RESUMO
Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.
Assuntos
Corantes , Eletroforese em Gel Bidimensional/métodos , Hipocampo/química , Microdissecção/métodos , Proteínas/isolamento & purificação , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Hipocampo/metabolismo , Processamento de Imagem Assistida por Computador , Lasers , Masculino , Proteínas/química , Proteínas/genética , Proteômica/métodos , Células Piramidais/química , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
A proteomics approach was used to identify liver proteins that displayed altered levels in mice following treatment with a candidate drug. Samples from livers of mice treated with candidate drug or untreated were prepared, quantified, labeled with CyDye DIGE Fluors, and subjected to two-dimensional electrophoresis. Following scanning and imaging of gels from three different isoelectric focusing intervals (3-10, 7-11, 6.2-7.5), automated spot handling was performed on a large number of gel spots including those found to differ more than 20% between the treated and untreated condition. Subsequently, differentially regulated proteins were subjected to a three-step approach of mass spectrometry using (a) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting, (b) post-source decay utilizing chemically assisted fragmentation, and (c) liquid chromatography-tandem mass spectrometry. Using this approach we have so far resolved 121 differentially regulated proteins following treatment of mice with the candidate drug and identified 110 of these using mass spectrometry. Such data can potentially give improved molecular insight into the metabolism of drugs as well as the proteins involved in potential toxicity following the treatment. The differentially regulated proteins could be used as targets for metabolic studies or as markers for toxicity.
