CHARACTERIZATION OF LOW-AFFINITY COMPLEXES BETWEEN CALMODULIN AND PYRAZINE DERIVATIVES BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Electrospray ionization mass spectrometry (ESIMS) was used to study th e weak non-covalent interactions occurring between droxymethyl)-8-(met hylamino)imidazo[1,2-a]pyrazine (1) and calmodulin, The formation of a 2:1 (ligand:protein) complex was observed. Using 2, a (diazomethyl)ca rbonyI derivative of 1 which under UV irradiation generates a highly r eactive carbene entity, calmodulin was photo-labeled and the mass spec trum of the covalent adduct was recorded. Under these circumstances, t wo species were detected, one corresponding to the binding of calmodul in to four carbenes derived from 2 and another corresponding to calmod ulin five carbenes after their loss of a bromine atom. These results s trongly confirm that ESIMS is a powerful technique for the characteriz ation of low-affinity complexes, even if part of the non-covalent inte ractions could be lost during the ESI process.