Urszula Jankowska1, Agnieszka Latosinska2, Bozena Skupien-Rabian2, Bianka Swiderska3, Marta Dziedzicka-Wasylewska2, Sylwia Kedracka-Krok4. 1. Department of Structural Biology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland. 2. Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. 3. Department of Structural Biology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland; Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. 4. Department of Structural Biology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland; Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. Electronic address: sylwia.kedracka-krok@uj.edu.pl.
Abstract
BACKGROUND: The cell nucleus is a highly dynamic subcellular compartment performing crucial processes for functioning and administration of the cell. Proteomic analysis of isolated nuclear fraction enables in-depth insight into these processes leading to better understanding of physiological and pathological state of the brain. There is no universal method for nuclear proteome investigation and each biological material needs individual optimization. An additional difficulty is the large amount of nucleic acids, which impairs isoelectrofocusing of proteins and shotgun mass spectrometry analysis of complex peptide samples. NEW METHOD: We performed the first comprehensive optimization of intact nuclei isolation from mouse brain in combination with nuclear protein purification prior to two-dimensional gel electrophoresis (2DE) and gel-free proteomic analysis. RESULTS: Application of sonication, digestion with nuclease and protein precipitation with acetone allowed to obtain high quality 2DE gels. Shotgun mass spectrometry analysis of isolated proteins proved an enrichment in nuclear proteins. The 66.4% of 265 identified proteins had assigned nucleus localization in UniProt database. Gene Ontology analysis using DAVID platform revealed the highest fold enrichment in spliceosome (24.5), nuclear periphery (12.4) and nuclear matrix (11.3). COMPARISON WITH EXISTING METHODS: The proposed procedure is tailored to mouse brain tissue nuclear subproteome investigation. The quality of isolated nuclei, the effectiveness of the protein purification, efficiency of protein recovery after precipitation and overall method reproducibility was taken into detailed consideration. CONCLUSIONS: The elaborated procedure could be further applied for in-depth proteomic analysis of molecular processes occurring in the mouse brain nucleus.
BACKGROUND: The cell nucleus is a highly dynamic subcellular compartment performing crucial processes for functioning and administration of the cell. Proteomic analysis of isolated nuclear fraction enables in-depth insight into these processes leading to better understanding of physiological and pathological state of the brain. There is no universal method for nuclear proteome investigation and each biological material needs individual optimization. An additional difficulty is the large amount of nucleic acids, which impairs isoelectrofocusing of proteins and shotgun mass spectrometry analysis of complex peptide samples. NEW METHOD: We performed the first comprehensive optimization of intact nuclei isolation from mouse brain in combination with nuclear protein purification prior to two-dimensional gel electrophoresis (2DE) and gel-free proteomic analysis. RESULTS: Application of sonication, digestion with nuclease and protein precipitation with acetone allowed to obtain high quality 2DE gels. Shotgun mass spectrometry analysis of isolated proteins proved an enrichment in nuclear proteins. The 66.4% of 265 identified proteins had assigned nucleus localization in UniProt database. Gene Ontology analysis using DAVID platform revealed the highest fold enrichment in spliceosome (24.5), nuclear periphery (12.4) and nuclear matrix (11.3). COMPARISON WITH EXISTING METHODS: The proposed procedure is tailored to mouse brain tissue nuclear subproteome investigation. The quality of isolated nuclei, the effectiveness of the protein purification, efficiency of protein recovery after precipitation and overall method reproducibility was taken into detailed consideration. CONCLUSIONS: The elaborated procedure could be further applied for in-depth proteomic analysis of molecular processes occurring in the mouse brain nucleus.
Authors: Alicia A Cutler; Eric B Dammer; Duc M Doung; Nicholas T Seyfried; Anita H Corbett; Grace K Pavlath Journal: Aging Cell Date: 2017-05-23 Impact factor: 9.304
Authors: Sónia Lucena; Ana V Coelho; Fernando Capela-Silva; Asta Tvarijonaviciute; Elsa Lamy Journal: Biomed Res Int Date: 2018-06-03 Impact factor: 3.411