Protein Footprinting by Carbenes on a Fast Photochemical Oxidation of Proteins (FPOP) Platform

摘要

Protein footprinting combined with mass spectrometry provides a method to study protein structures and interactions. To improve further current protein footprinting methods, we adapted a fast photochemical oxidation of proteins (FPOP) platform to utilize carbenes as the footprinting reagent. A Nd-YAG laser provides 355 nm laser for carbene generation in situ from photoleucine as the carbene precursor in a flow system with calmodulin as the test protein. Reversed-phase liquid chromatography coupled with mass spectrometry is appropriate to analyze the modifications produced in this footprinting. By comparing the modification extent of apo and holo calmodulin on the peptide level, we can resolve different structural domains of the protein. Carbene footprinting in a flow system is a promising strategy to investigate protein structures.