RESUMO
To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by one-dimensional PAGE. Total in-gel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways.
Assuntos
Bases de Dados de Proteínas , Proteínas Mitocondriais/química , Proteínas Mitocondriais/fisiologia , Proteoma/química , Proteoma/fisiologia , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Células Cultivadas , Eletroforese/métodos , Coração/fisiologia , Humanos , Armazenamento e Recuperação da Informação/métodos , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/fisiologia , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Peso Molecular , Miocárdio/química , Proteoma/genética , Proteômica/métodos , Análise de Sequência de Proteína/métodosRESUMO
Recent literature that highlights the power of using mass spectrometry (MS) for protein identification from preparations of highly purified organelles and other large subcellular structures is covered in this review with an emphasis on techniques that preserve the integrity of the functional protein complexes. Recent advances in distinguishing contaminant proteins from "bonafide" organelle-localized proteins and the affinity capture of protein complexes are reviewed, as well as bioinformatic strategies to predict protein organellar localization and to integrate protein-protein interaction maps obtained from MS-affinity capture methods with data obtained from other techniques. Those developments demonstrate that a revolution in cellular biology, fueled by technical advances in MS-based proteomic techniques, is well underway.
Assuntos
Espectrometria de Massas/métodos , Organelas/química , Proteoma/análise , Proteômica/métodos , Animais , HumanosRESUMO
An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using metrizamide gradients to remove cytosolic and other subcellular contaminant proteins; (b) separation of mitochondrial protein complexes using sucrose density gradients after solubilization with n-dodecyl-beta-D-maltoside; (c) 1D electrophoresis of the sucrose gradient fractions; (d) high-throughput proteomics using robotic gel band excision, in-gel digestion, MALDI target spotting and automated spectral acquisition; and (e) protein identification from mixtures of tryptic peptides by high-precision peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane proteins such as the adenine nucleotide translocase that are not recovered in 2D gels. The technique described here should also be useful for the identification of new protein-protein associations as exemplified by the validation of a recently discovered complex that involves proteins belonging to the prohibitin family.
Assuntos
Mitocôndrias/metabolismo , Miocárdio/metabolismo , Proteoma , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sacarose/farmacologia , Western Blotting , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Feminino , Glucosídeos/farmacologia , Humanos , Espectrometria de Massas , Peptídeos/química , Frações Subcelulares/metabolismoRESUMO
Recent evidence suggests that mitochondria are closely linked with the aging process and degenerative disorders such as Alzheimer's disease and Parkinson's disease. Thus, there has been increasing interest in cataloging mitochondrial proteomes to identify potential diagnostic and therapeutic targets. We have previously reported results of a one-dimensional electrophoresis/liquid chromatography MS/MS study to characterize the proteome of normal human heart mitochondria (Taylor et al. Nat. Biotechnol. 2003, 21, 281-286). We now report two subsequent studies where multidimensional liquid chromatography MS/MS was investigated as an alternative means for characterizing the same sample.