Radiolytic modification of acidic amino acid residues in peptides: probes for examining protein-protein interactions.

Hydroxyl radical-mediated footprinting coupled with mass spectroscopic analysis is a new technique for mapping protein surfaces, identifying structural changes modulated by protein-ligand binding, and mapping protein-ligand interfaces in solution. In this study, we examine the radiolytic oxidation of aspartic and glutamic acid residues to probe their potential use as structural probes in footprinting experiments. Model peptides containing Asp or Glu were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The radiolysis products were characterized by electrospray mass spectrometry including tandem mass spectrometry. Both Asp and Glu are susceptible to radiolytic oxidization by gamma-rays or synchrotron X-rays. Radiolysis results primarily in the oxidative decarboxylation of the side chain carboxyl group and formation of an aldehyde group at the carbon next to the original carboxyl group, giving rise to a characteristic product with a -30 Da mass change. A similar oxidative decarboxylation also takes place for amino acids with C-terminal carboxyl groups. The methylene groups in the Asp and Glu side chains also undergo oxygen addition forming ketone or alcohol groups with mass changes of +14 and +16 Da, respectively. Characterizing the oxidation reactions of these two acidic residues extends the number of useful side chain probes for hydroxyl radical-mediated protein footprinting from 10 (Cys, Met, Trp, Tyr, Phe, Arg, Leu, Pro, His, Lys) to 12 amino acid residues, thus enhancing our ability to map protein surface structure and in combination with previously identified basic amino acid probes can be used to examine molecular details of protein-protein interactions that are driven by electrostatics.