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1.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500632

RESUMO

In the Valpolicella area (Verona, Italy) Vitis vinifera cv. Corvina is the main grape variety used to produce Amarone wine. Before starting the winemaking process, the Corvina grapes are stored in a withering (i.e., dehydrating) warehouse until about 30% of the berry weight is lost (WL). This practice is performed to concentrate the metabolites in the berry and enrich the Amarone wine in aroma and antioxidant compounds. In compliance with the guidelines and strict Amarone protocol set by the Consorzio of Amarone Valpolicella, withering must be carried out by setting the grapes in a suitable environment, either under controlled relative air humidity (RH) conditions and wind speed (WS)-no temperature modification is to be applied-or, following the traditional methods, in non-controlled environmental conditions. In general, the two processes have different dehydration kinetics due to the different conditions in terms of temperature, RH, and WS, which affect the accumulation of sugars and organic acids and the biosynthesis of secondary metabolites such as stilbenes and glycoside aroma precursors. For this study, the two grape-withering processes were carried out under controlled (C) and non-controlled (NC) conditions, and the final compositions of the Corvina dried grapes were compared also to evaluate the effects on the organoleptic characteristics of Amarone wine. The findings highlighted differences between the two processes mainly in terms of the secondary metabolites of the dried grapes, which affect the organoleptic characteristics of Amarone wine. Indeed, by the sensory evaluation, wines produced by adopting the NC process were found more harmonious, elegant, and balanced. Finally, we can state how using a traditional system, grapes were characterised by higher levels of VOCs (volatile compounds), whilst wines had a higher and appreciable complexity and finesse.


Assuntos
Vitis/química , Compostos Orgânicos Voláteis/química , Frutas/química , Glicosídeos/química , Itália , Espectrometria de Massas/métodos , Odorantes , Sensação/fisiologia , Estilbenos/química , Vinho
2.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500648

RESUMO

Ethanol extracts of two types of pepper (sweet and hot) were separated into fractions with increasing lipophilicity. After drying the extracts and fractions, their chemical composition, anti-radical activity in the DPPH radical system, and cytotoxic activity against PC-3 and HTC-116 cells were determined. A detailed qualitative analysis of the fractions was performed with the LC-QTOF-MS method. It was found that the chemical composition of pepper fractions did not always reflect their biological activity. The highest antiradical activity was detected in the fraction eluted with 40% methanol from sweet pepper. The highest total content of phenolic compounds was found in an analogous fraction from hot pepper, and this fraction showed the strongest cytotoxic effect on the PC-3 tumour line. The LC-MS analysis identified 53 compounds, six of which were present only in sweet pepper and four only in hot pepper. The unique chemical composition of the extracts was found to modulate their biological activity, which can only be verified experimentally.


Assuntos
Capsicum/química , Extratos Vegetais/química , Antioxidantes/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Fenóis/química , Piper nigrum/química , Verduras/química
3.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445607

RESUMO

BACKGROUND: Transition metals play a crucial role in brain metabolism: since they exist in different oxidation states they are involved in ROS generation, but they are also co-factors of enzymes in cellular energy metabolism or oxidative defense. METHODS: Paired serum and cerebrospinal fluid (CSF) samples were analyzed for iron, zinc, copper and manganese as well as for speciation using SEC-ICP-DRC-MS. Brain extracts from Mn-exposed rats were additionally analyzed with SEC-ICP-DRC-MS. RESULTS: The concentration patterns of transition metal size fractions were correlated between serum and CSF: Total element concentrations were significantly lower in CSF. Fe-ferritin was decreased in CSF whereas a LMW Fe fraction was relatively increased. The 400-600 kDa Zn fraction and the Cu-ceruloplasmin fraction were decreased in CSF, by contrast the 40-80 kDa fraction, containing Cu- and Zn-albumin, relatively increased. For manganese, the α-2-macroglobulin fraction showed significantly lower concentration in CSF, whereas the citrate Mn fraction was enriched. Results from the rat brain extracts supported the findings from human paired serum and CSF samples. CONCLUSIONS: Transition metals are strictly controlled at neural barriers (NB) of neurologic healthy patients. High molecular weight species are down-concentrated along NB, however, the Mn-citrate fraction seems to be less controlled, which may be problematic under environmental load.


Assuntos
Encéfalo/metabolismo , Cromatografia em Gel/métodos , Espectrometria de Massas/métodos , Oligoelementos/sangue , Oligoelementos/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos
4.
Nat Commun ; 12(1): 4787, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373457

RESUMO

Label-free proteomics by data-dependent acquisition enables the unbiased quantification of thousands of proteins, however it notoriously suffers from high rates of missing values, thus prohibiting consistent protein quantification across large sample cohorts. To solve this, we here present IceR (Ion current extraction Re-quantification), an efficient and user-friendly quantification workflow that combines high identification rates of data-dependent acquisition with low missing value rates similar to data-independent acquisition. Specifically, IceR uses ion current information for a hybrid peptide identification propagation approach with superior quantification precision, accuracy, reliability and data completeness compared to other quantitative workflows. Applied to plasma and single-cell proteomics data, IceR enhanced the number of reliably quantified proteins, improved discriminability between single-cell populations, and allowed reconstruction of a developmental trajectory. IceR will be useful to improve performance of large scale global as well as low-input proteomics applications, facilitated by its availability as an easy-to-use R-package.


Assuntos
Espectrometria de Massas/métodos , Proteoma , Proteômica/métodos , Peptídeos , Espectrometria de Massas em Tandem , Fluxo de Trabalho
5.
Nat Commun ; 12(1): 4961, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400640

RESUMO

Esophageal cancer (EC) is a type of aggressive cancer without clinically relevant molecular subtypes, hindering the development of effective strategies for treatment. To define molecular subtypes of EC, we perform mass spectrometry-based proteomic and phosphoproteomics profiling of EC tumors and adjacent non-tumor tissues, revealing a catalog of proteins and phosphosites that are dysregulated in ECs. The EC cohort is stratified into two molecular subtypes-S1 and S2-based on proteomic analysis, with the S2 subtype characterized by the upregulation of spliceosomal and ribosomal proteins, and being more aggressive. Moreover, we identify a subtype signature composed of ELOA and SCAF4, and construct a subtype diagnostic and prognostic model. Potential drugs are predicted for treating patients of S2 subtype, and three candidate drugs are validated to inhibit EC. Taken together, our proteomic analysis define molecular subtypes of EC, thus providing a potential therapeutic outlook for improving disease outcomes in patients with EC.


Assuntos
Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Espectrometria de Massas/métodos , Proteômica , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ciclo Celular , Estudos de Coortes , Elonguina/genética , Elonguina/metabolismo , Humanos , Prognóstico , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
6.
Nat Commun ; 12(1): 4992, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404777

RESUMO

Liquid chromatography-mass spectrometry-based metabolomics studies are increasingly applied to large population cohorts, which run for several weeks or even years in data acquisition. This inevitably introduces unwanted intra- and inter-batch variations over time that can overshadow true biological signals and thus hinder potential biological discoveries. To date, normalisation approaches have struggled to mitigate the variability introduced by technical factors whilst preserving biological variance, especially for protracted acquisitions. Here, we propose a study design framework with an arrangement for embedding biological sample replicates to quantify variance within and between batches and a workflow that uses these replicates to remove unwanted variation in a hierarchical manner (hRUV). We use this design to produce a dataset of more than 1000 human plasma samples run over an extended period of time. We demonstrate significant improvement of hRUV over existing methods in preserving biological signals whilst removing unwanted variation for large scale metabolomics studies. Our tools not only provide a strategy for large scale data normalisation, but also provides guidance on the design strategy for large omics studies.


Assuntos
Metabolômica/métodos , Cromatografia Líquida , Humanos , Espectrometria de Massas/métodos , Modelos Biológicos , Fluxo de Trabalho
7.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361026

RESUMO

In the presented study, a capillary electrophoresis-mass spectrometry method combining high separation efficiency and sensitive detection has been developed and validated, for the first time, to quantify branched chain amino acids (valine, isoleucine, leucine) in commercial food and sport supplement samples and human plasma samples. The separations were performed in a bare fused silica capillary. The background electrolyte was composed of 500 mM formic acid with pH 2.0. The plasma sample pretreatment was realized by simple protein precipitation with acetonitrile. Injection of a short zone of highly basic electrolyte before the sample injection and application of the negative pressure on the separation were accompanied by enhanced resolution of the isobaric amino acids-isoleucine and leucine. The developed method was characterized by favorable validation parameters, such as linearity (r2 > 0.99), accuracy and precision, the limit of detection, lower limit of quantification, or robustness. These parameters were more than sufficient for the quantification of branched chain amino acids in various samples. The determined concentrations of branched chain amino acids in food and sports supplements were in very good agreement with the content declared by the manufacturer. The investigated concentrations of branched chain amino acids were in the range 294.68-359.24 µM for valine, 91.76-95.67 µM for isoleucine, and 196.78-251.24 µM for leucine. These concentrations fall within the physiological limits. The developed CE-MS/MS method represents a suitable alternative to traditional approaches used in branched chain amino acid quality control and bioanalysis.


Assuntos
Aminoácidos de Cadeia Ramificada/análise , Sangue/metabolismo , Suplementos Nutricionais , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Adulto , Aminoácidos de Cadeia Ramificada/sangue , Análise Química do Sangue/métodos , Humanos , Masculino
8.
Medicine (Baltimore) ; 100(30): e26588, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34397689

RESUMO

ABSTRACT: High cardiovascular disease risk in people living with HIV is partly attributed to antiretroviral therapy (ART). Lipid response to ART has been extensively studied, yet, little is known how small molecule lipids respond to Integrase inhibitor-based (INSTI-based) compared to Protease inhibitor-based (PI-based) ART regimens.Ancillary study to a phase 3, randomized, open-label trial [AIDS Clinical Trial Group A5257 Study] in treatment-naive HIV-infected patients randomized in a 1:1:1 ratio to receive ritonavir-boosted atazanavir (ATV/r), ritonavir-boosted darunavir (DRV/r) (both PI-based), or raltegravir with Tenofovir Disoproxil Fumarate-TDF plus emtricitabine (RAL, INSTI-based).We examined small molecule lipid response in a subcohort of 75 participants. Lipidomic assays of plasma samples collected pre- and post-ART treatment (48 weeks) were conducted using ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. The effect of ART regimens was regressed on lipid species response adjusting for the baseline covariates (lipids, age, sex, race, CD4 level, BMI, and smoking). Results were validated in the Centers for AIDS Research Network of Integrated Clinical Systems study (N = 16).Out of 417 annotated lipids, glycerophospholipids (P = .007) and sphingolipids (P = .028) had a higher response to ATV/r and DRV/r compared to RAL. The lysophosphatidylcholine (LPCs(16:1),(17:1),(20:3)) and phosphophatidylcholine species (PCs(40:7),(38:4)) had an opposite response to RAL versus ATV/r in the discovery and validation cohort. The INSTI-based regimen had an opposite response of ceramide species ((d38:1), (d42:2)), PCs((35:2), (38:4)), phosphatidylethanolamines (PEs(38:4), (38:6)), and sphingomyelin(SMd38:1) species compared with the PI-based regimens. There were no differences observed between 2 PI-based regimens.We observed differences in response of small molecule lipid species by ART regimens in treatment-naive people living with HIV.


Assuntos
Antirretrovirais/efeitos adversos , Lipidômica/métodos , Adulto , Antirretrovirais/uso terapêutico , Sulfato de Atazanavir/efeitos adversos , Sulfato de Atazanavir/uso terapêutico , Distribuição de Qui-Quadrado , Darunavir/efeitos adversos , Darunavir/uso terapêutico , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/fisiopatologia , Humanos , Lipidômica/estatística & dados numéricos , Lipídeos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Plasma/efeitos dos fármacos , Raltegravir Potássico/efeitos adversos , Raltegravir Potássico/uso terapêutico , Tenofovir/efeitos adversos , Tenofovir/uso terapêutico
9.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360890

RESUMO

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membranas Intracelulares/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Proteoma , Tilacoides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Filogenia , Processamento de Proteína Pós-Traducional , Proteômica/métodos
10.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445547

RESUMO

Gangliosides serve as antitumor therapy targets and aberrations in their composition strongly correlate with tumor growth and invasiveness. Anaplastic ganglioglioma is a rare, poorly characterized, malignant neuronal-glial tumor type. We present the first comparative characterization of ganglioside composition in anaplastic ganglioglioma vs. peritumoral and healthy brain tissues by combining mass spectrometry and thin-layer chromatography. Anaplastic ganglioglioma ganglioside composition was highly distinguishable from both peritumoral and healthy tissue despite having five to six times lower total content. Ten out of twelve MS-identified ganglioside classes, defined by unique glycan residues, were represented by a large number and considerable abundance of individual species with different fatty acid residues (C16-C24) in ceramide portions. The major structurally identified class was tumor-associated GD3 (>50%) with 11 species; GD3 (d18:1/24:0) being the most abundant. The dominant sphingoid base residue in ganglioside ceramides was sphingosine (d18:1), followed by eicosasphingosine (d20:1). The peritumoral tissue ganglioside composition was estimated as normal. Specific ganglioside composition and large variability of ganglioside ceramide structures determined in anaplastic ganglioglioma demonstrate realistic ganglioside expression patterns and correspond to the profile of high-grade malignancy brain tumors.


Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Carcinoma/patologia , Cromatografia em Camada Delgada/métodos , Ganglioglioma/patologia , Gangliosídeos/metabolismo , Espectrometria de Massas/métodos , Idoso , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Carcinoma/metabolismo , Feminino , Ganglioglioma/metabolismo , Gangliosídeos/análise , Humanos , Pessoa de Meia-Idade
11.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445776

RESUMO

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.


Assuntos
Anticorpos Monoclonais/química , Antineoplásicos Imunológicos/química , Cátions/química , Rituximab/química , Medicamentos Biossimilares/química , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Glicosilação , Espectrometria de Massas/métodos
12.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445296

RESUMO

To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R2 = 0.97; nasal, R2 = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.


Assuntos
Proteínas do Olho/metabolismo , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Proteínas do Olho/análise , Espectrometria de Massas/métodos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Traumatismos do Nervo Óptico/complicações , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Ratos , Ratos Wistar , Retina/química , Retina/metabolismo , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologia , Células Ganglionares da Retina/patologia
13.
Commun Biol ; 4(1): 934, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34345007

RESUMO

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using a LC-TOF mass spectrometer, requires no specialized knowledge of glycan analysis and exploits the differential resolving power of reverse phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. Rather than a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike glycopeptides. This database allows any accurate mass LC-MS laboratory to reliably identify and quantify Spike glycopeptides from a single overnight elastase digest in less than 90 minutes.


Assuntos
Glicopeptídeos/química , Espectrometria de Massas/métodos , Glicoproteína da Espícula de Coronavírus/química , Bases de Dados de Proteínas , Fatores de Tempo
14.
Nat Commun ; 12(1): 4912, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389721

RESUMO

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.


Assuntos
Vias Biossintéticas/genética , Proteínas de Caenorhabditis elegans/genética , Peptídeo Sintases/genética , Peptídeos/metabolismo , Policetídeo Sintases/genética , Policetídeos/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida/métodos , Enzimas/genética , Enzimas/metabolismo , Expressão Gênica , Espectrometria de Massas/métodos , Estrutura Molecular , Mutação , Neurônios/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos/química , Policetídeo Sintases/metabolismo , Policetídeos/química
15.
Nat Commun ; 12(1): 4608, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326324

RESUMO

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.


Assuntos
Anticorpos/química , Peptídeos/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Células Cultivadas , Cristalografia por Raios X/métodos , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Proteômica/métodos , Coelhos , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/imunologia , Ubiquitinação
16.
Molecules ; 26(14)2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34299492

RESUMO

Toads in the family Bufonidae contain bufadienolides in their venom, which are characterized by their chemical diversity and high pharmacological potential. American trypanosomiasis is a neglected disease that affects an estimated 8 million people in tropical and subtropical countries. In this research, we investigated the chemical composition and antitrypanosomal activity of toad venom from Rhinella alata collected in Panama. Structural determination using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy led to the identification of 10 bufadienolides. Compounds identified include the following: 16ß-hydroxy-desacetyl-bufotalin-3-adipoyl-arginine ester (1), bufotalin (2), 16ß-hydroxy-desacetyl-bufotalin-3-pimeloyl-arginine ester (3), bufotalin-3-pimeloyl-arginine ester (4), 16ß-hydroxy-desacetyl-bufotalin-3-suberoyl-arginine ester (5), bufotalin-3-suberoyl-arginine ester (6), cinobufagin-3-adipoyl-arginine ester (7), cinobufagin-3-pimeloyl-arginine ester (8), cinobufagin-3-suberoyl-arginine ester (9), and cinobufagin (10). Among these, three new natural products, 1, 3, and 5, are described, and compounds 1-10 are reported for the first time in R. alata. The antitrypanosomal activity assessed in this study revealed that the presence of an arginyl-diacid attached to C-3, and a hydroxyl group at C-14 in the structure of bufadienolides that is important for their biological activity. Bufadienolides showed cytotoxic activity against epithelial kidney Vero cells; however, bufagins (2 and 10) displayed low mammalian cytotoxicity. Compounds 2 and 10 showed activity against the cancer cell lines MCF-7, NCI-H460, and SF-268.


Assuntos
Antiprotozoários/farmacologia , Bufanolídeos/farmacologia , Bufonidae/metabolismo , Pele/metabolismo , Venenos de Anfíbios/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Células MCF-7 , Espectrometria de Massas/métodos , Panamá , Trypanosoma cruzi , Células Vero
17.
Molecules ; 26(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299543

RESUMO

The essential oils of hinoki (Chamaecyparis obtusa) leaves have anti-bacterial, anti-fungal, and relaxation properties that are likely associated with the major components such as sabinene, α-terpinyl acetate, limonene, elemol, myrcene, and hibaene. The present study describes the use of a cellulose-dissolving ionic liquid (IL) [C2mim][(MeO)(H)PO2] and low-toxicity solvents called betaine-based deep eutectic solvents (DESs) for the efficient extraction of hinoki essential oils. As a control method, organic solvent extraction was performed using either hexane, ethyl acetate (EtOAc), or acetone at 30 °C for 1 h. Both the experimental and control methods were conducted under the same conditions, which relied on partial dissolution of the leaves using the IL and DESs before partitioning the hinoki oils into the organic solvent for analysis. Quantitative analysis was performed using gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. The results indicated that extraction using the [C2mim][(MeO)(H)PO2]/acetone bilayer system improved the yields of limonene and hibaene, 1.5- and 1.9-fold, respectively, when compared with the control method. In addition, extraction using betaine/l-lactic acid (molar ratio 1:1) gave the greatest yields for both limonene and hibaene, 1.3-fold and 1.5-fold greater, respectively, than when using an organic solvent. These results demonstrate the effective extraction of essential oils from plant leaves under conditions milder than those needed for the conventional method. The less toxic and environmentally begin DESs for the extraction are also applicable to the food and cosmetic industries.


Assuntos
Chamaecyparis/química , Líquidos Iônicos/química , Limoneno/química , Solventes/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Espectrometria de Massas/métodos , Óleos Voláteis/química , Folhas de Planta/química , Óleos Vegetais/química , Sesquiterpenos/química
18.
Molecules ; 26(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299545

RESUMO

Red Delicious apple pomace was produced at laboratory scale with a domestic blender and different non-conventional extraction techniques were performed to isolate phenolic compounds, such as ultrasound-assisted extraction (UAE), ultraturrax extraction (UTE), accelerated solvent extraction (ASE) and pulsed electric field (PEF) extraction pre-treatment. Total phenolic content (TPC) was determined by Folin-Ciocalteu assay. Phloridzin, the main phenolic compound in apples, was determined by chromatographic analysis Q-TOF-LC/MS. The results obtained with these techniques were compared in order to identify the most efficient method to recover polyphenols. The highest value of TPC (1062.92 ± 59.80 µg GAE/g fresh apple pomace) was obtained when UAE was performed with EtOH:H2O (50:50, v/v), while ASE with EtOH:H2O (30:70, v/v) at 40 °C and 50% of flush was the most efficient technique in the recovery of phloridzin. The concentration of the main phenolic compounds ranged from 385.84 to 650.56 µg/g fresh apple pomace. The obtained results confirm that apple pomace represents an interesti-ng by-product, due to the presence of phenolic compounds. In particular, phloridzin could be considered a biomarker to determine the quality of numerous apple products. Therefore, this research could be a good starting point to develop a value-added product such as a functional food or nutraceutical.


Assuntos
Malus/química , Fenóis/química , Antioxidantes/química , Cromatografia Líquida de Alta Pressão/métodos , Etanol/química , Flavonoides/química , Espectrometria de Massas/métodos , Extratos Vegetais/química , Polifenóis/química
19.
Molecules ; 26(14)2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34299622

RESUMO

A method has been developed, optimised and validated to analyse protein powder supplements on an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS), with reference to ICH Guideline Q2 Validation of Analytical Procedures: Text and Methodology. This method was used in the assessment of twenty-one (n = 21) elements (Al, Au, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Pb, Pt, Sn, Ti, Tl, V) to evaluate the safety of thirty-six (n = 36) protein powder samples that were commercially available in the Irish marketplace in 2016/2017. Using the determined concentrations of elements in samples (µg·kg-1), a human health risk assessment was carried out to evaluate the potential carcinogenic and other risks to consumers of these products. While the concentrations of potentially toxic elements were found to be at acceptable levels, the results suggest that excessive and prolonged use of some of these products may place consumers at a slightly elevated risk for developing cancer or other negative health impacts throughout their lifetimes. Thus, the excessive use of these products is to be cautioned, and consumers are encouraged to follow manufacturer serving recommendations.


Assuntos
Espectrometria de Massas/métodos , Pós/química , Proteínas/química , Análise Espectral/métodos , Humanos , Irlanda , Metais , Medição de Risco , Oligoelementos/química
20.
Molecules ; 26(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34299444

RESUMO

We aimed to analyze the chemical compositions in Arabica coffee bean extracts, assess the relevant antioxidant and iron-chelating activities in coffee extracts and instant coffee, and evaluate the toxicity in roasted coffee. Coffee beans were extracted using boiling, drip-filtered and espresso brewing methods. Certain phenolics were investigated including trigonelline, caffeic acid and their derivatives, gallic acid, epicatechin, chlorogenic acid (CGA) and their derivatives, p-coumaroylquinic acid, p-coumaroyl glucoside, the rutin and syringic acid that exist in green and roasted coffee extracts, along with dimethoxycinnamic acid, caffeoylarbutin and cymaroside that may be present in green coffee bean extracts. Different phytochemicals were also detected in all of the coffee extracts. Roasted coffee extracts and instant coffees exhibited free-radical scavenging properties in a dose-dependent manner, for which drip coffee was observed to be the most effective (p < 0.05). All coffee extracts, instant coffee varieties and CGA could effectively bind ferric ion in a concentration-dependent manner resulting in an iron-bound complex. Roasted coffee extracts were neither toxic to normal mononuclear cells nor breast cancer cells. The findings indicate that phenolics, particularly CGA, could effectively contribute to the iron-chelating and free-radical scavenging properties observed in coffee brews. Thus, coffee may possess high pharmacological value and could be utilized as a health beverage.


Assuntos
Coffea/química , Sequestradores de Radicais Livres/análise , Proteínas de Ligação ao Ferro/análise , Alcaloides , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Ácido Clorogênico/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Coffea/toxicidade , Café/química , Café/toxicidade , Temperatura Alta , Humanos , Ferro/análise , Espectrometria de Massas/métodos , Fenóis/farmacologia , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/química , Extratos Vegetais/análise , Extratos Vegetais/química , Sementes/química
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