Organelle Proteomics

Abstract

Abstract-Functional proteome analysis is not restricted to the sequence information but includes the broad spectrum of structural modifications and quantitative changes of proteins to which they are subjected in different tissues and cell organelles and during the development of an organism. Cell biology has provided the means required for the analysis of the composition and properties of purified cellular elements. Subcellular fractionation is an approach universal across all cell types and tissues, including cardiac and vascular system. Subcellular fractionation and proteomics form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. Subcellular fractionation is a flexible and adjustable approach resulting in reduced sample complexity and is most efficiently combined with high-resolution 2D gel/mass spectrometry analysis as well as with gel-independent techniques. In this study we introduce state of the art subcellular fractionation techniques and discuss their suitability, advantages, and limitations for proteomics research.

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

“…In recent years, the tendency has been to focus on subcellular proteomes, either organelles or macromolecular structures of the cell [8,10,[12][13][14][15]. Separation of cell homogenates into organelles or other multiprotein fractions substantially increases the probability of detecting low abundance proteins.…”

“…Separation of cell homogenates into organelles or other multiprotein fractions substantially increases the probability of detecting low abundance proteins. In this context, terms such as "subcellular proteomics" [15] or "organelle proteomics" [12,13] have been established. The purity of organelles and other protein complexes is crucial to subcellular proteome research [13][14][15][16][17].…”

“…For the isolation of subcellular structures and organelles several methods, such as differential-and density-gradient centrifugation [18][19][20], immunoisolation [8], affinity purification, [21], and freeflow electrophoresis [22], have been applied. It has been shown that such fractionations improved identification of proteins from targeted subcellular structures [8,10,[12][13][14][15]. However, the copurification and identification of possible contaminants from other structures and compartments was still one of the major unresolved questions.…”

“…More and more researchers in the field of proteomics have started using methods for subcellular fractionation before proteomic analysis of the sample [12][13][14][15]. Using methods, such as differential centrifugation, electrophoresis, or affinity fractionation, cell or tissue lysates are preseparated into fractions containing particular organelles [8,12,14].…”

“…Using methods, such as differential centrifugation, electrophoresis, or affinity fractionation, cell or tissue lysates are preseparated into fractions containing particular organelles [8,12,14]. After this prefractionation, the "organelle proteome" can be analyzed by use of established proteomics techniques, such as 1-and 2-DE and chromatographic methods followed by MS [8,[12][13][14][15][25][26][27][28].…”