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1.
Methods Mol Biol ; 2414: 63-73, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34784032

RESUMO

Antigen selection is a critical step in subunit vaccine design, especially if the goal is to identify antigens that can be bound by neutralizing antibodies to prevent invasion of cells by intracellular bacteria. Here, we describe a method involving two dimensional gel electrophoresis (2-DE) coupled with western blotting (WB) and mass spectrometry (MS) to identify bacterial proteins that: (1) interact with the host target cell proteins, and (2) are targeted by antibodies from sera from infected animals. Subsequent steps would be performed to validate that the bacteria are targeted by neutralizing antibodies to prevent invasion of the eukaryotic cells.


Assuntos
Bactérias Gram-Negativas , Animais , Anticorpos Neutralizantes , Antígenos de Bactérias , Proteínas de Bactérias , Western Blotting , Eletroforese em Gel Bidimensional , Lawsonia (Bactéria)/imunologia
2.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 37(6): 673-677, 2021 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-34821104

RESUMO

Objective: To investigate the effects of long-term moderate-small intensity aerobic exercise on the differential expression of proteome in left ventricular muscle of rats, and to screen the target proteins sensitive to moderate-small intensity aerobic exercise stimulation. This study will enrich the basic theory of exercise and fitness and provide new ideas and experimental basis for the rehabilitation treatment of chronic cardiovascular disease. Methods: Twenty male SD rats were randomly divided into exercise group and control group (n=10). The treadmill training model of long-term moderate-small intensity aerobic exercise was established, and the whole protein samples of left ventricular muscle were extracted and separated by two-dimensional gel electrophoresis (2-DE). The two-dimensional gel electrophoresis map was analyzed by Bio PD quest image analysis software. The protein spots with differential expression more than 5 times or down-regulated over 80% after exercise were identified by tandem time-of-flight mass spectrometry (ULGRAFL-FLEX-TOF/TOF). Results: Compared with the group C, the heart weight index of the group E was increased by 32.0%, and the difference was significant (P<0.05). Compared with the group C, there were 71 protein spots expression were up-regulated≥2 times or down-regulated≥50% in the group E. 4 protein spots expression were up-regulated≥5 times or down-regulated≥80% were identified by mass spectrometry, 3 proteins and 1 unknown protein were identified. Conclusion: After long-term moderate-small intensity aerobic exercise, the rats heart had a good adaptive change, and the proteome of left ventricular muscle changed significantly. Long-term moderate-small intensity aerobic exercise can effective enhance the ability of myocardial antioxidation.


Assuntos
Condicionamento Físico Animal , Proteoma , Animais , Eletroforese em Gel Bidimensional , Masculino , Músculos , Ratos , Ratos Sprague-Dawley
3.
Exp Parasitol ; 230: 108159, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34563508

RESUMO

Trypanosoma rangeli is a non-virulent hemoflagellate parasite infecting humans, wild and domestic mammals in Central and Latin America. The share of genotypic, phenotypic, and biological similarities with the virulent, human-infective T. cruzi and T. brucei, allows comparative studies on mechanisms of pathogenesis. In this study, investigation of the T. rangeli Arginine Kinase (TrAK) revealed two highly similar copies of the AK gene in this taxon, and a distinct expression profile and activity between replicative and infective forms. Although TrAK expression seems stable during epimastigotes growth, the enzymatic activity increases during the exponential growth phase and decreases from the stationary phase onwards. No differences were observed in activity or expression levels of TrAK during in vitro differentiation from epimastigotes to infective forms, and no detectable AK expression was observed for blood trypomastigotes. Overexpression of TrAK by T. rangeli showed no effects on the in vitro growth pattern, differentiation to infective forms, or infectivity to mice and triatomines. Although differences in TrAK expression and activity were observed among T. rangeli strains from distinct genetic lineages, our results indicate an up-regulation during parasite replication and putative post-translational myristoylation of this enzyme. We conclude that up-regulation of TrAK activity in epimastigotes appears to improve proliferation fitness, while reduced TrAK expression in blood trypomastigotes may be related to short-term and subpatent parasitemia in mammalian hosts.


Assuntos
Arginina Quinase/metabolismo , Processamento de Proteína Pós-Traducional , Trypanosoma cruzi/enzimologia , Trypanosoma rangeli/enzimologia , Sequência de Aminoácidos , Animais , Arginina Quinase/biossíntese , Arginina Quinase/classificação , Arginina Quinase/genética , Western Blotting , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel Bidimensional , Feminino , Flagelos/enzimologia , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Alinhamento de Sequência , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidade , Trypanosoma rangeli/classificação , Trypanosoma rangeli/genética , Trypanosoma rangeli/patogenicidade , Regulação para Cima , Virulência
4.
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
5.
Molecules ; 26(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201770

RESUMO

Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes makes it also applicable to other areas of science. Milk is a highly heterogeneous and complex fluid, where there are numerous genetic variants and isoforms with post-translational modifications (PTMs). Due to the vast number of proteins and peptides existing in its matrix, proteomics is presented as a powerful tool for the characterization of milk samples and their products. The technology developed to date for the separation and characterization of the milk proteome, such as two-dimensional gel electrophoresis (2DE) technology and especially mass spectrometry (MS) have allowed an exhaustive characterization of the proteins and peptides present in milk and dairy products with enormous applications in the industry for the control of fundamental parameters, such as microbiological safety, the guarantee of authenticity, or the control of the transformations carried out, aimed to increase the quality of the final product.


Assuntos
Proteínas do Leite/metabolismo , Leite/química , Proteoma/metabolismo , Proteômica/métodos , Animais , Laticínios/análise , Eletroforese em Gel Bidimensional , Feminino , Espectrometria de Massas , Mastite/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Controle de Qualidade
6.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199160

RESUMO

Acadesine (ACA), a pharmacological activator of AMP-activated protein kinase (AMPK), showed a promising beneficial effect in a mouse model of colitis, indicating this drug as an alternative tool to manage IBDs. However, ACA displays some pharmacodynamic limitations precluding its therapeutical applications. Our study was aimed at evaluating the in vitro and in vivo effects of FA-5 (a novel direct AMPK activator synthesized in our laboratories) in an experimental model of colitis in rats. A set of experiments evaluated the ability of FA5 to activate AMPK and to compare the efficacy of FA5 with ACA in an experimental model of colitis. The effects of FA-5, ACA, or dexamethasone were tested in rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis to assess systemic and tissue inflammatory parameters. In in vitro experiments, FA5 induced phosphorylation, and thus the activation, of AMPK, contextually to the activation of SIRT-1. In vivo, FA5 counteracted the increase in spleen weight, improved the colon length, ameliorated macroscopic damage score, and reduced TNF and MDA tissue levels in DNBS-treated rats. Of note, FA-5 displayed an increased anti-inflammatory efficacy as compared with ACA. The novel AMPK activator FA-5 displays an improved anti-inflammatory efficacy representing a promising pharmacological tool against bowel inflammation.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Benzofuranos/uso terapêutico , Desenvolvimento de Medicamentos , Ativadores de Enzimas/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Animais , Benzofuranos/farmacologia , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Colo/efeitos dos fármacos , Colo/patologia , Dinitrofluorbenzeno/análogos & derivados , Eletroforese em Gel Bidimensional , Ontologia Genética , Doenças Inflamatórias Intestinais/patologia , Interleucina-10/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
7.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299329

RESUMO

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography-tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the 'Aroona' cultivar and 12 'Aroona' near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for 'Aroona' and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in 'Aroona' and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.


Assuntos
Glutens/análise , Glutens/metabolismo , Triticum/metabolismo , Alelos , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional/métodos , Haplótipos , Peso Molecular , Melhoramento Vegetal/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Triticum/química , Triticum/genética
8.
BMC Cancer ; 21(1): 865, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34320944

RESUMO

BACKGROUND: Cholangiocarcinoma (CCA) is an aggressive disease with poor prognosis. A molecular classification based on mutational, methylation and transcriptomic features could allow identifying tailored therapies to improve CCA patient outcome. Proteomic remains partially unexplored; here, we analyzed the proteomic profile of five intrahepatic cholangiocarcinoma (ICC) derived from Italian patients undergone surgery and one normal bile duct cell line. METHODS: Proteome profile was investigated by using 2D electrophoresis followed by Mass Spectrometry (MS). To validate proteomic data, the expression of four overexpressed proteins (CAT, SOD, PRDX6, DBI/ACBP) was evaluated by immunohistochemistry in an independent cohort of formalin fixed, paraffin-embedded (FFPE) ICC tissues. We also compared proteomic data with those obtained by transcriptomic profile evaluated by microarray analysis of the same tissues. RESULTS: We identified 19 differentially expressed protein spots, which were further characterized by MS; 13 of them were up- and 6 were down-regulated in ICC. These proteins are mainly involved in redox processes (CAT, SODM, PRDX2, PRDX6), in metabolism (ACBP, ACY1, UCRI, FTCD, HCMS2), and cell structure and organization (TUB2, ACTB). CAT is overexpressed in 86% of patients, PRDX6 in 73%, SODM in 100%, and DBI/ACBP in 81% compared to normal adjacent tissues. A concordance of 50% between proteomic and transcriptomic data was observed. CONCLUSIONS: This study pointed out that the impairment of the metabolic and antioxidant systems, with a subsequent accumulation of free radicals, might be a key step in CCA development and progression.


Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Biomarcadores Tumorais , Colangiocarcinoma/metabolismo , Metabolismo Energético , Oxirredução , Proteoma , Proteômica , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Espectrometria de Massas/métodos , Proteômica/métodos
9.
J Proteomics ; 248: 104331, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34303863

RESUMO

This study describes the association between meat tenderness and abundance of soluble muscle proteins in Nellore bulls (Bos indicus) using a proteomic approach. We evaluated shear force (SF) of Longissimus thoracis muscle 24 h after slaughter and selected three experimental groups of animals with moderately tender (TE; SF = 3.9 ± 0.7 kg), moderately tough (TO; SF = 5.6 ± 0.7 kg) and very tough meat (TO+; SF = 7.9 ± 1.4 kg). Proteome was investigated by two-dimensional electrophoresis (2D-PAGE) in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The metabolic proteins triosephosphate isomerase (TPI1) and phosphoglucomutase 1 (PGM1), the structural protein profilin 1 (PFN1), and cytosol aminopeptidase (LAP3) were up-regulated (P < 0.05) in the TE meat group when compared to the TO and TO+ groups. Actin structural proteins (ACTA1, ACTB, and ACTG1), the oxidative stress protein peroxiredoxin (PRDX6, PRDX2, PRDX1, and PARK7), heat shock protein isoforms, and co-chaperones (CDC37 and STIP1) were up-regulated (P < 0.05) in the TO and TO+ meat groups. In addition, we also identified proteins PFN1, LAP3, PRDX1, PRDX2, HSPD1, and ARHGDIA to be associated with beef tenderness. The results reported herein demonstrated that meat tenderness in Nellore cattle depends on the modulation and expression of a set of proteins involved in different biological pathways. SIGNIFICANCE: The manuscript entitled "Application of proteomic to investigate the different degrees of meat tenderness in Nellore breed" describes a classical proteomics work using two-dimensional gel electrophoresis (2D-PAGE), followed by mass spectrometry coupled to electrospray ionization ion trap (ESI-MS/MS) in order to understand the biochemical engineering involved in the process of meat tenderness. We evaluated shear force (SF) of Longissimus thoracis muscle samples of Nellore cattle (n = 90) and select three experimental groups of animals with moderately tender (TE; SF = 3.9 ± 0.7), moderately tough (TO; SF = 5.6 ± 0.7) and very tough meat (TO+; SF = 7.9 ± 1.4). The proteomic approach allowed observing that meat tenderness is influenced by structural proteins (ACTA1, ACTG1, ACTB, MYL1 and PFN1), co-chaperones (CDC37 and STIP1), heat shock proteins (HSP90AA1, HSP90AB1, HSPD1, HSPA1L, HSPA1A and HSPB1), regulatory protein (ARHGDIA), metabolic proteins (TPI1 and PGM1) and oxidative stress proteins (PRDX1, PRDX2, PRDX6, PARK7). Our results suggest that meat tenderness in Nellore depends on the modulation and expression of a set of proteins involved in different biological pathways.


Assuntos
Proteômica , Carne Vermelha , Animais , Bovinos , Eletroforese em Gel Bidimensional , Masculino , Carne/análise , Proteínas Musculares , Músculo Esquelético , Carne Vermelha/análise , Espectrometria de Massas em Tandem
10.
Aging (Albany NY) ; 13(11): 14709-14728, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074800

RESUMO

One of the genes which has been linked to the onset of juvenile/early onset Parkinson's disease (PD) is PINK1. There is evidence that supports the therapeutic potential of exercise in the alleviation of PD symptoms. It is possible that exercise may enhance synaptic plasticity, protect against neuro-inflammation and modulate L-Dopa regulated signalling pathways. We explored the effects of exercise on Pink1 deficient Drosophila melanogaster which undergo neurodegeneration and muscle degeneration. We used a 'power-tower' type exercise platform to deliver exercise activity to Pink1- and age matched wild-type Drosophila. Mitochondrial proteomic profiles responding to exercise were obtained. Of the 516 proteins identified, 105 proteins had different levels between Pink1- and wild-type non-exercised Drosophila. Gene ontology enrichment analysis and STRING network analysis highlighted proteins and pathways with altered expression within the mitochondrial proteome. Comparison of the Pink1- exercised proteome to wild-type proteomes showed that exercising the Pink1- Drosophila caused their proteomic profile to return towards wild-type levels.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Mitocondriais/metabolismo , Condicionamento Físico Animal , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eletroforese em Gel Bidimensional , Metabolismo Energético , Regulação da Expressão Gênica , Ontologia Genética , Espectrometria de Massas , Mitocôndrias/metabolismo , Anotação de Sequência Molecular , Mutação/genética , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteômica
11.
Methods Mol Biol ; 2276: 103-112, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060035

RESUMO

Native electrophoresis is a powerful tool to analyze the mitochondrial electron transport chain complexes (Cx) I-V and their assembly into supercomplexes. Valuable information regarding the composition and bioenergetic regulation in physiological and pathological conditions can be obtained. This chapter compares different types of native electrophoresis to analyze mitochondrial supercomplexes.


Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Immunoblotting/métodos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Humanos , Proteínas Mitocondriais/química
12.
Methods Mol Biol ; 2276: 383-396, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060056

RESUMO

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Immunoblotting/métodos , Espectrometria de Massas/métodos , Mitocôndrias Cardíacas/química , Proteínas Mitocondriais/análise , Ácido Peroxinitroso/química , Tirosina/análogos & derivados , Animais , Bovinos , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Tirosina/análise , Tirosina/metabolismo
13.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070710

RESUMO

8-Hydroxyguanine (8-oxoG) is the most common oxidative DNA lesion and unrepaired 8-oxoG is associated with DNA fragmentation in sperm. However, the molecular effects of 8-oxoG on spermatogenesis are not entirely understood. Here, we identified one infertile bull (C14) due to asthenoteratozoospermia. We compared the global concentration of 8-oxoG by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), the genomic distribution of 8-oxoG by next-generation sequencing (OG-seq), and the expression of sperm proteins by 2-dimensional polyacrylamide gel electrophoresis followed by peptide mass fingerprinting (2D-PAGE/PMF) in the sperm of C14 with those of a fertile bull (C13). We found that the average levels of 8-oxoG in C13 and C14 sperm were 0.027% and 0.044% of the total dG and it was significantly greater in infertile sperm DNA (p = 0.0028). Over 81% of the 8-oxoG loci were distributed around the transcription start site (TSS) and 165 genes harboring 8-oxoG were exclusive to infertile sperm. Functional enrichment and network analysis revealed that the Golgi apparatus was significantly enriched with the products from 8-oxoG genes of infertile sperm (q = 2.2 × 10-7). Proteomic analysis verified that acrosome-related proteins, including acrosin-binding protein (ACRBP), were downregulated in infertile sperm. These preliminary results suggest that 8-oxoG formation during spermatogenesis dysregulated the acrosome-related gene network, causing structural and functional defects of sperm and leading to infertility.


Assuntos
Acrossomo/metabolismo , Proteínas de Transporte/genética , Redes Reguladoras de Genes , Guanina/análogos & derivados , Infertilidade Masculina/genética , Tubulina (Proteína)/genética , Acrossomo/patologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Bovinos , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Guanina/metabolismo , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Metaboloma , Mapeamento de Interação de Proteínas , Proteômica/métodos , Análise do Sêmen , Espermatogênese/genética , Tubulina (Proteína)/metabolismo
14.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071777

RESUMO

In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of IPF, BALF proteomics is providing interesting new insights into its pathogenesis. To the best of our knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as the EVs isolation technique, and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider system involvement in the disease pathogenesis and support the importance of pre-fractioning of complex samples, such as BALF, in order to let low-abundant proteins-mediated pathways emerge.


Assuntos
Biomarcadores , Líquido da Lavagem Broncoalveolar , Vesículas Extracelulares/metabolismo , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , Proteoma , Proteômica , Idoso , Cromatografia Líquida , Suscetibilidade a Doenças , Eletroforese em Gel Bidimensional , Vesículas Extracelulares/ultraestrutura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Transdução de Sinais , Espectrometria de Massas em Tandem
15.
Methods Mol Biol ; 2228: 41-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950482

RESUMO

Two-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using two-dimensional gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods, (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of two-dimensional gels is performed.


Assuntos
Eletroforese em Gel Bidimensional , Proteínas/análise , Proteoma , Proteômica , Animais , Corantes Fluorescentes , Humanos , Medições Luminescentes , Projetos de Pesquisa , Coloração e Rotulagem
16.
Methods Mol Biol ; 2228: 53-62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950483

RESUMO

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH-gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures such as Coomassie, silver staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.


Assuntos
Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Proteínas/análise , Proteoma , Proteômica , Animais , Corantes Fluorescentes , Humanos , Concentração de Íons de Hidrogênio , Medições Luminescentes , Projetos de Pesquisa , Coloração e Rotulagem
17.
Methods Mol Biol ; 2228: 63-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950484

RESUMO

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. For its use in proteomics, two important additional features must be considered, compatibility with mass spectrometry and quantitative response. Both features are discussed in this chapter, and optimized silver staining protocols are proposed.


Assuntos
Eletroforese em Gel Bidimensional , Proteínas/análise , Proteoma , Proteômica , Coloração pela Prata , Animais , Humanos , Projetos de Pesquisa
19.
Insect Biochem Mol Biol ; 134: 103584, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34033897

RESUMO

In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the HopTum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity.


Assuntos
Drosophila melanogaster , Hemócitos/imunologia , Proteínas de Membrana/isolamento & purificação , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular/isolamento & purificação , Encapsulamento de Células , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/parasitologia , Eletroforese em Gel Bidimensional , Feminino , Hemócitos/metabolismo , Interações Hospedeiro-Parasita/imunologia , Proteínas de Insetos/isolamento & purificação , Integrinas/isolamento & purificação , Larva/imunologia , Larva/metabolismo , Larva/parasitologia , Espectrometria de Massas , Proteômica , Transdução de Sinais
20.
PLoS One ; 16(5): e0252450, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34048472

RESUMO

Except cells circulating in the bloodstream, most cells in vertebrates are adherent. Studying the repercussions of adherence per se in cell physiology is thus very difficult to carry out, although it plays an important role in cancer biology, e.g. in the metastasis process. In order to study how adherence impacts major cell functions, we used a murine macrophage cell line. Opposite to the monocyte/macrophage system, where adherence is associated with the acquisition of differentiated functions, these cells can be grown in both adherent or suspension conditions without altering their differentiated functions (phagocytosis and inflammation signaling). We used a proteomic approach to cover a large panel of proteins potentially modified by the adherence status. Targeted experiments were carried out to validate the proteomic results, e.g. on metabolic enzymes, mitochondrial and cytoskeletal proteins. The mitochondrial activity was increased in non-adherent cells compared with adherent cells, without differences in glucose consumption. Concerning the cytoskeleton, a rearrangement of the actin organization (filopodia vs sub-cortical network) and of the microtubule network were observed between adherent and non-adherent cells. Taken together, these data show the mechanisms at play for the modification of the cytoskeleton and also modifications of the metabolic activity between adherent and non-adherent cells.


Assuntos
Adesão Celular/fisiologia , Proteômica/métodos , Animais , Ciclo Celular , Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Hexoquinase/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Óxido Nítrico/metabolismo , Fagocitose , Células RAW 264.7
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