Application of Nanosecond Laser Photolysis Protein Footprinting to Study EGFR Activation by EGF in Cells

Mass
spectrometry-based protein footprinting emerged as a useful
technology to understand protein ligand interactions in vitro. We
have previously demonstrated the application of footprinting in live E. coli cells. Here, we further optimized an ultrafast laser
photolysis hydroxyl radical footprinting method and applied it to
study the interaction of EGF and EGFR in live mammalian cells. This
method used a nanosecond laser to photochemically generate a burst
of hydroxyl radicals in situ in-cell suspension to oxidize the amino
acids on the protein surface. Mass spectrometric analysis of the thus
modified peptides was interpreted to probe the solvent-accessible
surface areas of the protein in its native biological state with and
without EGF activation. Our footprinting data agreed with the two
relevant EGFR crystal structures, indicating that this in-cell laser
photolysis footprinting technique is a valid approach to study the
structural properties of integral membrane proteins directly in the
native environment.