Karolinska Institutet Scheeles väg 2 Tel +46-8-52487869

1. Combined pathway and multivariate data analysis on quantitative proteomics data; application on the impact of androgens on the rat hippocampus Pierre Le Grevès, Uwe Rossbach*, Roman Zubarev AAS 12 hours AAS 6 hours • Conclusions • Key node- and multivariate data analysis show relatively good agreement (high confidences) in the regulation of underlying hit proteins. • Some of the most regulated proteins (according to the OPLS-DA model) were not connected to a Key node in the ExPlain software. • Functional classification of the most up-regulated Key node scores in the multivariate analysis (S-plots) suggests that AAS stimulates hippocampal G-protein signaling, glycolysis and protein-protein interaction (the adaptor protein clathrin) at six and twelve hours after its administration • Functional classification of the most down-regulated Key node scores in the multivariate analysis (S-plots) suggests that AAS inhibits some energy and metabolic processes in the rat hippocampus at both time points. Background and purpose We have earlier presented interesting findings on the acute effects of 19-nortestosterone [an anabolic androgenic steroid (AAS)] on synaptic components in the rat hippocampus (1, 2). To further investigate the molecular mechanisms underlying the fast action of AAS we have applied quantitative proteomic involving LC-MS combined with pathway- and multivariate data analysis. The variable importance in the projection (VIP). VIP positions the overall contribution of the variables (here Key nodes) to the OPLS-DA model. VIP values more than 1.0 (listed here) are considered as statistical significant contribution to the model. Table 1. Key nodes regulated by AAS at 6 or 12h: The Key nodes are sorted by P-values (with Welch’s correction) calculated from each Key node scores generated in the ExPlain software. Values for confidence (p(corr)[1]), variable importance in the projection (VIP), its standard error (VIP SE) created in the OPLS-DA model and hits list from ExPlain are also shown. Hits are proteins found in Mascot searches based on LC-MS data. These hits underlie Key node scores and, as can be seen in the table, several Key nodes are based on the same protein hit. AAS 12 hours AAS 6 hours S-plot from the OPLS-DA model generated in the SIMCA-P+ software. The plots show contributors (w*c[1]) versus confidence (p(corr)[1]) of the model variables (proteins or Key nodes). Methods Tryptic fragments of a protein extract from rat hippocampal synaptoneurosomes were loaded onto fused silica microcapillary columns packed with C18 resin and analyzed in an online fashion, with peptides eluted directly into an Orbitrap mass spectrometer using nano-LC and a nano-ESI source. Data-dependent acquisition methods were employed, with both MS1 (30,000 RP) and MS2 (HCD; 7,500 RP) spectra acquired in the Orbitrap mass analyzer. The .raw files were converted to .mgf format (in-house software) for subsequent database searching using MASCOT. Resulting .dat files were processed using in-house quantitative software employing a label-free method (3, 4). The ExPlain software (Biobase, Germany) was used for upstream Key node score analysis and the Simca software (Umetrics, Sweden) for multivariate data analysis. References Rossbach, U.L., Le Greves, M., Nyberg, F., Zhou, Q. & Le Greves, P. (2010). Acute 19-nortestosterone transiently suppresses hippocampal MAPK pathway and the phosphorylation of the NMDA receptor. Mol Cell Endocrinol, 314, 143-9. Rossbach, U.L., Steensland, P., Nyberg, F. & Le Greves, P. (2007). Nandrolone-induced hippocampal phosphorylation of NMDA receptor subunits and ERKs. Biochem Biophys Res Commun, 357, 1028-33.Marin Marin-Vicente, C. & Zubarev, R.A. (2009). Search engine for proteomics. G. I. T. Laboratory Journal, 11-12. Marin-Vicente, C. & Zubarev, R.A. (2010). Search engine for proteomics. G. I. T. Laboratory Journal, 1-2. Karolinska Institutet Scheeles väg 2 Tel +46-8-52487869 Pierre Le Grevès 171 77 Stockholm E-mail: pierre.le-greves@ki.se Depatment of Medical Biochemistry and Biophysics, Div of Molecular Biometry