Project : PXD000379
DJ-1 as high-fat diet associated protein
Genetic and environmental factors mediate via different physiological and molecular processes a shifted energy balance leading to overweight and obesity. Significant progress in understanding the molecular mechanisms of appetite regulation and control of food consumption was made when the hypothalamus was identified as an important player in regulating energy and glucose homeostasis by sensing circulating hormones and nutrients. To get insights in the underlying processes involved in energy intake and weight gain, we compared hypothalamic tissue of mice kept on a high-fat or control diet for 10 days by a proteomic approach. Using 2D difference gel electrophoresis in combination with LC-MS/MS we observed significant abundance changes in 16 protein spots. One isoform of the protein DJ-1 was elevated in the high fat diet group in the analyzed three different mouse strains SWR/J, C57BL/6N and AKR/J. Large scale validation of DJ-1 isoforms in individual samples and tissues confirmed a shift in the pattern of DJ-1 isoforms towards more acidic isoforms in several brain and peripheral tissues after feeding a high-fat diet for 10 days. The identification of an oxidation of cysteine 106 as well as a 2-succinyl modification of the same residue by mass spectrometry not only explains the isoelectric shift of DJ-1 but also links our results to similar shifts of DJ-1 observed in neurodegenerative disease states under oxidative stress. We hypothesize that DJ-1 is a common physiological sensor involved in both nutrition-induced effects and neurodegenerative disease states. Peak lists of MS/MS spectra acquired on the HCT ion trap (Bruker Daltonics, Bremen, Germany) were generated using the software tool Data Analysis (version 4 SP4 Build 281, Bruker Daltonics) with default parameters. For peptide and protein identification using MASCOT 2.4.1, peak lists were correlated with the Swiss-Prot part of the UniProtKB release 12/2012 including a reverse decoy version of all entries resulting in a total of 33,160 mus musculus sequences. All searches were performed for peptides with charge states 2+, 3+ and 4+ with tryptic specificity allowing one missed cleavage. No fixed modification was set, oxidation of methionine was considered as variable modification. Ion trap MS/MS spectra were accepted with cut off scores of 30 as well as mass tolerance of 0.4 Da was applied for MS and MS/MS experiments. Only proteins with at least 2 identified peptides were considered to be identified. Raw files from the LTQ Orbitrap ELITE were further processed for protein and peptide identification and quantification using the MaxQuant software suite version 22.214.171.124 (Max Planck Institute of Biochemistry, Planegg, Germany) mainly with default parameters. Within the software suite database searches were carried out using 16,615 mouse sequences from the SwissProt part of UniProtKB (release June 2013) applying the following parameters: mass tolerance Fourier transformed mass spectra (Orbitrap) first/second search: 20 ppm / 6 ppm, mass tolerance fragment spectra (linear ion trap): 0.5 Da, no fixed modification was specified. For variable modifications methionine oxidation, acetylation at protein N-termini and carbamidomethylation were considered as well as one of the following modifications per search event: trioxidation of cysteines, 2-succinylation of cysteines. Peptides and proteins were accepted at a false discovery rate of 1 % and proteins identified with a minimum of two peptides.
Sample Processing Protocol
See details in reference PMID : 24646099
Data Processing Protocol
See details in reference PMID : 24646099
Gereon Poschmann, Molecular Proteomics Laboratory
Poschmann G, Seyfarth K, Besong Agbo D, Klafki HW, Rozman J, Wurst W, Wiltfang J, Meyer HE, Klingenspor M, Stühler K. High fat diet induced isoform changes of the Parkinson's disease protein DJ-1. J Proteome Res. 2014 Mar 19 PubMed(s) : 24646099
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