Abstract
The complexity of the plant cell proteome, exhibiting thousands of proteins whose abundance varies in several orders of magnitude, makes impossible to cover most of the plant proteins using standard shotgun-based approaches. Despite this general description of plant proteomes, the complexity is not a big issue (current protocols and instrumentation allow for the identification of several thousand proteins per injection), low or medium abundant proteins cannot be detected most of times, being necessary to fraction or perform targeted analyses in order to detect and quantify them. Among fractioning choices, cell fractioning in its different organelles is a good strategy for gaining not only a deeper coverage of the proteome but also the basis for understanding organelle function, protein dynamics, and trafficking within the cell, as nuclear and chloroplast communication. This approach is used routinely in many labs working with model species; however, the available protocols focusing on tree species are scarce. In this chapter, we provide a simple but robust protocol for isolating nuclei and chloroplasts in pine needles that is fully compatible with later mass spectrometry–based proteome analysis.
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Acknowledgments
Our research group is generously funded by Spanish Ministry of Science, Innovation, and Universities (AGL2016-77633-P and AGL2017-83988-R). M.M., and L.L. were also supported by Spanish Ministry of Science, Innovation, and Universities trough Ramón y Cajal (RYC-2014-14981) and Spanish PhD training (BES-2017-082092 to L.L.) programs. L.G. was supported by Government of Principado de Asturias, (Spain) trough Severo Ochoa program (BP19-146).
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Lamelas, L., García, L., Cañal, M.J., Meijón, M. (2020). Subcellular Proteomics in Conifers: Purification of Nuclei and Chloroplast Proteomes. In: Jorrin-Novo, J., Valledor, L., Castillejo, M., Rey, MD. (eds) Plant Proteomics. Methods in Molecular Biology, vol 2139. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0528-8_5
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DOI: https://doi.org/10.1007/978-1-0716-0528-8_5
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