gms | German Medical Science

Diffuse gliomas are the most frequent primary brain tumors and have an unpredictable rate of progression. It has been shown that malignant transformation of glial progenitor cells is associated with a number of genetic events, including p53 mutations, 1p/19q codeletion/translocation, as well as amplification of epidermal growth factor receptor. Recently, mutations in the isocitrate dehydrogenase genes 1 and 2 (IDH1 and IDH2) have been described in 85-90% of grade II–III gliomas, as well as in a minority of glioblastoma. IDH1 mutation is an early event in gliomagenesis and patients carrying this mutation exhibit a better overall prognosis.

For the quantitative labelfree analysis an immunoprecipitation of IDH1 was performed on three different human glioma cell lines in five replicates. Mass spectrometric analysis was performed on a Q Exactive Plus hybrid Quadrupole-Orbitrap mass spectrometer online coupled with a nanoUHPLC. Raw files were further processed for identification and quantification using MaxQuant and Perseus software. Only proteins that occured in four out of five replicates in at least one group were used for quantification. For the statistical analysis p-values <0.05 were considered to be significant and a fold change >1.8 had to be achieved. Interaction networks of IDH1 were generated using String software.

The goal of this study is to obtain the first detailed characterization of the interaction partners of mutated und wild-type IDH1 because until now, less is known about the interactome of IDH1 and its role in tumor biology. As no cell line model with mutated IDH1 was available we established three glioma cell lines overexpressing mutant or wild-type IDH1. The functional activity of recombinant mutant IDH1 was confirmed by increased concentration of 2-hydroxyglutarate, a specific metabolite of mutant IDH in the supernatant of the analysed human glioma cell lines. After immunoprecipitation with an IDH1 specific antibody a 20-fold enrichment of IDH1 based on the peptide spectrum matches could be demonstrated compared to the unenriched cell lysate using LC-MS analysis. Labelfree analysis led to the identification of 1043 proteins with quantitative data for 508 of these proteins. 94 proteins specifically interacting with wild-type IDH1 and 41 potential interaction partners of mutant IDH1 were detected. Between the wild-type and mutant IDH1 three significant differential interaction partners were found.