ETD and sequential ETD localize the residues involved in D2-A2A heteromerization

Abstract

Certain amino acid residues and posttranslational modifications play an important role in the formation of noncovalent complexes (NCXs) by electrostatic interactions. Electrospray ionization mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs, due to its softer ionization process and compatibility with the solution phase of NCX mixtures. In order to locate the site where interactions are forming in NCXs involving phosphopeptides and adjacent arginines, tandem mass spectrometry studies using collision-induced dissociation (CID) and electron transfer dissociation (ETD) were performed on NCXs at different charge states. CID fragmentation revealed two dissociation pathways: one in which the electrostatic interaction is disrupted and another in which the covalent bond attaching the phosphate group to the amino acid residue is cleaved, while the electrostatic interaction is maintained. ETD and sequential ETD/ETD, and CID/ETD allow the determination of the NCX interaction site. These results confirmed the involvement of the phosphorylated amino acid and at least two adjacent arginines as the binding site.