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1.
Theor Appl Genet ; 2018 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-30426174

RESUMO

KEY MESSAGE: Knocking down GW2 enhances grain size by regulating genes encoding the synthesis of cytokinin, gibberellin, starch and cell wall. Raising crop yield is a priority task in the light of the continuing growth of the world's population and the inexorable loss of arable land to urbanization. Here, the RNAi approach was taken to reduce the abundance of Grain Weight 2 (GW2) transcript in the durum wheat cultivar Svevo. The effect of the knockdown was to increase the grains' starch content by 10-40%, their width by 4-13% and their surface area by 3-5%. Transcriptomic profiling, based on a quantitative real-time PCR platform, revealed that the transcript abundance of genes encoding both cytokinin dehydrogenase 1 and the large subunit of ADP-glucose pyrophosphorylase was markedly increased in the transgenic lines, whereas that of the genes encoding cytokinin dehydrogenase 2 and gibberellin 3-oxidase was reduced. A proteomic analysis of the non-storage fraction extracted from mature grains detected that eleven proteins were differentially represented in the transgenic compared to wild-type grain: some of these were involved, or at least potentially involved, in cell wall development, suggesting a role of GW2 in the regulation of cell division in the wheat grain.

2.
Food Chem ; 268: 585-593, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30064801

RESUMO

The ethanol extract of the commercial tannin Tan'Activ C, (from Castanea sativa wood), employed in oenology, was subjected to chromatography on XAD-16 affording fractions X1-X5, evaluated for total phenols content (GAE), antioxidant activity (DPPH, ORAC), and hypoglycemic activity (α-glucosidase inhibition). Fraction X3 showed GAE, radical scavenging activity, and α-glucosidase inhibition higher than those of the Castanea sativa extract, and was subjected to chromatography on Sephadex LH-20 to obtain fractions S1-S7, analyzed by HPLC/ESI-MS/MS and 1H NMR to identify the main active constituents. In fractions with higher antioxidant activity, gallic acid (4), grandinin (5), valoneic acid dilactone (9), 1,2,3-tri-O-galloyl-ß-glucose (14), 3,4,6-tri-O-galloyl-ß-glucose (15) and galloyl derivative of valoneic acid dilactone (21) were identified as the major constituents. The highest hypoglycemic activity was found in fractions S6 and especially S7; the major constituents of these fractions are valoneic acid dilactone (9), three tetragalloyl glucose isomers (16-18) and 1,2,3,4,6-penta-O-galloyl-ß-glucose (23), previously reported as α-glucosidase inhibitors.

3.
Anal Chem ; 90(16): 9673-9676, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30044608

RESUMO

The material analyzed in this study is probably the most ancient archeological solid residue of cheese ever found to date. The sample was collected during the Saqqara Cairo University excavations in the tomb of Ptahmes dated to XIX dynasty ( El-Aguizy, O. Bulletin de l'Institut Française d'Archéologie Orientale (BIFAO) 2010 , 110 , 13 - 34 (ref (1) ); Staring, N. Bulletin de Institut Français d'Archéologie Orientale (BIFAO) 2015 , 114 , 455 - 518 (ref (2) )). Our biomolecular proteomic characterization of this archeological sample shows that the constituting material was a dairy product obtained by mixing sheep/goat and cow milk. The interactions for thousands of years with the strong alkaline environment of the incorporating soil rich in sodium carbonate and the desertic conditions did not prevent the identification of specific peptide markers which showed high stability under these stressing conditions. Moreover, the presence of Brucella melitensis has been attested by specific peptide providing a reasonable direct biomolecular evidence of the presence of this infection in the Ramesside period for which only indirect paleopathological evidence has been so far provided ( Pappas, G.; Papadimitriou P. Int. J. Antimicrob. Agents 2007 , 30 , 29 - 31 (ref (3) ); Bourke, J. B. Medical History 1971 , 15 ( 4 ), 363 - 375 (ref (4) )). Finally, it is worth noting that, although proteomic approaches are successfully and regularly used to characterize modern biological samples ( D'Ambrosio, C.; Arena, S.; Salzano, A. M.; Renzone, G.; Ledda, L.; and Scaloni, A. Proteomics 2008 8 , 3657 - 3666 (ref (5) ), their application in ancient materials is still at an early stage of progress, only few results being reported about ancient food samples ( Yang, Y.; Shevchenko, A.; Knaust, A.; Abuduresule, I.; Li, W.; Hu, X.; Wang, C.; Shevchenko, A. J. Archaeol. Sci. 2014 , 45 , 178 - 186 (ref (6) ). In the absence of previous relevant evidence of cheese production and/or use, this study, undoubtedly has a clear added value in different fields of knowledge ranging from archaeometry, anthropology, archeology, medicine history to the forensic sciences.

4.
Biochim Biophys Acta Bioenerg ; 1859(9): 806-816, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29890122

RESUMO

VDACs three isoforms (VDAC1, VDAC2, VDAC3) are integral proteins of the outer mitochondrial membrane whose primary function is to permit the communication and exchange of molecules related to the mitochondrial functions. We have recently reported about the peculiar over-oxidation of VDAC3 cysteines. In this work we have extended our analysis, performed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS, to the other two isoforms VDAC1 and VDAC2 from rat liver mitochondria, and we have been able to find also in these proteins over-oxidation of cysteines. Further PTM of cysteines as succination has been found, while the presence of selenocysteine was not detected. Unfortunately, a short sequence stretch containing one genetically encoded cysteine was not covered both in VDAC2 and in VDAC3, raising the suspect that more, unknown modifications of these proteins exist. Interestingly, cysteine over-oxidation appears to be an exclusive feature of VDACs, since it is not present in other transmembrane mitochondrial proteins eluted by hydroxyapatite. The assignment of a functional role to these modifications of VDACs will be a further step towards the full understanding of the roles of these proteins in the cell.


Assuntos
Cisteína/química , Mitocôndrias Hepáticas/metabolismo , Processamento de Proteína Pós-Traducional , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Cisteína/metabolismo , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 2 Dependente de Voltagem/química , Canal de Ânion 2 Dependente de Voltagem/genética
5.
Amino Acids ; 50(6): 735-746, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29572574

RESUMO

In the last years, donkey milk had evidenced a renewed interest as a potential functional food and a breast milk substitute. In this light, the study of the protein composition assumes an important role. In particular, ß-lactoglobulin (ß-LG), which is considered as one of the main allergenic milk protein, in donkey species consists of two molecular forms, namely ß-LG I and ß-LG II. In the present research, a genetic analysis coupled with a proteomic approach showed the presence of a new allele, here named F, which is apparently associated with a null or a severely reduced expression of ß-LG II protein. The new ß-LG II F genetic variant shows a theoretical average mass (Mav) of 18,310.64 Da, a value practically corresponding with that of the variant D (∆mass < 0.07 Da), but differs from ß-LG II D for two amino acid substitutions: Thr100 (variant F) → Ala100 (variant D) and Thr118 (variant F) → Met118 (variant D). Proteomic investigation of the whey protein fraction of an individual milk sample, homozygous FF at ß-LG II locus, allowed to identify, as very minor component, the new ß-LG II F genetic variant. By MS/MS analysis of enzymatic digests, the sequence of the ß-LG II F was characterized, and the predicted genomic data confirmed.

6.
J Proteome Res ; 16(12): 4319-4329, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28828861

RESUMO

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.


Assuntos
Mitocôndrias/química , Proteoma/fisiologia , Proteômica/normas , Linhagem Celular , Cromatografia Líquida , Humanos , Itália , Proteínas Mitocondriais/análise , Mapas de Interação de Proteínas/fisiologia , Espectrometria de Massas em Tandem
7.
Food Res Int ; 99(Pt 1): 41-57, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28784499

RESUMO

The legendary therapeutics properties of donkey milk have recently been supported by many clinical trials who have clearly demonstrated that, even if with adequate lipid integration, it may represent a valid natural substitute of cow milk for feeding allergic children. During the last decade many investigations by MS-based methods have been performed in order to obtain a better knowledge of donkey milk proteins. The knowledge about the primary structure of donkey milk proteins now may provide the basis for a more accurate comprehension of its potential benefits for human nutrition. In this aspect, experimental data today available clearly demonstrate that donkey milk proteins (especially casein components) are more closely related with the human homologues rather than cow counterparts. Moreover, the low allergenic properties of donkey milk with respect to cow one seem to be related to the low total protein content, the low ratio of caseins to whey fraction, and finally to the presence in almost all bovine IgE-binding linear epitopes of multiple amino acid differences with respect to the corresponding regions of donkey milk counterparts.

8.
J Proteomics ; 165: 102-112, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28625740

RESUMO

Gluten proteins are major determinants of the bread making quality of wheat, but also of important wheat-related disorders, including coeliac disease (CD), and allergies. We carried out a proteomic study using the total grain proteins from two low-gliadin wheat lines, obtained by RNAi, and the untransformed wild type as reference. The impact of silencing on both target and on non-target proteins was evaluated. Because of the great protein complexity, we performed separate analyses of four kernel protein fractions: gliadins and glutenin subunits, and metabolic and CM-like proteins, by using a classical 2D electrophoresis gel based approach followed by RP-HPLC/nESI-MS/MS. As a result of the strong down-regulation of gliadins, the HMW-GS, metabolic and chloroform/methanol soluble proteins were over-accumulated in the transgenic lines, especially in the line D793, which showed the highest silencing of gliadins. Basing on these data, and considering that metabolic proteins and chloroform/methanol soluble proteins (CM-like), such as the α-amylase/trypsin inhibitor family, ß-amylase and serpins, were related to wheat allergens, further in vivo analysis will be needed, especially those related to clinical trials in controlled patients, to determine if these lines could be used for food preparation for celiac or other gluten intolerant groups. BIOLOGICAL SIGNIFICANCE: Several enteropathies and allergies are related to wheat proteins. Biotechnological techniques such as genetic transformation and RNA interference have allowed the silencing of gliadin genes, providing lines with very low gliadin content in the grains. We report a proteomic-based approach to characterize two low-gliadin transgenic wheat lines obtained by RNAi technology. These lines harbor the same silencing fragment, but driven by two different endosperm specific promoters (γ-gliadin and D-hordein). The comprehensive proteome analysis of these transgenic lines, by combining two-dimensional electrophoresis and RP-HPLC/nESI-MS/MS, provided a large number of spots differentially expressed between the control and the transgenic lines. Hence, the results of this study will facilitate further safety evaluation of these transgenic lines.


Assuntos
Gliadina/genética , Plantas Geneticamente Modificadas , Proteômica/métodos , Triticum/química , Pão , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Inativação Gênica , Proteínas de Plantas/análise , Espectrometria de Massas em Tandem , Triticum/genética
9.
J Proteomics ; 152: 58-74, 2017 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-27784645

RESUMO

In the last years the amount of ovine milk production, mainly used to formulate a wide range of different and exclusive dairy products often categorized as gourmet food, has been progressively increasing. Taking also into account that sheep milk (SM) also appears to be potentially less allergenic than cow's one, an in-depth information about its protein composition is essential to improve the comprehension of its potential benefits for human consumption. The present work reports the results of an in-depth characterization of SM whey proteome, carried out by coupling the CPLL technology with SDS-PAGE and high resolution UPLC-nESI MS/MS analysis. This approach allowed the identification of 718 different protein components, 644 of which are from unique genes. Particularly, this identification has expanded literature data about sheep whey proteome by 193 novel proteins previously undetected, many of which are involved in the defence/immunity mechanisms or in the nutrient delivery system. A comparative analysis of SM proteome known to date with cow's milk proteome, evidenced that while about 29% of SM proteins are also present in CM, 71% of the identified components appear to be unique of SM proteome and include a heterogeneous group of components which seem to have health-promoting benefits. The data have been deposited to the ProteomeXchange with identifier .


Assuntos
Proteínas do Leite/análise , Proteoma/análise , Animais , Bovinos , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Imunidade , Proteômica/métodos , Ovinos , Especificidade da Espécie , Espectrometria de Massas em Tandem , Proteínas do Soro do Leite/análise
10.
Biochim Biophys Acta Biomembr ; 1859(3): 301-311, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27989743

RESUMO

Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane. In comparison with the most abundant isoform VDAC1, there is little knowledge about the functional role of VDAC3. Unlikely VDAC1, cysteine residues are particularly abundant in VDAC3. Since the mitochondrial intermembrane space (IMS) has an oxidative potential we questioned whether the redox state of VDAC3 can be modified. By means of SDS-PAGE separation, tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS analysis we investigated the oxidation state of cysteine and methionine residues of rat liver VDAC3. Our results demonstrate that the mitochondrial VDAC3, in physiological state, contains methionines oxidized to methionine sulfoxide. Furthermore, cysteine residues 36, 65, and 165 are oxidized to a remarkable extend to sulfonic acid. Cysteines 2 and 8 are observed exclusively in the carboxyamidomethylated form. Cys229 is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys122 could not be determined because peptides containing this residue were not detected. Control experiments ruled out the possibility that over-oxidation of cysteines might be due to artefactual reasons. The peculiar behavior of Met and Cys residues of VDAC3 may be related with the accessibility of the protein to a strongly oxidizing environment and may be connected with the regulation of the activity of this trans-membrane pore protein.


Assuntos
Cisteína/química , Metionina/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Espectrometria de Massas em Tandem , Canais de Ânion Dependentes de Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Oxirredução , Peptídeos/análise , Ratos , Tripsina/metabolismo , Canais de Ânion Dependentes de Voltagem/química
11.
Amino Acids ; 48(12): 2799-2808, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27550041

RESUMO

A comprehensive monosaccharide composition of the N-glycans of donkey milk lactoferrin, isolated by ion exchange chromatography from an individual milk sample, was obtained by means of chymotryptic digestion, TiO2 and HILIC enrichment, reversed-phase high-performance liquid chromatography, electrospray mass spectrometry, and high collision dissociation fragmentation. The results obtained allowed identifying 26 different glycan structures, including high mannose, complex and hybrid N-glycans, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476. Altogether, the N-glycan structures determined revealed that most of the N-glycans identified in donkey milk lactoferrin are neutral complex/hybrid. Indeed, 10 neutral non-fucosylated complex/hybrid N-glycans and 4 neutral fucosylated complex/hybrid N-glycans were found. In addition, two high mannose N-glycans, four sialylated fucosylated complex N-glycans and six sialylated non-fucosylated complex N-glycans, one of which containing N-glycolylneuraminic acid (Neu5Gc), were found. A comparison of the monosaccharide composition of the N-glycans of donkey milk lactoferrin with respect to that of human, bovine and goat milk lactoferrin is reported. Data are available via ProteomeXchange with identifier PXD004289.


Assuntos
Lactoferrina/química , Leite/química , Monossacarídeos/química , Polissacarídeos/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Equidae , Glicosilação , Cabras , Humanos , Lactoferrina/isolamento & purificação , Lactoferrina/metabolismo , Espectrometria de Massas , Leite/metabolismo , Monossacarídeos/isolamento & purificação , Monossacarídeos/metabolismo , Polissacarídeos/isolamento & purificação , Polissacarídeos/metabolismo
12.
Amino Acids ; 48(7): 1569-80, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27020775

RESUMO

Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg â†’ His), 328 (Thr â†’ Ile/Leu), 466 (Ala â†’ Gly), 642 (Asn â†’ Ser) and 668 (Ser â†’ Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476.


Assuntos
Lactoferrina/química , Leite/química , Polissacarídeos/química , Sequência de Aminoácidos , Animais , Equidae , Glicosilação , Lactoferrina/genética , Lactoferrina/metabolismo , Leite/metabolismo , Polissacarídeos/genética , Polissacarídeos/metabolismo
13.
Oncotarget ; 7(3): 2249-68, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26760765

RESUMO

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.


Assuntos
Cisteína/química , Fígado/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Transporte de Elétrons/fisiologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Fígado/citologia , Fígado/enzimologia , Espectrometria de Massas , Proteínas de Transporte da Membrana Mitocondrial/genética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Oxirredução , Isoformas de Proteínas/metabolismo , Ratos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Canais de Ânion Dependentes de Voltagem/genética
14.
J Proteomics ; 128: 69-82, 2015 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-26193489

RESUMO

The goat whey proteome has been explored in depth via capture with combinatorial peptide ligand libraries (CPLLs) at three different pH values. A total of 452 unique species have been tabulated, a proteome discovery so far unmatched in any single other investigation of milk from any mammalian species. This massive discovery is probably related to: i) the extraordinary load of proteins onto the CPLL beads (i.e. 2 g for each different pH captures) vs. barely 100 µL of beads; ii) the high resolution/high mass accuracy of mass spectral data; and iii) the use of two complementary tools, Mascot and PEAKS, each one contributing to a set of unique protein IDs. Due to the relative paucity of available protein annotations for goat, only 10% of the identified proteins belong to the capra, whereas 52% are specific of sheep and 37% are homologous to that of bovine milk. This work reports the largest description so far of the goat milk proteome, which has been compared with cow's milk proteome and would thus help to understand the importance of low-abundance proteins with respect to the unique biological properties of this nutrient.


Assuntos
Cabras/metabolismo , Proteínas do Leite/química , Leite/química , Proteoma/química , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular
15.
J Pharm Biomed Anal ; 105: 134-49, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25555262

RESUMO

A method based on liquid chromatography/high resolution tandem mass spectrometry coupled with electrophoretic separation, for determination and relative quantification of the protein composition of exhaled breath condensate (EBC), was developed. Application of the procedure to a sample of EBC, pooled from nine healthy subjects, resulted in the identification of 167 unique gene products, 113 of which not previously reported in EBC samples. The abundance of the protein identified was estimated by means of the exponentially modified protein abundance index protocol (emPAI). Cytokeratins were by far the most abundant proteins in EBC samples. Many of the identified proteins were associated with multiple cellular location with cytoplasm constituting the largest group. Cytosol, nucleus, membrane, cytoskeleton and extracellular were other abundantly represented locations. No amylase was detected, suggesting the absence of saliva protein contamination. The profile obtained represents the most comprehensive protein characterization of EBC so far reported and demonstrates that this approach provides a powerful tool for investigating the protein profile of EBC samples. Compared with analogous investigations, this study also shows that the protein profile of EBC is strongly affected by the sampling method adopted.


Assuntos
Testes Respiratórios/métodos , Cromatografia Líquida de Alta Pressão/métodos , Proteínas/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Adulto , Testes Respiratórios/instrumentação , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Proteômica
16.
J Mass Spectrom ; 49(9): 768-84, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25230173

RESUMO

In the last decades, the continuous and rapid evolution of proteomic approaches has provided an efficient platform for the characterization of food-derived proteins. Particularly, the impressive increasing in performance and versatility of the MS instrumentation has contributed to the development of new analytical strategies for proteins, evidencing how MS arguably represents an indispensable tool in food proteomics. Investigation of protein composition in foodstuffs is helpful for understanding the relationship between the protein content and the nutritional and technological properties of foods, the production of methods for food traceability, the assessment of food quality and safety, including the detection of allergens and microbial contaminants in foods, or even the characterization of genetically modified products. Given the high variety of the food-derived proteins and considering their differences in chemical and physical properties, a single proteomic strategy for all purposes does not exist. Rather, proteomic approaches need to be adapted to each analytical problem, and development of new strategies is necessary in order to obtain always the best results. In this tutorial, the most relevant aspects of MS-based methodologies in food proteomics will be examined, and their advantages and drawbacks will be discussed.


Assuntos
Análise de Alimentos/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Qualidade dos Alimentos
17.
Arch Microbiol ; 196(2): 79-85, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24346000

RESUMO

The L-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of D-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting L-alanine to the germination inhibitor D-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of L- to D-alanine. Unlike ATCC 14579 spores, L-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.


Assuntos
Alanina Racemase/metabolismo , Bacillus cereus/enzimologia , Bacillus cereus/fisiologia , Alanina/metabolismo , Alanina/farmacologia , Alanina Racemase/química , Alanina Racemase/genética , Sequência de Aminoácidos , Bacillus cereus/genética , Bacillus cereus/metabolismo , Ciclosserina/metabolismo , Ciclosserina/farmacologia , Microbiologia de Alimentos , Dados de Sequência Molecular , Proteômica , Esporos Bacterianos/citologia , Esporos Bacterianos/enzimologia , Esporos Bacterianos/fisiologia
18.
AMB Express ; 3(1): 35, 2013 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-23800329

RESUMO

The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.

19.
J Mass Spectrom ; 48(2): 148-53, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23378086

RESUMO

Donkey's milk (DM), representing a safe and alternative food in both IgE-mediated and non-IgE-mediated cow's milk protein allergy, can be categorized as precious pharma-food. Moreover, an economically relevant interest for the use of DM in cosmetology is also developing. The detection of adulterations and contaminations of DM is a matter of fundamental importance from both an economic and allergenic standpoint, and, to this aim, fast and efficient analytical approaches to assess the authenticity of this precious nutrient are desirable. Here, a rapid matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based method aimed to the detection of bovine or caprine milk in raw DM is reported. The presence of the extraneous milks was revealed by monitoring the protein profiles of the most abundant whey proteins, α-lactalbumin (α-LA) and ß-lactoglobulin, used as molecular markers. The possibility of obtaining a quantitative analysis of the level of cow or goat milk in DM based on the MALDI-TOF peak areas of α-LAs was also explored. The results showed that the experimental quantitative values were in good agreement with the real composition of each mixture. As pretreatment of the milk samples is not required, and owing to the speed and the high sensitivity of MALDI-MS, the protocol here reported could represent a reliable method for routine analyses aimed to assess the absence of contamination in raw fresh DM samples.


Assuntos
Contaminação de Alimentos/análise , Leite/química , Leite/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Equidae , Feminino , Cabras , Modelos Lineares , Proteínas do Leite/análise , Proteínas do Leite/química , Sensibilidade e Especificidade
20.
Eur J Mass Spectrom (Chichester) ; 19(4): 305-24, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24575629

RESUMO

Two citrus rootstocks, one sensitive to iron deficiency (Swingle Citrumelo (SCO)) and the other tolerant (Carrizo Citrange, (CC)), were studied to characterize variation in their root protein profile induced by iron-deficient conditions. Plants of both rootstocks were grown in two different soils, one volcanic (v) and the other calcareous (c), containing 0% and 10% active Lime, respectively. To evaluate the effects of the calcareous soil on the protein accumulation of both rootstocks, the root protein profiles (SCc vs. SCv and CCc vs. CCv) were characterized by two-dimensional gel electrophoresis, thus obtaining, for the first time, a reference map of this previously uncharacterized proteome. A total of 219 spots, significantly changed in abundance, were analyzed by high-performance Liquid chromatography nano electrospray ionization tandem mass spectrometry. The identified proteins were classified according to their putative function and known biosynthetic pathways. Principal component analysis, comparing the four sets of data, indicated that each group clustered together with low variance and that CCv and CCc data sets were well differentiated, whereas SCv and SCc were similar.


Assuntos
Citrus/metabolismo , Ferro/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Cromatografia Líquida de Alta Pressão , Citrus/química , Citrus/crescimento & desenvolvimento , Eletroforese em Gel Bidimensional , Raízes de Plantas/química , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
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