RESUMO
Proteins are very dynamic within the cell and their localization and trafficking between subcellular compartments are critical for their correct function. Indeed, the abnormal localization of a protein might lead to the pathogenesis of several diseases. The association of cell fractionation methods and mass spectrometry based proteomic methods allow both the localization and quantification of proteins in different sub-compartments. Here we present a detailed protocol for enrichment, identification, and quantitation of the nuclear proteome in cell lines combining nuclear subproteome enrichment by differential centrifugation and high-throughput proteomics.
Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Proteoma , Proteômica/métodos , Fracionamento Celular/métodos , Linhagem Celular , Núcleo Celular/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Ensaios de Triagem em Larga Escala , Humanos , Espectrometria de Massas/métodos , Transporte ProteicoRESUMO
The secretome is a sub-proteome of great interest in several fields of biomedical sciences, especially as a source of diagnostics and therapeutic targets. Proteomics has been contributing significantly to elucidate the secretome of a great diversity of cells, tissues, and organisms, turning profiles of thousands of proteins a usual practice. After elucidation of long protein lists, targeted proteomics also plays important roles in accurate quantification and validation of such secreted proteins. Here we present detailed protocols to explore and quantify the secretome of cancer cells, even though this protocol can be employed to any kind of biological material.
