1. Phosphoproteomic study of molecular mechanisms of drug dependence F. Sucharski 1, P.Suder 1, G.Schroeder 2, J.Silberring 1,3 1 Department of Biochemistry and Neurobiology, Faculty of Materials Sciences and Ceramics, AGH University of Science and Technology, Mickiewicza 30 Ave, 30-059 Krakow, Poland 2 Department of Chemistry, Adam Mickiewicz University, Poznan, Poland 3 Center of Polymer and Carbon Materials, Polish Academy of Sciences, Sowinskiego 5 Str, 44-121 Gliwice, Poland Introduction High dynamism and reversible character, make phosphorylation a main regulator of signaling networks. Therefore, a global wide-scale analysis of phosphoproteome however, troublesome, gives hope on detailed recognition of complicated molecular mechanisms and mutual connections between metabolic pathways. It is known, that drug dependence has a huge impact on many cell functions as well as biological processes, for which the relation with the drug dependence seems to be indirect. Thus, only a global analysis of a state of phosphoproteins, in particular kinases with their mutual relations, can contribute to solving a puzzle. The project is pioneer work in neuroscience, involving the newest, fully quantitative, methods for phosphoproteome analysis. In particular, phosphoproteome analysis is based on the methods for phosphopeptides isolation, and mass spectrometry as an analytical tool for identification and quantification. This approach consists in identification of all possible phosphoproteins, specific peptides fragmentation method, and annotation of phosphorylation sites. The project is pioneer work in neuroscience, involving the newest, fully quantitative, methods for phosphoproteome analysis. In particular, phosphoproteome analysis is based on the methods for phosphopeptides isolation, and mass spectrometry as an analytical tool for identification and quantification. This approach consists in identification of all possible phosphoproteins, specific peptides fragmentation method, and annotation of phosphorylation sites. Here we present a workflow for quantitative phosphopeptides analysis and preliminary results from phosphoproteomic study of prefrontal cortices of morphine dependent rats. The aim of the study The aim of the present project is the search for, and identification of the markers of drug dependence. The research is focused on the identification of molecular mechanisms involved in these processes and clarification of the role of particular proteins in drug dependence. Project puts special emphasis on phosphoproteins, and in general on phosphorylation, as a main mechanism involved in control of essentially any biological process and many diseases. Materials and Methods Isolation and treatment of rat tissues 5 Male Wistar rats were administered morphine for 3 days in comparison to respective control group. Rats were sacrificed and fragments of brain such as prefrontal cortex (PFC) were manually excised. Tissues were homogenized in buffer containing proteases and phosphatases inhibitors. Samples were ultracentrifuged for 90 min at 130,000g. Supernatat was subjected for aceton precipitation. Protein chemistry Protein pellets were dissolved in denaturation buffer, reduced with TCEP, carbamidomethylated with IA and digested by trypsin. Morphine treated samples were methylated with d 3 methanol whereas control samples were methylated with d 0 methanol (Fig.1). Samples were mixed equimollary according to Bradford protein assay. brain structures extract TiO 2 Phosphopeptides cytoplasmatic fraction tryptic peptides MS differential labeling with CD 3 OD LC-MS and database analysis All data was acquired on an Amazon ETD mass spectrometer (Bruker Daltonik, Bremen, Germany). Chromatographic separation of peptides was achieved on a Proxeon EASY-nLC system, equipped with 10 cm long, 3μm ReproSil-Pur C18 resin, 100 μm fused silica column, and 2 cm long, 5μm ReproSil-Pur C18 resin, 100 μm ID pre-column. The LC system operated with mobile phases: solvent A (98:2 water:ACN (v/v) supplemented with 0.1% formic acid), and solvent B (10:80 water:ACN (v/v) supplemented with 0.1% formic acid). Samples were loaded from a cooled (7°C) autosampler and separated with a linear gradient, that was formed at a flow rate of 300 nL/min. It consisted of 120 min linear ramp up to 55% solvent B, subsequently 10-min isocratic run at 100% B and eventually 10-min isocratic run at 100% solvent A. The scan range was set to 200-2000m/z in MS mode and increased in MS2 mode up to 3000m/z. The instrument was operated in auto-MSn mode. Three highest peaks within the range of 450-2000 were automatically fragmented, when their intensity exceeded threshold value. Each precursor ion was fragmented with CID and ETD method. When phospho-loss was detected additional CID MS3 spectrum of neutral loss have been done without previous isolation. All the spectra were collected in positive mode. LC-MS/MS spectra were analyzed using Trans Proteomic Pipeline. Peak lists were submitted to the Mascot Server 2.1 (Matrix Science, London, UK). Searches were performed using a database IPI_RAT. Trypsin was selected as a site-specific enzyme used in experiment. The precursor ion tolerance was set to 0.8 Da and fragment mass tolerance was set to 0.6 Da. In addition, cysteine was defined with fixed carboxyamidomethylation modification as well as methyl esters of aspartic and glutaminic acids. Serine/Threonine and Tyrosine were set with phosphorylation and heavy methyl methyl esters of glutaminic and aspartic acid were selected as variable modification. Finally, y and b fragment ions were defined for all CID data, while c and z fragment ions were defined for ETD. Identified peptides were quantified with ASAPRatio (Fig. 2) and XPRESS software from Trans Proteomic Pipeline. Figure 1. Differential labeling. Figure 2. ASAPRatio quantitative analysis of Creatine Kinase B peptide LAVEALSSLDGDLSGR at three different charge states. Results Drug dependence has a huge impact not only on a function of reward’s system but on many other systems e.g. yet not understood “psychological” mechanism of addiction. Only a reward system engages few kinds of brain structures, cell types, receptors, finally many molecular systems. In this study we present quantitative phosphoproteomics analysis of prefrontal cortex in morphine dependence. We have found several proteins to be substantially regulated. Up-regulated proteins:Down-regulated proteins: Pgam1 Phosphoglycerate mutase 11.97 Tubulin beta - 2A chain/ 2B chain1.71 Tubulin beta-3 chain2.41 Tubulin alpha-3 chain2.12 Gene symbol=Zfp259 Uncharacterized2.35 Gene symbol=Dv12 LOC100361515 dishevelled 21.91 Trembl:D3ZQ99 Uncharacterized protein5.23 Creatine Kinase B-type0.86 Gene symbol=Ywhaz 14-3-3 protein zeta/delta0.72 Gene_Symbol=Ywhag 14-3-3 protein gamma0.48 Gene_Symbol=Eno3 Beta-enolase0.60 Gene_Symbol=Ywhah 14-3-3 protein eta0.56