas/química , Cromatografia Líquida de Alta Pressão/instrumentação , Morfolinas/química , Nitrilas/química , Ácidos Polimetacrílicos/química , Politetrafluoretileno/análogos & derivados , Propiofenonas/química , Acetofenonas/efeitos da radiação , Caseínas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Herbicidas/isolamento & purificação , Metacrilatos/química , Morfolinas/efeitos da radiação , Nitrilas/efeitos da radiação , Fragmentos de Peptídeos/isolamento & purificação , Compostos de Fenilureia/isolamento & purificação , Polimerização , Ácidos Polimetacrílicos/síntese química , Politetrafluoretileno/química , Propiofenonas/efeitos da radiação , Raios Ultravioleta
18.
Sensors (Basel) ; 19(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744130

RESUMO

Heart failure is a class of cardiovascular diseases that remains the number one cause of death worldwide with a substantial economic burden of around $18 billion incurred by the healthcare sector in 2017 due to heart failure hospitalization and disease management. Although several laboratory tests have been used for early detection of heart failure, these traditional diagnostic methods still fail to effectively guide clinical decisions, prognosis, and therapy in a timely and cost-effective manner. Recent advances in the design and development of biosensors coupled with the discovery of new clinically relevant cardiac biomarkers are paving the way for breakthroughs in heart failure management. Natriuretic neurohormone peptides, B-type natriuretic peptide (BNP) and N-terminal prohormone of BNP (NT-proBNP), are among the most promising biomarkers for clinical use. Remarkably, they result in an increased diagnostic accuracy of around 80% owing to the strong correlation between their circulating concentrations and different heart failure events. The latter has encouraged research towards developing and optimizing BNP biosensors for rapid and highly sensitive detection in the scope of point-of-care testing. This review sheds light on the advances in BNP and NT-proBNP sensing technologies for point-of-care (POC) applications and highlights the challenges of potential integration of these technologies in the clinic. Optical and electrochemical immunosensors are currently used for BNP sensing. The performance metrics of these biosensors-expressed in terms of sensitivity, selectivity, reproducibility, and other criteria-are compared to those of traditional diagnostic techniques, and the clinical applicability of these biosensors is assessed for their potential integration in point-of-care diagnostic platforms.


Assuntos
Técnicas Biossensoriais , Insuficiência Cardíaca/diagnóstico , Peptídeo Natriurético Encefálico/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Biomarcadores/análise , Humanos , Peptídeos Natriuréticos/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito
19.
Analyst ; 144(21): 6270-6275, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31566639

RESUMO

Enhanced-fluidity, reversed-phase liquid chromatography was developed using custom instrumentation for separation and characterization of intact KRas proteins and tryptic peptides. The KRas, HRas and NRas function as GDP-GTP regulated binary switches in many signalling pathways, and mutations in Ras proteins are frequently found in human cancers and represent poor prognosis markers for patients. Mutations of the KRas isoform constitute some of the most common aberrations among all human cancers and intensive drug discovery efforts have been directed toward targeting the KRas protein. Separation and characterization of the KRas protein and tryptic peptides are helpful for exploring targeting, which has not been fully investigated using liquid chromatography-tandem mass spectrometry. EFLC-MS provided improved chromatographic performance compared to traditional HPLC-MS in terms of shorter analysis time, increased ion intensity and a shift to higher charge states for intact KRas proteins.


Assuntos
Cromatografia Líquida/métodos , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Solventes/química
20.
Int J Mol Sci ; 20(20)2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31635129

RESUMO

Velvet antler has a long history in traditional medicine. It is also an important healthy ingredient in food as it is rich in protein. However, there has been no report about antioxidant peptides extracted from velvet antler by enzymatic hydrolysis. Thus, the objective of this study was to hydrolyze velvet antler using different commercial proteases (Acalase, Neutrase, trypsin, pepsin, and α-chymotrypsin). Antioxidant activities of different hydrolysates were investigated using peroxyl radical scavenging assay by electron spin resonance spectrometry. Among all enzymatic hydrolysates, Alcalase hydrolysate exhibited the highest peroxyl radical scavenging activity. Alcalase hydrolysate was then purified using ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. The purified peptide was identified to be Trp-Asp-Val-Lys (tetrapeptide) with molecular weight of 547.29 Da by Q-TOF ESI mass spectroscopy. This purified peptide exhibited strong scavenging activity against peroxyl radical (IC50 value, 0.028 mg/mL). In addition, this tetrapeptide showed significant protection ability against AAPH-induced oxidative stress by inhibiting of reactive oxygen species (ROS) generation in Chang liver cells in vitro and in a zebrafish model in vivo. This research suggests that the tetrapeptide derived from Alcalase-proteolytic hydrolysate of velvet antler are excellent antioxidants and could be effectively applied as functional food ingredients and pharmaceuticals.


Assuntos
Antioxidantes/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Subtilisinas/química , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Chifres de Veado/química , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Humanos , Hidrólise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra
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