The molecular mechanisms underlying the changes in the nigrostriatal pathway in

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinsons disease (PD) are not completely understood. whether the protein displayed significant abundance changes following each neurotoxin treatment. A two-sample test was applied to detect abundance differences between the two models. A protein is considered to have a significant abundance change when the protein has a value less than 0.05 with at least two different peptides, Salinomycin sodium salt manufacture with both showing a relatively good peptide level value (average value < 0.2). Microarray Analysis Gene expression data were obtained from the pooled left and right striata of the three groups of mice: MPTP, METH, and Con (test was applied for transcript abundance ratios against a mean value of zero to determine whether the transcript was significantly different, following each neurotoxin treatment. A two-sample test was applied to detect transcript abundance differences between the two models. A false discovery rate (FDR) was used to adjust for multiple hypothesis testing in R Salinomycin sodium salt manufacture (,26 Gene Set Enrichment Analysis To detect groups of proteins having statistically significant concordant changes, we used gene set enrichment analysis (GSEA).27 The data set was first screened against Gene Ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and other annotations using For proper handling of the missing values and estimation of the confidence of the changes for the selected groups, we performed a sign test using the BSDA package of the R environment for statistical computing ( Results Dopamine Depletion in Neurotoxin-Treated Striata C57/BL6J mice were treated with MPTP, METH, or saline (control) (test 1.81 10?4, df 1, < 0.0002) and METH-treated animals (71% decrease, test 2.21 10?4, df 1, < 0.0003), while levels of a different neurotransmitter, serotonin, and its metabolites remained unchanged (Supplementary Figure 1 in the Supporting Information). Proteomic Abundance Profiling To Rabbit polyclonal to AGAP9 analyze the potential neurotoxin-induced protein abundance changes, we applied a global quantitative proteomic approach (Figure 1), which integrates 16O/18O labeling and Cys-peptide fractionation with the AMT tag strategy to achieve relatively good proteome coverage with quantification.21 Striata from MPTP, METH, and control mice were individually processed and labeled, which led to the generation of 10 16O/18O-paired samples. Each sample was fractionated into Cys and non-Cys samples, which were individually analyzed Salinomycin sodium salt manufacture by LCCMS to identify statistically significant changes. An extensive mouse brain peptide/protein database was recently developed from a global characterization of the mouse brain proteome by liquid chromatography coupled with tandem mass spectrometry (LCCMS/MS).10 Figure 1 Flowchart showing the experimental strategy The analyses resulted in the identification of ~4600 unique peptides corresponding to 1614 proteins, with all proteins quantified in at least 4 of the 10 paired biological samples. Relative protein abundance is expressed as a ratio of neurotoxin-treated to control sample levels. Figure 2A shows the overall reproducibility of the analyses by comparing the correlation of the raw peptide intensities (only 18O intensities were used) between any two biological samples. As shown, the Pearson correlation coefficient is 0.94 0.02 within Salinomycin sodium salt manufacture the same METH or MPTP models (intratoxin), while the observed correlation coefficient 0.89 0.03 between the two models (intertoxin) is slightly lower, as expected. The results suggest overall good reproducibility of the quantitative data. Figure 2 Reproducibility of proteomic analysis Statistical analyses confidently identified 912 proteins with at least two unique peptides among the total 1614 proteins. Using a Students test, 199.