RESUMO
Excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial cells is a common pathogenetic pathway in various blinding retinal diseases including age-related macular degeneration, which is now the most common cause of registerable blindness in the industrialized nations. To better understand the role of lipofuscin accumulation and to manipulate the pathogenetic mechanisms on both experimental and therapeutic levels we analyzed the proteome of isolated human ocular lipofuscin granules from human RPE cells. After homogenization and fractionation by gradient ultracentrifugation of the RPE/choroid complex from 10 pairs of human donors, protein compounds were separated by 2D gel electrophoresis and analyzed using matrix-assisted laser desorption/ionization mass spectrometry and HPLC-coupled electrospray tandem mass spectrometry. Besides a better understanding of downstream pathways, this approach may provide new targets for therapeutic interventions in a currently untreatable disease.
Assuntos
Lipofuscina/isolamento & purificação , Epitélio Pigmentado Ocular/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Humanos , Lipofuscina/metabolismo , Degeneração Macular/metabolismo , Proteoma , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Four years after the original sequence submission, we have re-annotated the genome of Mycoplasma pneumoniae to incorporate novel data. The total number of ORFss has been increased from 677 to 688 (10 new proteins were predicted in intergenic regions, two further were newly identified by mass spectrometry and one protein ORF was dismissed) and the number of RNAs from 39 to 42 genes. For 19 of the now 35 tRNAs and for six other functional RNAs the exact genome positions were re-annotated and two new tRNA(Leu) and a small 200 nt RNA were identified. Sixteen protein reading frames were extended and eight shortened. For each ORF a consistent annotation vocabulary has been introduced. Annotation reasoning, annotation categories and comparisons to other published data on M.pneumoniae functional assignments are given. Experimental evidence includes 2-dimensional gel electrophoresis in combination with mass spectrometry as well as gene expression data from this study. Compared to the original annotation, we increased the number of proteins with predicted functional features from 349 to 458. The increase includes 36 new predictions and 73 protein assignments confirmed by the published literature. Furthermore, there are 23 reductions and 30 additions with respect to the previous annotation. mRNA expression data support transcription of 184 of the functionally unassigned reading frames.
Assuntos
Genes Bacterianos/genética , Genoma Bacteriano , Mycoplasma pneumoniae/genética , Fases de Leitura Aberta/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional , Espectrometria de Massas , Dados de Sequência Molecular , Mycoplasma pneumoniae/química , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de SequênciaRESUMO
A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions.
