Identification of the Interactome and Novel Phosphatase Substrates for the Putative Oncogene hYVH1/DUSP12 using Soft Elution Affinity Chromatography and GeLC-MS/MS

摘要

The soft elution mechanism efficiently liberated the protein target from the antibody conjugated beads without eluting any significant amount of conjugated antibody. Numerous unique protein bands in the trapping mutant sample not observed in the wild type sample were resolved (representing putative substrate candidates). Furthermore, a large amount of unique bands were seen in the trapping sample and the wild type sample which were not detected in the empty vector transfected sample. GeLC-MS/MS identified greater than 100 proteins that represent either candidate substrates (unique to the substrate trap mutant) or putative direct or indirect associating proteins (unique to the substrate trap mutant and wild type hYVH1). Numerous ribosomal proteins were identified which was expected given the fact that YVH1 in yeast has been shown to associate with the 60S ribonucleoprotein (RNP) particle. Interestingly, proteins known to be present on additional RNP particles were identified including proteins from the pre-mRNA splicesome, RNP stress granules, mRNPs, and the nucleosome. Representative RNP proteins identified in the study were validated by a combination of sub-organelle fractionation, co-immunoprecipitation, and co-localization experiments. These results suggest that hYVH1 may regulate the assembly of numerous RNP particles important in mRNA processing.