Proteomic Analysis of Rat Liver Peroxisome

Abstract

Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macromolecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. Because of the high sensitivity achieved by the present proteomics technology, the purity of samples to be analyzed is important for the interpretation of the results obtained. In the present study, peroxisomes isolated from rat liver by usual cell fractionation were further purified by immunoisolation using a specific antibody raised against a peroxisomal membrane protein, PMP70. The isolated peroxisomes were analyzed by SDS-PAGE combined with liquid chromatography/mass spectrometry. Altogether 34 known peroxisomal proteins were identified in addition to several mitochondrial and microsomal proteins. Some of the latter may reside in the peroxisomes as well. Analysis of membrane fractions identified all known peroxins except for Pex7. Two new peroxisomal proteins of unknown function were of high abundance. One is a bi-functional protein consisting of an aminoglycoside phosphotransferase-domain and an acyl-CoA dehydrogenase domain. The other is a newly identified peroxisome-specific isoform of Lon protease, an ATP-dependent protease with chaperonelike activity. The peroxisomal localization of the protein was confirmed by immunological techniques. The peroxisome-type Lon protease, which is distinct from the mitochondrial isoform, may play an important role in the peroxisomal biogenesis.

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

“…The next step was usually tryptic digestion, followed by chromatographic separation and mass spectrometric analysis of digested peptides [1][2][3][4]. Alternatively, a protein mixture was separated by use of 1-or 2-DE, followed by excision of polypeptide bands or spots, tryptic digestion, and again by MS analysis [4][5][6][7][8]. However, without fractionation and enrichment of biological samples, the highly abundant proteins often masked lessabundant ones, and made their detection difficult or even impossible [9][10][11].…”

“…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.…”

“…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].…”

“…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.…”

“…However, the copurification and identification of possible contaminants from other structures and compartments was still one of the major unresolved questions. It was often difficult, and sometimes impossible, to conclude whether proteins, typically localized in other organelles but now identified in a targeted sample, represent true endogenous partners or were products of artificial association induced by cell disruption or incomplete purification [8,17].…”