(582dp) Targeted Chemical Biotinylation of Protein Complexes Using Monomeric Streptavidin

The majority of proteins in the cell interact with other proteins as part of their natural function. Identifying the components of stable and transient protein complexes is therefore important to understand protein function. To this end, mass spectrometry (MS) can significantly accelerate the rate of discovery by analyzing complex protein mixtures through high resolution peptide sequencing. A critical step during MS analysis is sample preparation. The protein complex of interest needs to be separated from other cellular components, many of which are present in high abundance and interact nonspecifically with the target protein. We used monomeric streptavidin (mSA), which was recently designed in our lab, to achieve selective chemical biotinylation of the proteins that interact with a protein of interest. Biotinylation allows the interacting proteins to be subsequently purified on streptavidin beads utilizing well known streptavidin-biotin interaction. Since biotinylation is irreversible, even weakly or transiently associated complexes can be purified using the technique. This technique differs from other existing purification schemes commonly used for MS analysis, e.g. tandem affinity purification (TAP), in that it does not require any prior modification to any part of the protein complex that is being studied. Rather, the chemical crosslinking is catalyzed by mSA, which brings the crosslinking reagent to close proximity of the target molecule. We demonstrate that this technique is specific and works both in vitro and in vivo, and show that it can be implemented in a number of different ways without loss of efficiency.