sample_processing
Cells can be fractionated into their structural and functional components for preparative and analytical purposes. Although strategies for cell disruption and separation of organelles depend on the final goal of a study, the nature of the starting material determines the choice of the utilized methods. The subcellular fractionation process involves the essential stages of homogenization and the following separation of the cellular components. Whereas disruption of cultured cells can be achieved in different ways, including hypotonic lysis and sonification, tissue material has to be treated mechanically by shearing or grinding. Gentle treatment leaves nuclei, mitochondria, and some other membranous structures intact and enables subsequent fractionation of organelles. Based on variation in physical properties of various organelles, the subcellular components can be separated by various methods, however, differential sedimentation and isopycnic centrifugations are most commonly used.
In a large group of proteomics approaches the obvious advantages of subcellular fractionation are often ignored. Although it is possible to perform a global proteome profiling using non-fractionated (whole lysates) or roughly fractionated but not purified cell components, these approaches are limited to identification of almost exclusively highly abundant proteins. Further reduction of protein complexity by applying different types of protein and peptide fractionation might enable identification of some proteins present in minute amounts. Unfortunately, this mutidimensional separation approach usually leads to a generation of high number of fractions that have to be analyzed individually which requires extensive analytical capabilities in terms of instrumentation and time. Even circumventing these technical limitations, large differences between different proteins in individual fractions could make it impossible to profile and map low abundant proteins.
Subcellular fractionation offers an efficient way to reduce sample complexity but also can provide information on intra-cellular distribution of proteins and or their forms. Most types of organelles can be efficiently purified or enriched in this way. Combination of differential and gradient centrifugation enables separation of various organelles by their size and buoyant density. The extent of enrichment of a particular organelle can be quantitatively monitored by measuring enzymatic activities accompanying them, the so- called marker activity. Mitochondria prepared in this way were used for a comprehensive study on mitochondrial and plasma membranes.
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