Application of affinity-purification mass spectrometry based predictive mapping for identification of RBM45 protein interactors in amyotrophic lateral sclerosis

摘要

The combination of cross-linked co-IP and regular co-IP coupled to the robust ability of LC-MS/MS allowed for identification of 138 putative RBM45 protein interactors after manual thresholding analysis. 135 of the 138 proteins identified were also predicted interactors using the SAINTexpress algorithm. Of these, 62 proteins reported a SAINTexpress probability score >0.7 increasing the confidence in interaction. 23 proteins with a probability score >0.7 were identified by cross-linked co-IP and regular co-IP, as well as high confidence by both manual and SAINTexpress analysis. RBM45 interactions are heavily enriched toward nuclear ribonucleoprotein and RNA-binding protein partners. 7 proteins were verified as true RBM45 binding partners by immunoblot, all of which have been previously reported in ALS literature. IPA identified 28 significantly enriched pathways (p-value < 0.05). Examination of the top pathways identified by IPA show that RBM45 has a high affinity for proteins involved in elongation initiation factor signaling. Though it has not been detailed in literature as directly participating in these pathways, RMB45 is a strong interactor of their components, specifically EIF4. The pathway analysis results further demonstrate the functional range of RBM45 both in the nucleus and cytoplasm.