Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

Christian Stephan,Wilhelm W. Just ,Heiko Hayen,Bettina Warscheid,Lukas Schollenberger,Rob Ofman,Ronald J. A. Wanders ,Sebastian Wiese,Martin Eisenacher,Markus Kunze,Ralf Erdmann,Thomas Gronemeyer,J. A. Afonso De Almeida ,Hans R. Waterham,Wolfgang Schliebs,Cláudia P. Grou,Helmut E. Meyer,Jorge E. Azevedo,Thomas Korosec,Johannes Berger

doi:10.1074/mcp.m700169-mcp200

Abstract

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Highlights

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by ␣- and ␤-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals
Peroxisomes are small organelles present in virtually all eukaryotic cells. They are surrounded by a single membrane and harbor a large set of enzymes that enables them to execute an array of metabolic functions, such as ␣- and ␤-oxidation of fatty acids, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals [1]
Proteomics Investigation of Mouse Kidney Peroxisomes—Peroxisomes from mouse kidney were isolated by density and gradient centrifugation of a light mitochondrial fraction prepared by differential centrifugation using established protocols [13]

Summary

Introduction

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by ␣- and ␤-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. At about the same time, strategies were developed to enable the profiling of hundreds of proteins through various fractions of a density gradient using quantitative MS in combination with [14] or without [15] stable isotope labels These quantitative profiling approaches combined with statistical analyses were shown to allow for the reliable cellular location of proteins in a global manner, thereby providing an excellent means by which new insights into the proteomes and functions of subcellular structures can be obtained [15,16,17,18,19]

Results

Discussion

Conclusion