Abstract
This chapter discusses the use of alternating hydrostatic pressure (Pressure Cycling Technology, or PCT) and optimized solvents for detergent-free disruption of white adipose tissue samples for proteomic analyses. This technique can increase efficiency of protein recovery from mouse fat tissue specimens as compared to conventional homogenization and dissolution techniques. Protein extracts generated using PCT were analyzed by SDS-PAGE, 2D-electrophoresis, and nanoflow HPLC coupled to high resolution, high mass accuracy tandem mass spectrometry. As a result, a novel pressure cycling-assisted liquid-liquid extraction and fractionation method has been developed to achieve nearly complete tissue dissolution and fractionation of lipids and proteins into distinct liquid phases. This method allows for efficient extraction of highly hydrophobic proteins typically underrepresented in the extracts obtained using conventional methods. A pilot analysis of several genetically distinct model mouse lines and diets has revealed trends in protein expression which may be linked to obesity and metabolic syndrome progression, or also serve as potential drug targets. Proteomic analysis using 2D-gels and 1D-gel LC MS/MS revealed greater representation of proteins involved in lipid, glucose, and energy metabolism, as well as acute phase response and oxidative stress response signaling pathways linked to obesity. The pilot results of comparative proteomic analyses of adipose tissue harvested from animals of different genotypes and exposed to different diets were analyzed further using gene ontology and functional pathway analysis bioinformatics tools. Lipid fractions obtained from the tissue samples using the newly developed sample preparation and fractionation platform were collected for future lipid mass spectrometry-based profiling studies.
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Acknowledgements
We would like to thank Pressure Biosciences and Drs. Haiming Cao and Gökhan Hotamisligil for providing pilot fat adipose tissue samples. We are thankful for the financial support of the Department of Genetics and Complex Diseases at the Harvard School of Public Health.
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Freeman, E.A., Gross, V., Romanovsky, I., Lazarev, A.V., Ivanov, A.R. (2011). Adipose Tissue Lysis and Protein Extraction Followed by MS-based Proteomic Profiling Reveals Constituents of Oxidative Stress in Obesity. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_30
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DOI: https://doi.org/10.1007/978-94-007-0828-0_30
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