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1.
J Agric Food Chem ; 68(7): 2263-2275, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31986019

RESUMO

The growth conditions and age of Panax ginseng are vital for determining the quality of the ginseng plant. However, the considerable difference in price according to the cultivation method and period of P. ginseng leads to its adulteration in the trade market. We herein focused on ginseng peptides and the possibility of these peptides to be used as biomarker(s) for discrimination of P. ginseng. We applied an ultraperformance liquid chromatography-high resolution mass spectrometry-based peptidomics approach to characterize ginseng peptides and discover novel peptide biomarkers for authentication of mountain-cultivated ginseng (MCG). We identified 52 high-confidence peptides and screened 20 characteristic peptides differentially expressed between MCG and cultivated ginseng (CG). Intriguingly, 6 differential peptides were expressed significantly in MCG and originated from dehydrins that accumulated during cold or drought conditions. In addition, 14 other differential peptides that were significantly expressed in CG derived from ginseng major protein, an essential protein for nitrogen storage. These biological associations confirmed the reliability and credibility of the differential peptides. Additionally, we determined several robust peptide biomarkers for discrimination of MCG through a precise selection process. These findings demonstrate the potential of peptide biomarkers for identification and quality control of P. ginseng in addition to ginsenoside analysis.


Assuntos
Panax/química , Peptídeos/química , Sequência de Aminoácidos , Biomarcadores/química , Cromatografia Líquida de Alta Pressão , Análise Discriminante , Contaminação de Alimentos/análise , Espectrometria de Massas , Panax/crescimento & desenvolvimento , Mapeamento de Peptídeos , Raízes de Plantas/química , Raízes de Plantas/crescimento & desenvolvimento , Controle de Qualidade
2.
J Agric Food Chem ; 68(7): 2091-2098, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31927882

RESUMO

In this study, we used reversed-phase high performance liquid chromatography (LC) to isolate three novel peptides with calcium-chelating capacity from tilapia bone collagen hydrolysate. Using LC-tandem mass spectrometry, we determined the amino acid sequences to be GPAGPHGPVG, FDHIVY, and YQEPVIAPKL. We then synthesized the three peptides and verified their calcium-chelating activity. Results showed that the calcium-chelating activity of GPAGPHGPVG, FDHIVY, and YQEPVIAPKL reached 18.80 ± 0.49, 35.73 ± 0.74, and 28.4 ± 0.94 mg/g, respectively. We next investigated how each peptide enhanced intestinal calcium absorption using Caco-2 cell monolayers. Compared with the control group, GPAGPHGPVG, FDHIVY, and YQEPVIAPKL potently enhanced calcium transport within 30 min by 89 ± 9, 202 ± 12, and 130 ± 7%, respectively. Results suggest that these peptides isolated from tilapia bone hydrolysate can be used as dietary supplements to increase calcium absorption.


Assuntos
Osso e Ossos/química , Cálcio/química , Cálcio/metabolismo , Quelantes/química , Colágeno/química , Proteínas de Peixes/química , Mucosa Intestinal/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Humanos , Mapeamento de Peptídeos , Hidrolisados de Proteína/química , Tilápia
3.
J Sci Food Agric ; 100(3): 1246-1255, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31696520

RESUMO

BACKGROUND: Chinese mutton ham is a dry-cured meat product with a long ripening time. The aim of this study was to identify and characterize antioxidant peptides from Chinese mutton ham. RESULTS: Mutton ham peptides (MHPs) were purified by gel filtration, anion exchange and reversed-phase high-performance liquid chromatography steps. The 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging capacity was used to guide the purification of MHPs. Three antioxidant peptides were identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) as Met-Trp-Thr-Asp (MWTD), Ala-Pro-Tyr-Met-Met (APYMM) and Phe-Trp-Ile-Ile-Glu (FWIIE), with molecular weights 551.61, 611.76, and 706.84 Da, respectively. Among them, APYMM exhibited the highest ABTS radical scavenging activity. The three peptides had the ability to inhibit lipid oxidation and Fenton's reagent-induced protein oxidation and DNA damage. After simulated gastrointestinal digestion, FWIIE and APYMM showed increased antioxidant activity, while MWTD showed decreased activity. CONCLUSION: Three novel peptides isolated from Chinese mutton ham had strong biological activity. Chinese mutton ham is potentially a functional food and an excellent source of natural antioxidants. © 2019 Society of Chemical Industry.


Assuntos
Antioxidantes/química , Produtos da Carne/análise , Peptídeos/química , Sequência de Aminoácidos , Animais , China , Cromatografia Líquida , Lipídeos/química , Oxirredução , Mapeamento de Peptídeos , Suínos , Espectrometria de Massas em Tandem
4.
Artigo em Inglês | MEDLINE | ID: mdl-31881514

RESUMO

Peptide mapping by liquid chromatography-mass spectrometry (LC-MS) is a common method for characterizing primary sequences of monoclonal antibodies (mAbs) and their post-translational modifications (PTMs). Most methods prepare digests by incubating samples with proteases from several hours to overnight. This often induces artifacts of some modifications such as deamidation and isomerization, resulting in overestimated product-related modifications levels. Hour-long-digestion can generate complicate chromatographic profiles due to semi-cleavages or other unnecessary reactions, interfering with the quantitation of peaks of interest. On the other hand, shortening digestion-time can cause incomplete peptide cleavages, thus low sequence coverage and poor repeatability. This study applied pressure cycling technology (PCT) to tryptic digestion and PNGase F deglycosyaltion. A 0.5-h PCT assistant tryptic digestion, by alternating cycles of 10-s atmospheric pressure and 50-s high pressure (30 kpsi) at 37 °C, was evaluated and compared with two conventional digestions, 4-h and 18-h (i.e., overnight) incubations at 37 °C under atmospheric pressure. The 0.5-h PCT assistant deglycosylation was also assessed using the same conditions as those by PCT tryptic digestion. The results demonstrated the application of a 0.5-h PCT to tryptic digestion minimized modification artifacts and reduced interference with quantitation by providing a clean chromatographic profile. The 0.5-h PCT assistant deglycosylation completely removed the N-glycans from the Asn301 in the heavy chains of monoclonal antibody with no impact on the chromatographic profile of the tryptic digests.


Assuntos
Anticorpos Monoclonais/análise , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Humanos , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional
5.
Anal Bioanal Chem ; 412(3): 561-575, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31872272

RESUMO

Statically adsorbed or covalently coupled capillary coatings are of crucial importance in capillary electrophoresis-mass spectrometry for the separation of peptides and proteins. So far, published coating strategies and commercially available coated capillaries have a limited pH-stability so that the analysis at strongly acidic pH is limited, or harsh rinsing procedures for biological sample analysis cannot be applied. We here present a capillary coating based on Si-C linkages to N-acryloylamido ethoxyethanol (AAEE) with a new synthetic strategy including LiAlH4 surface reaction. We optimized the coating method with emphasis on stability and reproducibility applying harsh rinsing procedures (strong acid, strong base and organic solvent), using the electroosmotic mobility and separation efficiency of tryptic peptides as performance measure. Complete synthesis is performed in less than 2 days for up to 8 capillaries in parallel of more than 16 m total length. Intra- and inter-batch reproducibility were determined regarding electroosmotic mobility, separation efficiency and migration time precision in CE-MS separations of tryptically digested bovine serum albumin. Coating stability towards rinsing with strong acid (1 mol/L HCl), organic solvent (acetonitrile) and strong base (1 mol/L NaOH) was investigated. Outstanding performance was found for single capillaries. However, inter-capillary reproducibility is discussed critically. The new coating was successfully applied for reproducible CE-MS separation of large proteins in diluted serum, medium-sized peptides and small and highly charged polyamines in fish egg extracts using a very acidic background electrolyte containing 0.75 mol/L acetic acid and 0.25 mol/L formic acid (pH 2.2).


Assuntos
Eletroforese Capilar/métodos , Etanol/análogos & derivados , Espectrometria de Massas/métodos , Concentração de Íons de Hidrogênio , Mapeamento de Peptídeos , Tripsina/química
6.
J Agric Food Chem ; 67(45): 12452-12460, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31674183

RESUMO

Increasing cases of infections by foodborne pathogenic bacteria resulted in a great demand to find safe and novel antimicrobial compounds that can be used in the food industry. The isolation and application of antimicrobial peptides including lipopeptides has been increasing tremendously in the past years. In this study, a new bacterial strain called Brevibacillus laterosporus fmb70 (fmb70) was isolated and exhibited strong antimicrobial activities against Gram-positive, Gram-negative bacteria, and fungi. Two major antimicrobial components produced by fmb70 were respectively identified as lipopeptide: brevibacillin V (MW: 1570.12 Da) and brevibacillin (MW: 1583.75 Da), of which brevibacillin V was a new compound. Both of them consisted of 13 amino acids and C6 fatty acyl (FA) chain. Brevibacillin V and brevibacillin showed significant antimicrobial activities against most foodborne pathogenic bacteria and phytopathogenic fungi. They stayed activity at 100 °C and remained 50% of their antimicrobial activities at pH 3 for 22 h. Hemolytic activities of them were lower than 8%. They effectively eliminated the S. aureus GIM 1.142 and L. monocytogenes ATCC 21633 in skim milk. In conclusion, the Brevibacillus laterosporus fmb70 and its major antimicrobial components has remarkable potentials in the food industry.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Brevibacillus/química , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Leite/microbiologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Brevibacillus/metabolismo , Bovinos , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Lipopeptídeos/metabolismo , Testes de Sensibilidade Microbiana , Mapeamento de Peptídeos
7.
Molecules ; 24(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408988

RESUMO

As a folk medicine of the Jingpo minority in Yunnan province, the venom of Vespa magnifica has been commonly used for the treatment of rheumatoid arthritis. Quality standardization of the wasp venom is a necessary step for its pharmaceutical research and development. To control the quality of the wasp venom, a method based on high-performance liquid chromatography (HPLC) was developed for chemical fingerprint analysis. In the chromatographic fingerprinting, chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), and principal component analysis (PCA), were applied to classify 134 batches (S1-S134) of wasp venom from different origins. The HPLC fingerprint method displayed good precision (Relative standard deviation, RSD < 0.27%), stability (in 16 h, RSD < 0.34%), and repeatability (RSD < 1.00%). Simultaneously, four compounds (VMS1, VMS2, VMS3, and VMS4) in the wasp venom were purified and identified. VMS1 was 5-hydroxytryptamine, and the other compounds were three peptides that were sequenced as follows: Gly-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Asp-NH2 (VMS2), Ile-Asn-Leu-Lys-Ala-Ile-Ala-Ala-Leu-Ala-Lys-Lys-Leu-Leu-NH2 (VMS3), and Phe-Leu-Pro-Ile-Ile-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Leu-NH2 (VMS4). The quantifications for these components were 110.2 mg/g, 26.9 mg/g, 216.3 mg/g, and 58.0 mg/g, respectively. The results of this work indicated that the combination of the chemical fingerprint and quantitative analysis offers a reasonable way to evaluate the quality of wasp venom.


Assuntos
Anti-Inflamatórios/isolamento & purificação , Peptídeos/isolamento & purificação , Serotonina/isolamento & purificação , Venenos de Vespas/química , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Artrite Reumatoide/tratamento farmacológico , Cromatografia Líquida de Alta Pressão/normas , Humanos , Medicina Tradicional Chinesa , Medicina Tradicional/métodos , Mapeamento de Peptídeos/métodos , Peptídeos/química , Análise de Componente Principal , Controle de Qualidade , Serotonina/química , Vespas
8.
Chem Commun (Camb) ; 55(67): 9979-9982, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31367719

RESUMO

A chemical tag enhances peptide detection by multiple orders in mass spectrometry. The substantial improvement in the peptide mapping along with simplified and enhanced fragmentation pattern enables the unambiguous sequencing of a protein and antibody. The chemoselective sensitivity booster provides a tool for remarkably improved analysis of protein bioconjugates.


Assuntos
Peptídeos/análise , Proteínas/análise , Citocromos c/análise , Lisina/química , Mapeamento de Peptídeos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
9.
Food Funct ; 10(9): 5426-5435, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31402368

RESUMO

In this study, oyster (Crassostrea gigas) proteins were digested under in vitro gastrointestinal conditions to screen potential antithrombotic peptides. The sequences of the released peptides in the intestinal digestion phase were identified by ultra-performance liquid chromatography coupled to quadrupole time-of-flight MS (UPLC-Q-TOF-MS/MS). According to the antithrombotic activity analysis, the inhibitory ratio of oyster peptides showed an increasing trend, reaching up to 35.80% for a digestion period of 4 h. The APTT (activated partial thromboplastin time) and TT (thromboplastin time) were increased by oyster peptides for human serum in vitro. Oyster peptides showed a competitive inhibition effect on thrombin, based on Lineweaver-Burk plot analysis. Molecular docking between the antithrombotic peptides and thrombin (PDB: ) was conducted using Discovery Studio 2017. Potential inhibitors against thrombin and the mechanism of antithrombotic activity were predicted using the algorithm of CDOCKER. There are fourteen potential antithrombotic peptides, whose affinity with thrombin is higher than that of hirudin, as indicated by the "-CDOCKER energy" score (181.491). Peptide LSKEEIEEAKEV is similar in sequence to thrombin inhibitors. The binding sites of potential antithrombotic peptides against thrombin at the S1 pocket were compared with hirudin variant-2 (GDFEEIPEEYLQ). In addition, the peptides containing the RG/RGD sequence were identified, which can be hydrolyzed by thrombin as a substrate. Consequently, the oyster peptides released in simulated gastrointestinal digestion probably inhibit thrombin in two ways, not only as the inhibitor against the active site, but also as the substrate of thrombin. These results maybe be attributed to the potentially strong antithrombotic activity in the human digestive system.


Assuntos
Antitrombinas/química , Trato Gastrointestinal/metabolismo , Ostreidae/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Antitrombinas/metabolismo , Digestão , Humanos , Ostreidae/metabolismo , Tempo de Tromboplastina Parcial , Mapeamento de Peptídeos , Peptídeos/metabolismo , Espectrometria de Massas em Tandem , Trombina/antagonistas & inibidores , Trombina/química
10.
Adv Exp Med Biol ; 1140: 27-43, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347040

RESUMO

Matrix assisted laser desorption ionization (MALDI) mass spectrometry allows for the rapid profiling of different biomolecular species from both biofluids and tissues. Whilst originally focused upon the analysis of intact proteins and peptides, MALDI mass spectrometry has found further applications in lipidomic analysis, genotyping, microorganism identification, biomarker discovery and metabolomics. The combining of multiple profiles data from differing locations across a sample furthermore, allows for spatial distribution of biomolecules to be established utilizing imaging MALDI analysis. This chapter discusses the MALDI process, its usual applications in the field of protein identification via peptide mass fingerprinting before focusing upon advances in the application of the profiling potential of MALDI mass spectrometry and its' various applications in biomedicine.


Assuntos
Peptídeos/análise , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Humanos , Mapeamento de Peptídeos
11.
Adv Exp Med Biol ; 1140: 225-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347050

RESUMO

Selection of high-producing lead and backup cell lines with high-fidelity primary structure is a major goal of cell line development of protein therapeutics. Conventional techniques for sequence variant analysis, such as mass spectrometry (MS) and next-generation sequencing (NGS) have limitations on the sample number and turnaround time, thus often are only applied at the final stages of development, where an undesired lead or backup clone could cause a significant delay in project timeline. Here we presented a high-throughput (HT) peptide mapping workflow which can be applied at early stages of cell line selection for testing of a batch of 30-40 clones within 2-week turnaround while reporting valuable information on sequence variants and posttranslational modifications (PTMs). The successful application of this workflow was demonstrated for two mAb programs. Multiple clones were removed from a total of 33 mAb-1 clones using various criteria: nine clones contained at least one >1% upregulated unknown peptide ions, 11 clones contained at least eight >0.1% upregulated unknowns, and six clones contained upregulated critical PTMs. For mAb-2, light chain (LC) sequence extension of approximately 30 amino acids were detected in 6 out of 36 clones at levels up to 11%. Besides, a Q to H mutation at ~30% was detected in the heavy chain (HC) of a single clone. Q to H mutation has mass change of 9.00 Da and failed to be detected by intact mass analysis. Rapid PTM quantitation also facilitated the selection of clones with desirable quality attributes, such as N-glycan profile. Hence, we demonstrated a risk-reducing strategy where abnormal clones could be detected at earlier stages of cell line selection, which should result in reduced and more predictable timeline of cell line development.


Assuntos
Anticorpos Monoclonais/química , Sequenciamento de Nucleotídeos em Larga Escala , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Animais , Células CHO , Cricetinae , Cricetulus , Espectrometria de Massas
12.
Immunology ; 158(2): 94-103, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31323138

RESUMO

Transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of Cryptomeria japonica (Japanese cedar; JC), have been developed as immunotherapeutic candidates for JC pollinosis. Because the transgenic rice (TG-rice) seeds express allergens containing whole amino acid sequences of Cry j 1 and Cry j 2 in the endosperm tissue (edible part of rice grain), they can potentially target all Cry j 1- and Cry j 2-specific T-cells. However, it was unknown whether antigenicity of Cry j 1 and Cry j 2 could be completely preserved in TG-rice seeds. We verified the antigenicity of TG-rice seeds to T-cells through the analysis of the proliferative responses of T-cells in Cry j 1- or Cry j 2-immunized mice or T-cell lines to TG-rice seed extract. First, four mouse strains were immunized with Cry j 1 or Cry j 2. T-cells in the immunized mice proliferated on treatment with TG-rice seed extract, but not non-transgenic wild-type rice (WT-rice) seed extract. Furthermore, T-cell lines were established from the spleen cells of the immunized mice. Each T-cell line resulted in a proliferative response to TG-rice seed extract, but not to WT-rice seed extract, suggesting that TG-rice seeds certainly express T-cell epitopes corresponding to T-cell lines. Considering the modified amino acid sequences of Cry j 1 and Cry j 2 in TG-rice seeds, the expression of specific T-cell epitopes suggested that TG-rice seeds express all possible T-cell epitope repertoires of Cry j 1 and Cry j 2.


Assuntos
Alérgenos/farmacologia , Antígenos de Plantas/imunologia , Epitopos de Linfócito T/imunologia , Oryza/química , Proteínas de Plantas/imunologia , Rinite Alérgica Sazonal/imunologia , Linfócitos T/efeitos dos fármacos , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Plantas/química , Antígenos de Plantas/genética , Proliferação de Células/efeitos dos fármacos , Cryptomeria/genética , Cryptomeria/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Expressão Gênica , Imunização , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Oryza/genética , Oryza/imunologia , Mapeamento de Peptídeos , Extratos Vegetais/imunologia , Extratos Vegetais/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/imunologia , Cultura Primária de Células , Rinite Alérgica Sazonal/induzido quimicamente , Rinite Alérgica Sazonal/genética , Rinite Alérgica Sazonal/patologia , Sementes/química , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Transgenes
13.
J Agric Food Chem ; 67(24): 6757-6764, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31184153

RESUMO

In the present study we purified and identified peptides from broccoli protein hydrolysates and evaluated their angiotensin-converting enzyme (ACE) inhibitory activity in vitro and hypotensive effect in vivo. Three ACE inhibitory peptides were isolated and identified as IPPAYTK, LVLPGELAK, and TFQGPPHGIQVER, and their inhibitory IC50 values were 23.5, 184.0, and 3.4 µM, respectively. We then investigated the effect of gastrointestinal digestion on ACE inhibitory activity. We detected almost two times the ACE inhibitory activity of the peptide LVLPGELAK following simulated digestion than prior to digestion. LVLPGE and LAK, two novel peptides exhibiting high ACE inhibitory activity, were discovered following digestion and possessed IC50 values of 13.5 and 48.0 µM, respectively. The hypotensive effect of the peptides was assessed after oral administration to spontaneous hypertensive rats (SHRs). We found that LVLPGE and LAK demonstrated a significant hypotensive effect in vivo. Protein from broccoli may thus constitute a potential antihypertensive peptide source.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Anti-Hipertensivos/administração & dosagem , Brassica/química , Hipertensão/tratamento farmacológico , Peptídeos/administração & dosagem , Proteínas de Plantas/química , Administração Oral , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Pressão Sanguínea/efeitos dos fármacos , Feminino , Humanos , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Mapeamento de Peptídeos , Peptídeos/isolamento & purificação , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Hidrolisados de Proteína/química , Ratos , Ratos Endogâmicos SHR
14.
J Agric Food Chem ; 67(29): 8149-8159, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31246442

RESUMO

Ganoderma lucidum (G. lucidum) has been widely used in Asia to treat hypertension, but the active substances responsible for its antihypertensive effects remain unclear. Using the well-established angiotensin I-converting enzyme (ACE) as a target, we identified three ACE inhibitory peptides (ACEIPs), Gln-Leu-Val-Pro (QLVP), Gln-Asp-Val-Leu (QDVL), and Gln-Leu-Asp-Leu (QLDL), which account for the antihypertensive activity of G. lucidum. Notably, QLVP worked in a mixed-type manner against ACE with an IC50 value of 127.9 µmol/L. Molecular dynamics simulation suggested that the potent charge energy of QLVP, which interacted with Gln242 and Lys472 of ACE via a hydrogen bond and a salt bridge, potentially contributed to ACE inhibitory activity. Moreover, QLVP markedly activated angiotensin I-mediated phosphorylation of endothelial nitric oxide synthase in human umbilical vein endothelial cells and partly reduced mRNA and protein expression of the vasoconstrictor factor endothelin-1. This is the first report of the antihypertensive activity of small ACEIPs originating from G. lucidum mycelia, paving the way for the possible application of these peptides as potent drug candidates for treating hypertension.


Assuntos
Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Reishi/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Linhagem Celular , Humanos , Cinética , Espectrometria de Massas , Simulação de Dinâmica Molecular , Micélio/química , Mapeamento de Peptídeos , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo
15.
PLoS One ; 14(6): e0218951, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31247021

RESUMO

Fast and reliable detection coupled with accurate data-processing and analysis of antibiotic-resistant bacteria is essential in clinical settings. In this study, we use MALDI-TOF on intact cells combined with a refined analysis framework to demonstrate discrimination between methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus. By combining supervised and unsupervised machine learning methods, we firstly show that the mass spectroscopy data contains strong signal for the clustering of MSSA and MRSA. Then we concentrate on applying supervised learning to extract and verify the important features. A new workflow is proposed that allows for extracting a fixed set of reference peaks so that any new data can be aligned to it and hence consistent feature matrices can be obtained. Also note that by doing so we are able to examine the robustness of the important features that have been found. We also show that appropriate size of the benchmark data, appropriate alignment of the testing data and use of an optimal set of features via feature selection results in prediction accuracy over 90%. In summary, as proof-of-principle, our integrated experimental and bioinformatics study suggests a novel intact cell MALDI-TOF to be of great promise for fast and reliable detection of MRSA strains.


Assuntos
Staphylococcus aureus Resistente à Meticilina/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus aureus/classificação , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Biologia Computacional , Humanos , Meticilina/farmacologia , Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Mapeamento de Peptídeos/métodos , Mapeamento de Peptídeos/estatística & dados numéricos , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Aprendizado de Máquina Supervisionado , Máquina de Vetores de Suporte , Fluxo de Trabalho
16.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180067

RESUMO

Proteins in a proteome can be identified from a sequence of K integers equal to the digitized volumes of subsequences with L residues from the primary sequence of a stretched protein. Exhaustive computations on the proteins of Helicobacter pylori (UniProt id UP000000210) with L and K in the range 4-8 show that approx. 90% of the proteins can be identified uniquely in this manner. This computational result can be translated into practice with a nanopore, an emerging technology that does not require analyte immobilization, proteolysis or labeling. Unlike other methods, most of which focus on a specific target protein, nanopore-based methods enable the identification of multiple proteins from a sample in a single run. Recent work by Kennedy, Kolmogorov and associates shows that the blockade current due to a protein molecule translocating through a nanopore is roughly proportional to one or more contiguous residues. The present study points to a modified version in which the volumes of subsequences (rather than of single residues) may be obtained by integrating the blockade current due to L contiguous residues. The advantages arising from this include lower detector bandwidth, elimination of the homopolymer problem and reduced noise. Because an identifier is based on near as well as distant (up to 2KL-L) residues, this approach uses more global information than an approach based on single residues and short-range correlations. The results of the study, which are available in a data supplement, are discussed in detail. Potential implementation issues are addressed.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Helicobacter pylori/genética , Modelos Estatísticos , Mapeamento de Peptídeos/estatística & dados numéricos , Proteoma/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos , Proteínas de Bactérias/genética , Bases de Dados de Proteínas , Helicobacter pylori/química , Nanoporos , Fragmentos de Peptídeos/análise , Mapeamento de Peptídeos/métodos , Proteoma/genética
17.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180068

RESUMO

Laminins are a major constituent of the extracellular matrix (ECM). Laminin-111, the most extensively studied laminin isoform, consists of the α1, the ß1 and the γ1 chain, and is involved in many cellular processes, like adhesion, migration and differentiation. Given the regulatory role of phosphorylation in protein function, it is important to identify the phosphorylation sites of human laminin ß1-chain sequence (LAMB1). Therefore, we computationally predicted all possible phosphorylation sites in LAMB1. For the first time, we identified the possibly responsible kinases for already in vitro experimentally observed phosphorylated residues in LAMB1. All known functional (active) sites of LAMB1, were recorded after an extensive literature search and combined with the experimentally observed and our predicted phosphorylated residues. This generated a detailed phosphorylation map of LAMB1. Five kinases (PKA, PKC, CKII, CKI and GPCR1) were indicated important, while the role of PKA, PKC and CKII, kinases known for ectophosphorylation activity, was highlighted. The activity of PKA and PKC was associated with the active site RIQNLLKITNLRIKFVKLHTLGDNLLDS. Also, predicted phosphorylations inside two amyloidogenic (DSITKYFQMSLE, VILQHSAADIAR) and two anti-cancerous (YIGSR and PDSGR) sites suggested a possible role in the development of the corresponding diseases.


Assuntos
Biologia Computacional/métodos , Laminina/química , Mapeamento de Peptídeos/métodos , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Caseína Quinase I/química , Caseína Quinase I/metabolismo , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/química , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Expressão Gênica , Humanos , Laminina/genética , Laminina/metabolismo , Fosforilação , Proteína Quinase C/química , Proteína Quinase C/metabolismo
18.
Food Chem ; 295: 456-465, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174782

RESUMO

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date.


Assuntos
Kefir/análise , Proteínas do Leite/análise , Peptídeos/análise , Peptídeos/farmacologia , Animais , Caseínas/análise , Caseínas/metabolismo , Fermentação , Cabras , Proteínas do Leite/metabolismo , Proteínas do Leite/farmacologia , Mapeamento de Peptídeos/métodos , Peptídeos/metabolismo , Probióticos , Proteólise , Proteômica/métodos , Fatores de Tempo
19.
Am J Physiol Endocrinol Metab ; 317(2): E374-E387, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31211616

RESUMO

Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxidoreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosynthetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function.


Assuntos
Metabolismo Energético/fisiologia , Fígado/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Proteoma/metabolismo , Animais , Feminino , Humanos , Fígado/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/análise , Músculo Esquelético/química , Especificidade de Órgãos , Mapeamento de Peptídeos/métodos , Proteoma/análise
20.
Plant Physiol Biochem ; 141: 183-192, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31174035

RESUMO

The present study is focused on the characterization of yacon [Smallanthus sonchifolius (Poepp. et Endl.) H. Robinson] accessions from different geographic origins (Bolivia, Ecuador, and Peru) by iPBS markers and metabolomic fingerprinting. The results showed that the number of amplified polymorphic fragment levels ranged from 20 up to 27 with a level of polymorphism ranging from 80 to 100%. Five of the iPBS primers used in this study provided no specific banding pattern able to discriminate between the different yacon accessions. However, two iPBS primer pairs were able to separate Peru accessions from those of Ecuador and Bolivia. The UPLC-HRMS/MS-based metabolomic fingerprinting showed highly similar metabolomic fingerprints characterized by the accumulation of high quantities of sesquiterpene lactones and diterpenes, but no apparent geographic clustering. The present study demonstrates that yacon accessions from different geographical origins maintained ex situ (in the Czech Republic) present a rather low chemical and genetic diversity.


Assuntos
Antioxidantes/química , Asteraceae/química , Diterpenos/química , Lactonas/química , Extratos Vegetais/química , Sesquiterpenos/química , Asteraceae/genética , Bolívia , Análise por Conglomerados , República Tcheca , Equador , Variação Genética , Geografia , Glicosilação , Espectrometria de Massas , Metabolômica , Análise Multivariada , Mapeamento de Peptídeos , Peru , Raízes de Plantas/química , Retroelementos
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