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Celem projektu była identyfikacja biomarkerów uzależnienia od morfiny. Badania opierały się na określeniu molekularnych mechanizmów uzależnienia, a także na wyjaśnieniu roli poszczególnych białek zaangażowanych w ten proces. Projekt kładzie szczególny nacisk na rolę fosfobiałek w procesie uzależnienia, ze względu na fakt, że ich fosforylacja jest głównym regulatorem poszczególnych ścieżek w sygnalizacji komórkowej. Projekt zakładał opracowanie metodyki pozwalającej na ilościową analizę fosfoproteomu na szeroką skalę. W tym celu opracowane zostały: metoda znakowania stabilnymi izotopami, metody izolacji fosfopeptydów (TiO2, IMAC), metoda akwizycji danych za pomocą spektrometrii mas (Data-dependent CID/ETD), a także platforma bioinformatyczna, niezbędna do analizy danych. Realizacja projektu pozwoliła na identyfikację i określenie stosunków ilościowych dla kilku tysięcy białek. W rezultacie otrzymaną listę białek poddano specyficznej filtracji i otrzymane w ten sposób wybrane białka, różnicujące grupy badawcze, zostały szerzej omówione. Quantitative analysis of phosphoproteome of the selected brain structures in morphine dependence. The aim of the project was the search for, and identification of the markers of drug dependence. The research was focused on the identification of molecular mechanisms involved in these processes and clarification of the role of particular proteins in morphine 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. 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 involves the newest, fully quantitative, methods for phosphoproteome analysis. In particular a special work-flow based on two methods of phosphopeptides enrichment (TiO2, IMAC), data- dependent mass spectrometry acquisition and specific bioinformatic platform were developed. This approach allowed for simultaneous analysis of CID/ETD data from the Bruker ion-trap mass spectrometer with Mascot as identification tool and finally with the Trans-proteomic pipeline. This work describes methods for phosphoproteomics and gives a detailed protocols for sample preparation, phosphopeptides enrichment and mass spectrometry analysis. However it puts special emphasis on data analysis including self-programmed python scripts for data conversion, which is crucial in such wide-scale experiments. The project resulted in identification and quantitation of a large number of phosphoproteins, which may be considered as potential biomarkers for morphine dependence. A few of them were discussed in details, however further research, especially based on MRM (Multiple reaction monitoring) approach is required.