An interaction network of RNA-binding proteins involved in Drosophila oogenesis

Prashali Bansal; Johannes Madlung; Kristina Schaaf; Boris Macek; Fulvia Bono

To detect fold changes in protein abundances with high precision, we used dimethyl labeling MS. This approach is advantageous for Drosophila, where metabolic isotopic labeling is still challenging. To confirm the results of our label-free analysis, we carried out dimethyl labeling MS for the Hrp48-and Vas-GFP samples. We chose Hrp48 and Vas as these proteins express well and they are involved in the translational regulation of the same set of transcripts (35, 36, 40, 45-47). We carried out the experiments in duplicates and purified the samples the same way as for label-free MS (Fig. S2b ). After in-gel digestion, we labeled the peptides with heavy, medium or light isotopes and inverted the labels in the replicate, to minimize the variability due to the labeling procedures. As before, the raw data were processed with the MaxQuant software (71), providing confident identification of proteins (1% FDR) and normalized protein-abundance ratios. The analysis of the Vas-and Hrp48-associated proteomes resulted in the identification of 4027 peptides, mapping to 615 protein groups. The replicates showed high correlation and the abundance ratios calculated could be well duplicated. We considered as a hit those proteins that we identified with an abundance ratio of >2 in both replicates. Consistent with the label-free analysis, we found several known interactors, most of them reproducibly enriched (Fig. 2b ). To check how the two analyses relate to each other, we mapped the proteins identified in labeled MS onto the label-free MS data. As shown in Fig. 3 , the proteins that were significantly enriched in the labeled MS followed the same distribution profile and showed up to 47% overlap (for Vas) with those enriched in the label-free MS analysis. Background proteins identified in labeled MS (<2 fold in both replicates) showed a similar profile when graded on the corresponding label-free MS data (Fig. 3 ). To get a comprehensive view of the proteomes associated with Hrp48 and Vas, we combined the enriched proteins from both analyses.