Desenvolvimento da técnica de MALDI Imaging utilizando tecidos animais

Currently the study of important molecular compounds present in low abundance in some tissues has been a challenge for proteomic analysis classic. An analysis requires more exploratory investigation of small regions of a tissue or a group of cells. MALDI Imaging Technology (MSI) is an application of mass spectrometry facing the chemical analysis of intact tissues. Thus, advances in mass spectrometry MALDI being obtained by the integration of histology, the best methods and automation are the main tools of data analysis. This tool has become essential to analyze the spatial distribution of peptides and proteins throughout the tissue sections, providing an enormous amount of data with minimum sample preparation. Thus, the aim of this study was to develop the technique of MALDI Imaging using tissue from glioblastoma multiforme (GBM), a form of most common malignant tumor in the brain. For this we used the printer chemical ChIP-1000 (Chemical Inkjet Printer, Shimadzu) and mass spectrometer type Maldi-ToF-ToF (Axima Performance, Shimadzu), a search of the identifications were performed in databases such as SwissProt. We identified more than forty proteins with diverse functions such as proteins F-actin-capping and Thymosin to the structure and organization cellular and proteins such several Tumor necrosis factor receptor development-related pathology. The development of this technique will permit to carry-out proteomic analysis directly into the tissue, enabling earlier diagnosis of diseases, as well as the identification and characterization of potential biomarkers of disease.Atualmente, o estudo de importantes compostos moleculares, presentes em baixa abundância em alguns tecidos animais, tem sido um grande desafio para as estratégias clássicas das análises proteômica. Uma análise mais exploratória exige a investigação mais aprofundada e direcionada em pequenas regiões do tecido estudado ou de um pequeno grupo de células. A tecnologia MALDI Imaging (MSI) é uma aplicação da espectrometria de massas voltada para a análise química de tecidos intactos. Desta forma, os avanços na espectrometria de massas do tipo MALDI estão sendo obtidos pela integração da histologia, com os melhores métodos de automação e os principais instrumentos de análise de dados. Esta ferramenta tem se tornado essencial para analisar a distribuição espacial de peptídeos e proteínas ao longo de cortes de tecidos, proporcionando uma enorme quantidade de dados com um tempo mínimo de manipulação e preparação da amostra. Dessa forma, o objetivo desse estudo foi desenvolver a técnica de MALDI Imaging utilizando tecido de Glioblastoma multiforme (GBM), uma forma de tumor maligno muito comum no cérebro humano. Neste trabalho, a impressora química CHIP-1000 (Chemical Inkjet Printer, Shimadzu) e o espectrômetro de massas do tipo Maldi-ToF-ToF (Axima Performance, Shimadzu) foram utilizados, sendo que as proteínas identificadas foram obtidas utilizando o servidor público SwissProt. Aproximadamente, foram identificadas quarenta proteínas com diversas funções, dentre elas as proteínas F-actina e Thimosina, as quais estão relacionadas com a estrutura e organização celular, e proteínas como diversos Fatores de necroses tumorais, as quais estão relacionadas diretamente com a patologia analisada nesse estudo. O desenvolvimento dessa técnica proporcionou uma análise proteômica diretamente no tecido, favorecendo o desenvolvimento de testes diagnósticos...(Resumo completo, clicar acesso eletrônico abaixo

Desenvolvimento da técnica de MALDI Imaging utilizando tecidos animais

Currently the study of important molecular compounds present in low abundance in some tissues has been a challenge for proteomic analysis classic. An analysis requires more exploratory investigation of small regions of a tissue or a group of cells. MALDI Imaging Technology (MSI) is an application of mass spectrometry facing the chemical analysis of intact tissues. Thus, advances in mass spectrometry MALDI being obtained by the integration of histology, the best methods and automation are the main tools of data analysis. This tool has become essential to analyze the spatial distribution of peptides and proteins throughout the tissue sections, providing an enormous amount of data with minimum sample preparation. Thus, the aim of this study was to develop the technique of MALDI Imaging using tissue from glioblastoma multiforme (GBM), a form of most common malignant tumor in the brain. For this we used the printer chemical ChIP-1000 (Chemical Inkjet Printer, Shimadzu) and mass spectrometer type Maldi-ToF-ToF (Axima Performance, Shimadzu), a search of the identifications were performed in databases such as SwissProt. We identified more than forty proteins with diverse functions such as proteins F-actin-capping and Thymosin to the structure and organization cellular and proteins such several Tumor necrosis factor receptor development-related pathology. The development of this technique will permit to carry-out proteomic analysis directly into the tissue, enabling earlier diagnosis of diseases, as well as the identification and characterization of potential biomarkers of disease.Atualmente, o estudo de importantes compostos moleculares, presentes em baixa abundância em alguns tecidos animais, tem sido um grande desafio para as estratégias clássicas das análises proteômica. Uma análise mais exploratória exige a investigação mais aprofundada e direcionada em pequenas regiões do tecido estudado ou de um pequeno grupo de células. A tecnologia MALDI Imaging (MSI) é uma aplicação da espectrometria de massas voltada para a análise química de tecidos intactos. Desta forma, os avanços na espectrometria de massas do tipo MALDI estão sendo obtidos pela integração da histologia, com os melhores métodos de automação e os principais instrumentos de análise de dados. Esta ferramenta tem se tornado essencial para analisar a distribuição espacial de peptídeos e proteínas ao longo de cortes de tecidos, proporcionando uma enorme quantidade de dados com um tempo mínimo de manipulação e preparação da amostra. Dessa forma, o objetivo desse estudo foi desenvolver a técnica de MALDI Imaging utilizando tecido de Glioblastoma multiforme (GBM), uma forma de tumor maligno muito comum no cérebro humano. Neste trabalho, a impressora química CHIP-1000 (Chemical Inkjet Printer, Shimadzu) e o espectrômetro de massas do tipo Maldi-ToF-ToF (Axima Performance, Shimadzu) foram utilizados, sendo que as proteínas identificadas foram obtidas utilizando o servidor público SwissProt. Aproximadamente, foram identificadas quarenta proteínas com diversas funções, dentre elas as proteínas F-actina e Thimosina, as quais estão relacionadas com a estrutura e organização celular, e proteínas como diversos Fatores de necroses tumorais, as quais estão relacionadas diretamente com a patologia analisada nesse estudo. O desenvolvimento dessa técnica proporcionou uma análise proteômica diretamente no tecido, favorecendo o desenvolvimento de testes diagnósticos...(Resumo completo, clicar acesso eletrônico abaixo

Uma abordagem peptidômica do venedo do escorpião Tityus serrulatus

O aumento populacional de animais peçonhentos em áreas urbanizadas ou de atividades relacionadas à exploração de áreas naturais vem favorecendo um aumento significativo na ocorrência de acidentes devido ao contato do homem com estes animais. Dessa forma, o envenenamento por toxinas animais tem sido objeto de muitos estudos pelo homem por se revelarem úteis no desenvolvimento de novos fármacos e de novas abordagens metodológicas ou técnicas resolutivas. Os acidentes por escorpiões são os que apresentam o maior número de registro de casos dentre todos os animais peçonhentos no Brasil. Ao longo dos mais de cem anos de pesquisas com venenos destes animais, o foco toxinológico tem se concentrado quase que exclusivamente nas proteínas, responsáveis pela difusão do veneno nos tecidos das presas e/ou vítimas de ferroadas, e principalmente nos peptídeos longos (apresentando entre 30 e 70 resíduos de amino ácidos) responsáveis pelas ações neurotóxicas. A despeito da existência de pequenos peptídeos lineares nesses venenos são raros os estudos que visam elucidar a estrutura molecular e funcional dos pequenos peptídeos. O presente estudo teve como objetivo investigar peptídeos deste grupo (negligenciados) no veneno do escorpião Tityus serrulatus. Para isto, o veneno bruto deste escorpião foi submetido a extração diretamente em acetonitrila 50 % (v/v), e a fração solúvel foi fracionada, e cada fração individual foi submetida à análise por espectrometria de massas ESI-IT-TOF-MS e MSn a fim obter as sequências completas de novos peptídeos. A estratégia utilizada permitiu a identificação de cinco novos peptídeos, estruturalmente diferenciados daqueles já descritos na literatura. Os peptídeos caracterizaram-se por serem lineares e não possuírem cisteínas. Desta forma, eles ampliam a classe...The increasing population of venomous animals in urbanized areas or activities related to the exploitation of natural areas has favored significant accidents due to human contact with these animals. Thus, the toxins from animal venoms has been the subject of many studies prove useful in developing new drugs and new methodological approaches or high-resolution techniques. Scorpions’s accidents are those with the highest number of reported cases among all venomous animals. More than a hundred years of research with these poisons animals, the principal focus toxinology is on proteins, responsible for the spread of the venom in tissues prey or victims, especially in neurotoxic peptides (presenting between 30 and 70 amino acid residues). Despite on the existence of small and linear peptides in these venoms are few studies exploring the molecular structure and function of small peptides. The present study aimed to the study of this peptides group (at moment is neglected) from scorpion venom Tityus serrulatus. For this, the venom were fractionated by chromatography, posteriorly each fraction was subjected to individual analysis by mass spectrometry (LC-MS IT-ToF) in order to obtain the complete sequence about novel peptides. This strategy promoted the detection of five undiscovered peptide sequences highly differentiated those previously described at literature. The peptides were characterized like non-disulfide-bridged peptides it is contributed to expanding the class of scorpions peptides with similar physicochemical characteristic. Two peptides were synthesized on solid phase (fraction 14 and fraction 18) and tested by biological activity in vivo and in vitro. The fraction 18 showed us antinociceptive and edematogenic effect, in addition to antibiotic effect. These results has been demonstrated the large biotechnology... (Complete abstract click electronic access below)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

MALDI Imaging Analysis of Neuropeptides in the Africanized Honeybee (Apis mellifera) Brain: Effect of Ontogeny

The occurrence and spatial distribution of the neuropeptides AmTRP-5 and AST-1 in the honeybee brain were monitored via MALDI spectral imaging according to the ontogeny of Africanized Apis mellifera. The levels of these peptides increased in the brains of 0-15 day old honeybees, and this increase was accompanied by an increase in the number of in-hive activities performed by the nurse bees, followed by a decrease in the period from 15 to 25 days of age, in which the workers began to perform activities outside the nest (guarding and foraging). The results obtained in the present investigation suggest that AmTRP-5 acts in the upper region of both pedunculi of young workers, possibly regulating the cell cleaning and brood capping activities. Meanwhile, the localized occurrence of AmTRP-5 and AST-1 in the antennal lobes, subesophageal ganglion, upper region of the medulla, both lobula, and alpha- and beta-lobes of both brain hemispheres in 20 to 25 day old workers suggest that the action of both neuropeptides in these regions may be related to their localized actions in these regions, regulating foraging and guarding activities. Thus, these neuropeptides appear to have some functions in the honeybee brain that are specifically related to the age-related division of labor.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee (<i>Apis mellifera</i>) Brain: Effect of Aggressiveness

Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging analysis, and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59–76) (AYT­YVS­EYK­RLP­VYN­FGL-NH2), AmAST A (69–76) (LPV­YNF­GL-NH2), AmTRP (88–96) (APM­GFQ­GMR-NH2), and AmTRP (254–262) (ARM­GFH­GMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biologically validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69–76) or AmTRP (88–96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista

The phospholipases A(1) (PLA(1)s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1), combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2)s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.FAPESP[05/00982-1]BIOprospecTA/FAPESP[06/57122-7]CNPqCAPE

Proteomic profiling of the molecular targets of interactions of the mastoparan peptide Protopolybia MP-III at the level of endosomal membranes from rat mast cells

It is well known that the activation of mast cells due to the binding of mastoparan to the Ga subunit of trimeric G proteins involves exocytosis regulation. However, experimental evidence in the literature indicates that mastoparan can also activate certain regulatory targets of exocytosis at the level of the mast cell endosomal membranes that have not yet been identified. Therefore, the aim of the present investigation was the proteomic identification of these targets. To achieve these objectives, mast cells were activated by the peptide Protopolybia MP-III, and the proteins of the endosomal membranes were converted to proteoliposomes using sonication. Proteins were separated from one another by affinity chromatography using proteoliposomes as analytes and Protopolybia MP III-immobilized Sepharose 4B resin as the ligand. This experimental approach, which used SDS-PAGE, in-gel trypsin digestion and proteomic analysis, permitted the identification of five endosomal proteins: Rho GTPase Cdc 42 and exocyst complex component 7 as components of the Ca2+-independent FceRI-mediated exocytosis pathway, synaptosomal-associated protein 29, and GTP-binding protein Rab3D as components of the Ca2+-dependent FceRI-mediated exocytosis pathway and Ras-related protein M-Ras, a protein that is related to the mediation of cell shaping and proliferation following exocytosis. The identification of the five proteins as targets of mastoparans may contribute in the near future to the use of this family of peptides as novel tools for dissecting the mechanism of exocytosis in mast cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

A simple, rapid method for the extraction of whole fire ant venom (Insecta: Formicidae: Solenopsis)

The invasive fire ant Solenopsis invicta is medically important because its venom is highly potent. However, almost nothing is known about fire ant venom proteins because obtaining even milligram-amounts of these proteins has been prohibitively challenging. We present a simple and fast method of obtaining whole venom compounds from large quantities of fire ants. For this, we separate the ants are from the nest soil, immerse them in dual-phase mixture of apolar organic solvent and water, and evaporate each solvent phase in separate. The remaining extract from the aqueous phase is largely made up of ant venom proteins. We confirmed this by using 2D gel electrophoresis while also demonstrating that our new approach yields the same proteins obtained by other authors using less efficient traditional methods. © 2013 Elsevier Ltd