Cu(II) partially protects three histidine residues and the N-terminus of amyloid-beta peptide from diethyl pyrocarbonate (DEPC) modification.

Diethyl pyrocarbonate (DEPC) has been primarily used as a residue-specific modifying agent to study the role of His residues in peptide/protein and enzyme function; however, its action is not specific, and several other residues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid beta (Abeta) peptides and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and determined the modification sites by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI Q-TOF MS/MS). Our results indicate that five residues in Abeta1-42 are modified in the presence of 30 fold molar excess of DEPC. After hydroxylamine treatment (which removes modifications from three His residues), two labels remain bound at the peptide N-terminus and Lys16. DEPC treatment of Abeta1-42 promotes peptide aggregation, as monitored through the loss of soluble Abeta42 in a semi-quantitative MALDI-TOF MS assay. It has been previously proposed that Cu(II) ions protect Abeta1-16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of Abeta1-16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects Abeta from DEPC-modification. Sequencing of obtained Cu(II)-protected Abeta1-16 samples showed that Cu(II) does not protect any residues completely, but partially protects all three His residues and the N-terminus. Thus, the protection by Cu(II) ions is not related to specific metal binding to a particular residue (for example His6), but rather all His residues and the N-terminus are involved in binding of Cu(II) ions. These results allow us to elucidate the effect of DEPC modification on amyloidogenity of human Abeta and to speculate about the role of His residues in these processes.