Probing the Function of Alternative Proteins in Cell Reprogramming by Large Scale Proteomics and Cross - Linking Mass Spectrometry

Many proteins are lacking from reference databases because they are issued from alternative ORFs neither respecting the rules decreed for the genome annotation or for the protein translation from mRNA. It was evidenced that not only a single protein is therefore translated from an mRNA. However, the function of these Alternative Proteins (AltProts) remain largely unknown. Here, we were interested by looking to the function of AltProts in the context of cancer cell reprogramming. We have developed a large scale approach based on shot-gun proteomics and cross-linking mass spectrometry (XL-MS) to identify the regulation of the reference proteins (RefProts) versus the AltProts and find the their interaction partners. The study was conducted from NCH82 human glioma cells which were stimulated by the protein kinase A (PKA) activator Forskolin upon 16H, 24H and 48H to induce cell differentiation and epithelial-mesenchymal transition (EMT). The data have shown to enable tracing back the function of the AltProts by combining experimental data to in silico analysis using cytoscape with ClueGo for GO Term annotation and enrichment of pathways with String. Very interestingly many AltProts demonstrate to be involved in the regulation of tRNA through their interaction with aaRS proteins and of the cellular mobility.