Affinity Purification and Mass Spectomtery
Cellular signaling processes are mediated by multi-molecular complexes that form or disassemble under regulatory stimuli. These complexes have mostly been discovered by low-throughput experiments that interrogate the possibility of binding between two molecules of interest.. The Jackson Lab has used affinity purification mass spectrometry (AP/MS) to make unbiased identifications of protein complexes involved in a wide variety of diseases, including Bardet-Biedl Syndrome, Nephronophthisis, Joubert Syndrome, other ciliopathies and Rotavirus. Proteomics work continues on these diseases as well as on obesity and non-small cell lung cancer.
The AP/MS experiment begins with the expression of the relevant “bait” protein in a relevant cell line. We introduce this protein by either a FLP/FRT recombinase system or lentiviral transduction. The engineered cells are then expanded to a large scale (109-1010cells), and expression of the bait protein is induced. We then lyse the cells and isolate the bait protein, in any naturally-occurring complexes, by a tandem-affinity purification process. The final eluate is analyzed by tandem mass spectrometry, which identifies bait-associated proteins and their post-translational modifications. From these identifications, we can derive a list of candidate proteins that may be involved in similar cellular processes as the bait protein. For example, we identified the entire Intra-Flagellar Transport B (IFT-B) complex in an experiment with mutant RABL2B as the bait protein, pointing toward a novel mechanism by which IFT-B is introduced into cilia. We can explore this data further using our bioinformatics capabilities.