Protein partners identification in Pyrococcus abyssi by nanoLC-MSMS

Recombinant aRNase J from P. abyssi, with C-terminal (His)6-tag (aRNase J-His), was produced, purified and used as baits for AP-MS experiments. In order to discriminate specific protein interaction from column background, we performed mock AP-MS analyses with P. abyssi cell extracts in absence of protein bait. In addition, to determine if some interactions are mediated through RNA or DNA, we implemented a nuclease treatment after incubating the cell extract with the bait. The co-purified partners were identified by bottom-up proteomic techniques coupled with mass spectrometry.