It is time for top-down venomics

The protein composition of animal venoms is usually determined by peptide-centric proteomics approaches (bottom-up proteomics). However, this technique cannot, in most cases, distinguish among toxin proteoforms, herein called toxiforms, because of the protein inference problem. Top-down proteomics (TDP) analyzes intact proteins without digestion and provides high quality data to identify and characterize toxiforms. Denaturing top-down proteomics is the most disseminated subarea of TDP, which performs qualitative and quantitative analyzes of proteoforms up to ~30 kDa in high-throughput and automated fashion. On the other hand, native top-down proteomics provides access to information on large proteins (> 50 kDA) and protein interactions preserving non-covalent bonds and physiological complex stoichiometry. The use of native and denaturing top-down venomics introduced novel and useful techniques to toxinology, allowing an unprecedented characterization of venom proteins and protein complexes at the toxiform level. The collected data contribute to a deep understanding of venom natural history, open new possibilities to study the toxin evolution, and help in the development of better biotherapeutics.(AU)

It is time for top-down venomics

Abstract The protein composition of animal venoms is usually determined by peptide-centric proteomics approaches (bottom-up proteomics). However, this technique cannot, in most cases, distinguish among toxin proteoforms, herein called toxiforms, because of the protein inference problem. Top-down proteomics (TDP) analyzes intact proteins without digestion and provides high quality data to identify and characterize toxiforms. Denaturing top-down proteomics is the most disseminated subarea of TDP, which performs qualitative and quantitative analyzes of proteoforms up to ~30 kDa in high-throughput and automated fashion. On the other hand, native top-down proteomics provides access to information on large proteins (> 50 kDA) and protein interactions preserving non-covalent bonds and physiological complex stoichiometry. The use of native and denaturing top-down venomics introduced novel and useful techniques to toxinology, allowing an unprecedented characterization of venom proteins and protein complexes at the toxiform level. The collected data contribute to a deep understanding of venom natural history, open new possibilities to study the toxin evolution, and help in the development of better biotherapeutics.

Differencial proteome of clear-cell renal cell carcinoma (ccRCC) tissues

Purpose We attempted to detect, for the first time in a Brazilian cohort, differences in protein expression between clear-cell renal cell carcinoma (ccRCC) and their normal adjacent tissues, aiming to identify biomarkers and/or therapeutic target candidates for this disease. Material and Methods Twenty-four ccRCC and adjacent normal tissues were collected after surgery and their protein extracts were quantified, pooled and separated by two-dimensional polyacrylamide gel electrophoresis (2DE), followed by statistical analysis of the stained gels. Spots of interest were excised from the gels, digested with trypsin and identified by MALDI-TOF-TOF mass spectrometry. Results Twenty-six differential spots were detected between the two classes of tissues, among which twenty were identified by mass spectrometry and sixteen were found to be non-redundant. Eleven proteins were either underexpressed or undetected in the ccRCC extracts, such as prohibitin and peroxiredoxin-3, whereas five were found to be overexpressed or exclusively detected in the ccRCC extract, including αβ crystalin and heat shock protein 27. CONCLUSIONS Several proteins were detected at differential levels when compared to normal adjacent tissues, and, moreover, many have been previously described by their relationship with RCC. Therefore, this work corroborates previous reports on the search for biomarkers for ccRCC, as well as it points out new candidates that may be validated in future studies. .

Obstáculos epistemológicos entre pós-graduandos de bioquímica / Epistemological obstacles amongst graduate students in biochemistry

A partir do estudo da epistemologia de Bachelard na disciplina Lógica e Filosofia da Ciência da Pós-Graduação em Bioquímica do Instituto de Química/Universidade Federal do Rio de Janeiro (IQ/UFRJ), buscou-se identificar obstáculos epistemológicos entre pós-graduandos em bioquímica e áreas correlatas. Um questionário com perguntas e excertos de artigos científicos de revistas de alto fator de impacto foi respondido, anonimamente, por pós-graduandos de diferentes cursos da UFRJ e de outras universidades, que nunca cursaram disciplina relacionada à epistemologia. Foi possível identificar concepções vitalistas (animismo) tanto nas respostas às perguntas como na aceitação ou não identificação deste obstáculo nos excertos. O obstáculo pragmático e unitário foi identificado através de uma concepção teleológica dos processos evolutivos, em afirmações como a existência de objetivos/finalidades na adaptação dos organismos. Verificou-se a presença de figuras de linguagem, metáforas e analogias (obstáculo verbal) na explicação da evolução e do sistema imune, também encontradas nos excertos dos artigos. Foram também identificados obstáculos associados à observação primeira e generalização prematura. A partir deste diagnóstico verificou-se a necessidade de enfatizar o caráter objetivo, material, não teleológico da bioquímica, em disciplinas oferecidas desde a graduação.

Proteômica na sepse: estudo piloto / Proteomics in sepsis: a pilot study

Na sepse ocorre desregulação da expressão gênica. Os marcadores genéticos revelam apenas o genótipo do indivíduo, não sendo afetados pelos processos biológicos decorrentes da ação do ambiente, estes expressos nas proteínas. Este estudo teve como objetivo alcançar maior compreensão sobre as bases moleculares da sepse. Para tal realizou a identificação e análise da expressão diferencial de proteínas no soro de paciente séptico em diferentes estágios de gravidade (sepse, sepse grave e choque séptico) através de técnicas proteômicas. Amostras de soro referentes a cada estágio da sepse foram colhidas e submetidas à eletroforese unidimensional em fitas com gradiente de pH imobilizado seguida de eletroforese bidimensional em gel de poliacrilamida 12,5 por cento. Os géis obtidos foram corados, escaneados e analisados através do programa ImageMasterPlatinum. As proteínas expressas diferencialmente nos géis foram excisadas, digeridas com tripsina e identificadas através de espectrometria de massa. Foram identificadas 14 proteínas expressas diferencialmente entre os estágios da sepse, assim como uma proteína não expressa em todos os estágios, sugerindo a existência de um possível biomarcador. Foram elas: amilóide sérico A, apolipoproteína A-1 (2 isoformas), proteína dedo de zinco 222, albumina humana, PRO 2619, imunoglobulina de cadeia leve kappa região VLJ, imunoglobulina M monoclonal de aglutinação a frio e 7 inibidoras de proteases - alfa-1 antitripsina. Os resultados obtidos neste estudo piloto demonstram a participação das vias do complemento e coagulação, do metabolismo lipídico e da informação genética na sepse. A grande maioria de proteínas identificadas está envolvida no sistema imune com predomínio das proteínas inibidoras de proteases.

Gene expression is disrupted by sepsis. Genetic markers can only reveal a patient's genotype, and they are not affected by environmental biological processes. These processes are expressed by proteins. This study was aimed to advance the insight into the molecular foundations of sepsis. It employed proteomic techniques to identify and analyze differential serum protein expressions taken from a patient throughout the stages of sepsis (sepsis, severe sepsis and septic shock). Serum samples were collected at each stage of sepsis and submitted to one-dimensional electrophoresis, on gradient strips of immobilized pH, followed by two-dimensional 12.5 percent polyacrylamide gel electrophoresis. The gels obtained were stained, scanned and analyzed by the ImageMasterPlatinum program. Proteins that were differentially expressed in the gels were excised, digested with trypsin and identified through mass spectrometry. Fourteen differentially expressed proteins were identified throughout the stages of sepsis, as well as a protein that was not expressed in all stages, suggesting the potential existence of a biomarker. The differentially expressed proteins identified were: serum amyloid A, apolipoprotein A-1 (2 isoforms), zinc finger protein 222, human albumin, PRO 2619, immunoglobulin kappa light chain VLJ region, monoclonal immunoglobulin M cold agglutinin, 7 proteinase inhibitors - alpha-1 antitrypsin. The findings of this pilot study demonstrate the involvement of the complement and coagulation pathways, of the lipid metabolism and of genetic information in sepsis. The vast majority of proteins identified are involved in the immune system and the proteinase inhibitor proteins are predominant.

Proteômica e sepse: novas perspectivas para o diagnóstico / Proteomics and sepsis: new perspectives for diagnosis

JUSTIFICATIVA E OBJETIVOS: O diagnóstico e o tratamento da sepse continuam a desafiar a todos; e desenvolver formas mais precisas de abordagem são absolutamente necessárias. O objetivo deste estudo foi empregar técnicas proteômicas, eletroforese bidimensional e espectrometria de massa, para verificar a expressão diferencial de proteínas, em soro de pacientes com sepse comparado com controles saudáveis. MÉTODO: Amostras de soro de 30 pacientes com sepse, causada por vários tipos de microorganismos e de 30 controles saudáveis foram obtidas para análise. A seguir, foram submetidas a 2D-SDS-PAGE, comparação entre géis, seleção de spots para excisão e digestão com tripsina, sendo os peptídeos analisados por MALDI TOF-TOF. Os espectros obtidos foram processados (Mascot-matrixscience) para identificação de proteínas no NCBInr Data Bank. RESULTADOS: A análise das imagens mostrou vários spots com expressão diferencial nos géis dos pacientes com sepse em relação aos controles. A identificação de proteínas em alguns destes spots encontrou: precursor Orosomucoide 1, Apolipoproteína A-IV, precursor Apolipoproteína A-IV, precursor Haptoglobina, Haptoglobina, proteína Zinc finger, Amilóide sérico A-1, Transtiretina, Nebulin, Complemento C4, Alfa1-Antitripsina, produto protéico não nominado e outros. CONCLUSÕES: Soros de pacientes com diferentes tipos de sepse expressam padrão protéico característico por 2D-SDS-PAGE comparado com controles. A maior expressão foi de proteínas de fase aguda e lipoproteínas. É possível que no futuro, com a proteômica, criar painel diagnóstico de proteínas, encontrar novos biomarcadores e alvos para intervenção terapêutica na sepse. Esta é a primeira descrição, com a proteômica, das alterações na expressão protéica, no soro de pacientes com sepse.

BACKGROUND AND OBJECTIVES: The diagnostic and treatment of sepsis continue to challenger all, and, more specific forms to approach are absolutely necessary. The objective of this study was to use proteomics techniques, two-dimensional electrophoresis and mass spectrometry, to verify the differential protein expression between serum of patients with sepsis and health controls. METHODS: Samples of serum the 30 patients with sepsis, caused for different types of microorganisms and serum of 30 health controls were obtained for analysis. Next, were submitted to 2D-SDS-PAGE, gels compared, selection of spots for excision and digestion with trypsin, being the peptides analyzed for MALDI TOF-TOF. The obtained spectrums were processed (Mascot-matrix science) for protein identification in NCBInr Data Bank. RESULTS: Image analyses showed several spots with differential expressions in the gels of the patients with sepsis in relation to the controls. The protein identification of some of these spots founded: Orosomucoid 1 precursor, Apolipoprotein A-IV, Apolipoprotein A-IV precursor, Haptoglobin protein precursor, Haptoglobin, Zinc finger protein, Serum amyloid A-1, Transthyretin, Nebulin, Complement C4, Alpha1-Antitrypsin, Unnamed protein product and others. CONCLUSIONS: Serum of the patients with different types of sepsis express characteristic protein profiles by 2D-SDS-PAGE compared with controls. The most expressed were from acute phase proteins and lipoproteins. It is possible in the future, with proteomics, create diagnostic panel of proteins, finding news biomarkers and targets for therapeutic interventions in sepsis. This is a first description, with proteomics, of the alterations in protein expression, in serum of the patients with sepsis.