Comparative profiling of the peroxisomal proteome of wildtype and Pex7 knockout mice by quantitative mass spectrometry

We present a label-free quantitative proteomic approach for the study of kidney peroxisomes of Pex7 knockout mice which is a bona-fide model for the human disease rhizomelic chondrodysplasia punctata (RCDP). RCDP is an autosomal recessive human disorder caused by mutations in the PEX7 gene encoding for Pex7, the cytosolic receptor protein that is essential for the import of proteins containing a functional peroxisomal targeting signal (PTS)-type 2. In this work, we quantitatively followed hundreds of proteins through high density gradient fractions of wildtype (WT) and Pex7 knockout (Pex7−/−) mice by high resolution mass spectrometry. A set of candidate proteins with altered abundance was defined via statistical and quantitative assessment of protein profiles obtained from WT and Pex7−/− mice. The results obtained demonstrate the feasibility of this approach to identify proteins specifically affected in abundance by the deletion of Pex7. All three known PTS2 proteins, including acetyl-Coenzyme A acyltransferase, alkylglycerone phosphate synthase and phytanoyl-CoA hydroxylase were determined to be virtually absent in these fractions whereas KIAA0564, a so far uncharacterized protein, was barely detectable in peroxisomal fractions of Pex7−/− mice. Furthermore, we report numerous PTS1 proteins with increased abundance levels in Pex7−/− mice that fulfill essential functions in the β-oxidation of very long-chain fatty acids or the biosynthesis of ether-phospholipids in peroxisomes.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (141 K)Download as PowerPoint slideHighlights► MS-based protein profiling across several density gradients enabled to comprehensively characterize peroxisomal proteomes of Pex7−/− and WT mice. ► Comparative analysis of hundreds of protein profiles generated by label-free quantitative MS allowed to identify proteins specifically affected by the deletion of Pex7. ► All proteins known to contain a functional peroxisomal targeting signal 2 (PTS2) were determined to be virtually absent in high density gradient fractions of Pex7−/− mice. ► KIAA0564 was further identified as new PTS2 candidate protein and co-localization studies of KIAA0564 fragments confirmed a peroxisomal localization. ► In addition, numerous PTS1 proteins fulfilling important functions in peroxisomal metabolism were found to be considerably increased in abundance in Pex7−/− mice.