Seqüenciamento de peptídeos usando espectrometria de massas: um guia prático

This paper introduces the basics of peptide mass spectra interpretation applied to proteomics and is directed to chemists, biochemists and biologists. The manuscript presents a well detailed protocol aiming to serve as a first choice guide for understanding peptide sequencing. The tutorial was elaborated based on both a thorough bibliographic revision and the author's experience. In order to prove the applicability of the proposed guide, spectra obtained on different instruments have been successfully interpreted by applying the presented rational.

Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.

Revisiting cangitoxin, a sea anemone peptide: Purification and characterization of cangitoxins II and III from the venom of Bunodosoma cangicum

Sodium channel toxins from sea anemones are employed as tools for dissecting the biophysical properties of inactivation in voltage-gated sodium channels. Cangitoxin (CGTX) is a peptide containing 48 amino acid residues and was formerly purified from Bunodosoma cangicum. Nevertheless, previous works reporting, the isolation procedures for such peptide from B. cangicum secretions are controversial and may lead to incorrect information. In this paper, we report a simple and rapid procedure, consisting of two chromatographic steps, in order to obtain a CGTX analog directly from sea anemone venom. We also report a substitution of N16D in this peptide sample and the co-elution of an inseparable minor isoform presenting the R14H substitution. Peptides are named as CGTX-II and CGTX-III, and their effects over Nav1.1 channels in patch clamp experiments are demonstrated. (c) 2008 Elsevier Ltd. All rights reserved.

Protein identification pipeline for the homology-driven proteomics

Homology-driven proteomics is a major tool to characterize proteomes of organisms with unsequenced genomes. This paper addresses practical aspects of automated homology-driven protein identifications by LC-MS/MS on a hybrid LTQ orbitrap mass spectrometer. All essential software elements supporting the presented pipeline are either hosted at the publicly accessible web server, or are available for free download. (C) 2008 Elsevier B.V. All rights reserved.

Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.

Characterization of protein hydrolysates of cosmetic use by CE-MS

Protein hydrolysates have been used as active principles in cosmetic products conferring different properties to the final formulations, which are mostly controlled by the peptide size and its amino acid sequence. In this work, capillary electrophoresis coupled to mass spectrometry analyses were carried out in order to investigate such characteristics of protein hydrolysates. Samples of different origins (milk, soy and rice) were obtained from a local company, and were analyzed without a previous preparation step. The background electrolyte (BGE) and sheath liquid compositions were optimized for each sample. The best BGE composition (860 mmol/L formic acid - pH 1.8 - in 70: 30 v/v water/methanol hydro-organic solvent) was chosen based on the overall peak resolution whereas the best sheath liquid was selected based on increased sensitivity and presented different compositions to each sample (10.9-217 mmol/L formic acid in 75: 25-25: 75 v/v water/methanol hydro-organic solvent). Most of the putative peptides in the hydrolysate samples under investigation presented molecular masses of 1000 Da or less. De novo sequencing was carried out for some of the analytes, revealing the hydrophobicity/polarity of the peptides. Hence, the technique has proved to be an advantageous tool for the quality control of industrial protein hydrolysates.

Novo protocolo para as ações de saúde bucal coletiva: padronização no armazenamento, distribuição e uso do material de higiene bucal

Este trabalho avaliou o material de higiene bucal usado em escolas para estabelecer um protocolo às ações de higiene bucal coletiva. O estudo foi dividido em duas etapas: 1ª- 20 responsáveis pelos procedimentos coletivos com escolares de Bauru e São José dos Campos - SP responderam a dois questionários sobre o uso de cinco kits de higiene bucal coletiva. A análise estatística foi realizada através do teste Wilcoxon (p < 0,05); 2ª - 178 escolares de 4 a 8 anos de Bauru e Bariri-SP dispensaram na escova uma quantidade de creme dental e dentifrício líquido para a prática da escovação, a qual foi pesada através de uma balança portátil. A análise estatística foi obtida através do coeficiente de correlação de Pearson e a análise de covariância (p< 0,05). O kit 5 obteve graus de satisfação e muita satisfação quando comparado aos kits 1 a 4. A quantidade de creme dental dispensada pelos escolares foi em média 0,41g (Bauru) e 0,48g (Bariri). Não houve diferença estatística entre os escolares de Bauru e Bariri em relação ao dentifrício líquido (média de 0,15g). O dentifrício líquido, através da "técnica da gota", foi considerado prático, dispensando uma pequena quantidade padronizada. O kit 5 demonstrou ser uma boa alternativa ao estabelecimento de um protocolo de ações em saúde bucal coletiva no SUS.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania) infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L.(L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

Novo gênero de Calliini (Coleoptera, Cerambycidae, Lamiinae)

Paradrycothaea gen. nov. (Calliini, Lamiinae) é proposto para duas espécies mexicanas: P. pilosicornis sp. nov., espécie-tipo, de Quintana Roo e P. jamesi sp. nov. de Chiapas.

Termogravimetria: um novo enfoque para a clássica determinação de cálcio em cascas de ovos

The use of thermoanalytical data in sample preparation is described as a tool to catch the students' attention to some details that can simplify both the analysis and the analytical procedure. In this case, the thermal decomposition of eggshells was first investigated by thermogravimetry (TGA). Although the classical procedures suggest long exposure to high temperatures, the TGA data showed that the decomposition of organic matter takes place immediately when the sample is heated up to 800 °C under air atmosphere. After decomposition, the calcium content was determined by flame atomic emission photometry and compared with the results obtained using classical volumetric titration with EDTA.

Eranina Monné (Coleoptera, Cerambycidae, Lamiinae) e novo gênero de Hemilophini

Novas espécies descritas em Eranina Monné, 2005: E. moysesi e E. esquinas da Costa Rica; E. rondonia do Brasil (Rondônia) e E. hovorei do Equador. Acrescenta-se chave para as 34 espécies de Eranina. O gênero monotípico Eraninella gen. nov. é criado para Eranina longiscapus (Bates, 1881).

Contribuição para o estudo dos Rhinotragini (Coleoptera, Cerambycidae). III: Oxyommata Zajciw, 1970 e novo gênero oriundo da divisão de Xenocrasis Bates, 1873

Oxyommata Zajciw, 1970 e O. collaris (Audinet-Serville, 1833) são redescritos e comentados. Um novo gênero proveniente da divisão de Xenocrasis Bates, 1873 é descrito e três espécies alocadas nele: X. fulvicollis(Lacordaire, 1868); X. pubipennis (Fisher, 1952); e X. vestitipennis Zajciw, 1963. É apresentada chave para as espécies do novo gênero.

Novas espécies, nova sinonímia e novo registro em Cerambycidae (Coleoptera) do México e da América do Sul

São descritos três novos táxons de Cerambycinae do Brasil: em Cerambycini, Iuati gen. nov., espécie-tipo, I. spinithorax sp. nov. (Rondônia); em Trachyderini, Ancylosternus annulicorne sp. nov. (Mato Grosso). Em Lamiinae são descritas três espécies novas: em Apomecynini, Ptericoptus avanyae sp. nov. (Brasil, Paraíba); em Desmiphorini, Estoloides (Estoloides) aurantius sp. nov. (México, Quintana Roo); Mimestoloides fasciatus sp. nov. (México, Guerrero). Butherium scabricolle Nonfried, 1895 é considerada sinônimo de Coleoxestia corvina (Germar, 1824). Trachyderomorpha notabilis Tippmann, 1960 é registrada para o Peru.

Novos táxons e novo registro em Hemilophini (Coleoptera: Cerambycidae: Lamiinae)

Novas espécies descritas e ilustradas: Adesmus nigrolineatus sp. nov. do México (Oaxaca). Da Costa Rica: A. moruna sp. nov. (Heredia); Corcovado bezarki sp. nov. (Guanacaste); Alampyris fuscus sp. nov. (Guanacaste), Cariua gen. nov. espécie-tipo C. sulphurea sp. nov., (Guanacaste). Da Bolívia: Phoebemima albomaculata sp. nov. (Cochabamba); Ipepo gen. nov. espécie-tipo I. dilatatus sp. nov. (Santa Cruz). Do Brasil: Adesmus facetus sp. nov. e Canarana arguta sp. nov. (Rondônia). É acrescentado novo registro na Costa Rica para Piruanycha pitilla Galileo & Martins, 2005. As três espécies novas de Adesmus distinguem-se: A. nigrolineatus sp. nov. pelas faixas longitudinais de tegumento preto nos élitros; A. moruna sp. nov. pelos élitros inteiramente pretos; A. facetus pelas faixas oblíquas de pubescência branca após o meio dos élitros além das manchas do quarto apical e das epipleuras. Phoebemima albomaculata sp. nov. caracteriza-se pela mancha basal de pubescência branca dos élitros estendendo-se sobre a sutura. Corcovado bezarki sp. nov. distingue-se pelo escapo preto e antenômeros esbranquiçados. Canarana arguta sp. nov. tem o protórax e os urosternitos I a IV cobertos por densa pubescência amarelada. Alampyris fusca sp. nov. difere de A. cretaria principalmente pelo antenômero III mais longo que o escapo. Cariua sulphurea sp. nov. separa-se pela presença de urosternitos revestidos por pubescência branca compacta e Ipepo dilatatus sp. nov. caracteriza-se pelos élitros tri-carenados.

Ucai, novo gênero de Anacolini (Coleoptera, Cerambycidae, Prioninae) do Brasil

Novos táxons descritos: Ucai gen. nov. espécie-tipo, Ucai nascimentoi sp. nov. de São Paulo.