Quantitative proteomics of a presymptomatic A53T alpha-synuclein Drosophila model of Parkinson disease

A global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry was used for quantitative proteome analysis of a presymptomatic A53T alpha-synuclein Drosophila model of Parkinson disease (PD). Multiple internal standard proteins at different concentration ratios were spiked into samples from PD-like and control animals to assess quantification accuracy. Two biological replicates isotopically labeled in forward and reverse directions were analyzed. A total of 253 proteins were quantified with a minimum of two identified peptide sequences (for each protein); 180 ( approximately 71%) proteins were detected in both forward and reverse labeling measurements. Twenty-four proteins were differentially expressed in A53T alpha-synuclein Drosophila; up-regulation of troponin T and down-regulation of fat body protein 1 were confirmed by Western blot analysis. Elevated expressions of heat shock protein 70 cognate 3 and ATP synthase are known to be directly involved in A53T alpha-synuclein-mediated toxicity and PD; three up-regulated proteins (muscle LIM protein at 60A, manganese-superoxide dismutase, and troponin T) and two down-regulated proteins (chaoptin and retinal degeneration A) have literature-supported associations with cellular malfunctions. That these variations were observed in presymptomatic animals may shed light on the etiology of PD. Protein interaction network analysis indicated that seven proteins belong to a single network, which may provide insight into molecular pathways underlying PD. Gene Ontology analysis indicated that the dysregulated proteins are primarily associated with membrane, endoplasmic reticulum, actin cytoskeleton, mitochondria, and ribosome. These associations support prior findings in studies of the A30P alpha-synuclein Drosophila model (Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Protein expression in a Drosophila model of Parkinson's disease. J. Proteome Res. 6, 348-357; Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Lifetime proteomic profiling of an A30P alpha-synuclein Drosophila model of Parkinson's disease. J. Proteome Res. 6, 3729-3738) that defects in cellular components such as actin cytoskeleton and mitochondria may contribute to the development of later symptoms.     

Lifetime proteomic profiling of an A30P alpha-synuclein Drosophila model of Parkinson's disease

A survey of the proteome changes in an A30P alpha-synuclein Drosophila model of Parkinson's disease (PD) in comparison to age-matched controls is presented for seven different ages across the adult lifespan. The data were acquired by a shotgun proteomic approach that involves multidimensional liquid chromatographies coupled to mass spectrometry and database searching techniques. Semiquantitative analysis to assess relative changes in protein expression between the Drosophila PD model and age-matched controls provides evidence that 28, 19, 12, 5, 7, 23, and 17 proteins are significantly differentially expressed at days 1, 10, 20, 30, 40, 50, and 60, respectively. From the experimental approach employed, it appears that most dysregulated proteins are associated with narrow distributions of ages, such that disease-associated differences change substantially across the lifespan. Previous measurements [J. Proteome Res. 2007, 6, 348] at days 1, 10, and 30 showed dysregulation of actin cytoskeletal proteins at day 1 and mitochondrial proteins at day 10, suggesting that defects in the actin cytoskeleton and the mitochondria are associated with dopaminergic neuron degeneration in PD. Analysis of the day 20, 40, 50, and 60 animals supports the finding that these cytoskeletal and mitochondrial changes predominate in the youngest (pre-symtomatic and early disease stages) animals. Although studies across many time points appear to be important for characterizing disease state, an understanding of molecular changes at the youngest ages should be most important for addressing causation.     

金针菇成熟期和伸长期菌盖的转录组与蛋白组比较分析

菌盖是大型真菌的重要组成部分,也是其产生有性孢子的部位,但是其发育机制仍不明确。本研究以金针菇Flammulina filiformis为材料,采用转录组和蛋白组联合分析的方法,比较分析了金针菇成熟期和伸长期菌盖的差异基因与蛋白,并对其进行GO (gene ontology)功能聚类分析、KEGG (Kyoto encyclopedia of genes and genomes)富集分析和蛋白互作网络分析。本研究筛选到差异表达基因有1 391个,差异表达蛋白147个,均以上调表达为主。GO功能聚类分析结果表明,催化活性(catalytic activity)条目富集基因最多,其次是细胞组分(cell part)、细胞过程(cellular process)和细胞器(organelle)。KEGG富集分析结果表明,差异表达基因和蛋白主要富集在碳水化合物代谢通路(carbohydrate metabolism)和氨基酸代谢通路(amino acid metabolism)等。本研究选取了9个关键的差异表达基因,使用实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)对其表达量进行了验证。RT-qPCR验证结果与转录组测序结果相一致。蛋白互作网络分析表明,水解酶类、结构域类和转录调节类蛋白为互作网络的主要结点。本研究联合转录组、蛋白组测序数据,通过分析差异基因与蛋白,为深入了解金针菇菌盖发育机制提供数据参考。