Binding position analysis of target proteins with the use of amidopyrene probes as LA-LDI enhancing tags

Amidopyrene-conjugated compounds can be detected by label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) without matrixes. When actin, a cytoskeletal protein, was labeled with an excess amount of amidopyrene N-hydroxysuccinate (apy–OSu), eight apy-labeled actin peptides were predominantly detected by LA-LDI MS. Then actin was labeled with an amidopyrene NHS ester of the antitumor marine macrolide aplyronine A (ApA–apy–OSu) to form a 1 : 1 conjugate. The sequence of an apy-labeled peptide was established as A108PLNPKANR116 by MS/MS analysis, in which the NHS ester moiety specifically reacted with the e-amino group of K113. While the fragmentation at the linker part reduces the detection sensitivity of apy-labeled peptides on LA-LDI MS, our chemical probe method is useful for analyzing the binding modes of various ligands and target biomacromolecules that include multiple and weak interactions.