Cu(II) partially protects three histidine residues and the N-terminus of amyloid-β 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 (A beta) 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 MS (ESI Q-TOF MS/MS). Our results indicate that five residues in A beta 1-42 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 A beta 1-42 promotes peptide aggregation, as monitored through the loss of soluble A beta 42 in a semi-quantitative MALDI-TOF MS assay. It has been previously proposed that Cu(II) ions protect A beta 1-16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of A beta 1-16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects A beta from DEPC modification. Sequencing of obtained Cu(II)-protected A beta 1-16 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 (e.g. 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 A beta and to speculate about the role of His residues in these processes.