RESUMO
Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.
Assuntos
Mapeamento Encefálico/métodos , Encéfalo/metabolismo , Conectoma/métodos , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Aprendizado de Máquina , Mamíferos/fisiologia , Espectrometria de Massas/métodos , Camundongos , Mutação/genéticaRESUMO
BP/MS is a new experimental proteomic platform for systematic study of native protein complexes and global protein-protein interaction networks based on deep biochemical purification of soluble protein extracts and mass spectrometry identification of coeluting proteins. Herein, we describe the application of this methodology to draft a high-confidence protein interaction network for Candida albicans.
Assuntos
Candida albicans/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Proteínas Fúngicas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia Líquida/métodosRESUMO
A high-resolution two-dimensional (2-D) proteomic fractionation technique for the systematic purification and subsequent mass spectrometry-based identification of endogenous protein macromolecular complexes is described. The method hyphenates preparative isoelectric focusing (IEF) with mixed-bed ion exchange chromatography (IEX) to efficiently separate cell- or tissue- derived soluble protein mixtures, allowing for more effective and less biased physiochemical characterization of stable multiprotein assemblies. After comprehensive 2D fractionation of cell-free lysates, each fraction is subjected to quantitative tandem mass spectrometry (MS/MS) and subsequent computational analysis to map high-confidence protein-protein interactions (PPIs). Herein, the experimental component (workflow protocols) for this global "interactome" network mapping platform is described.
Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Proteoma , Proteômica , Proteínas de Bactérias , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Biologia Computacional/métodos , Imunoprecipitação , Focalização Isoelétrica , Proteólise , Proteômica/métodos , Solubilidade , Espectrometria de Massas em TandemRESUMO
A proteomic platform for global analysis of protein complexes and protein-protein interactions (PPIs) is described. Briefly, after comprehensive physiochemical separation of soluble protein extracts using non-denaturing ion exchange chromatography (IEX), each fraction is subjected to quantitative tandem mass spectrometry analysis.
