Copper(II) partially protects three histidine residues and the N-terminus of amyloid-b peptide from diethyl pyrocarbonate (DEPC) modification Merlin Friedemann , Vello T~ ougu and Peep Palumaa Department of Chemistry and Biotechnology, Tallinn University of Technology, Estonia Keywords amyloid-beta peptide; Cu(II) ions; diethyl pyrocarbonate; ESI Q-TOF MS; insulin; MALDI-TOF MS; MS Correspondence M. Friedemann, Department of Chemistry and Biotechnology, Tallinn University of Technology, Akadeemia 15, 12618, Tallinn, Estonia Tel: (+372) 6204412 E-mail: merlin.friedemann@taltech.ee (Received 15 October 2019, revised 9 March 2020, accepted 31 March 2020) doi:10.1002/2211-5463.12857 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 resi- dues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid-beta (Ab) 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 quad- rupole time-of-flight tandem MS (ESI Q-TOF MS/MS). Our results indi- cate that five residues in Ab1–42 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 Ab1–42 promotes pep- tide aggregation, as monitored through the loss of soluble Ab42 in a semi- quantitative MALDI-TOF MS assay. It has been previously proposed that Cu(II) ions protect Ab1–16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of Ab1–16 modification with the excess of DEPC being lower as compared to the con- trol, which indicates that Cu(II) protects Ab from DEPC modification. Sequencing of obtained Cu(II)-protected Ab1–16 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 modifi- cation on amyloidogenity of human Ab and to speculate about the role of His residues in these processes. Alzheimer disease (AD) is the most common neurodegenerative disease, which is believed to start with the pathological build-up of cerebral extracellular amyloid plaques, comprised of aggregated amyloid-b peptides (Ab) [1]. Enormous efforts have been directed to unravelling the factors initiating the formation of amyloid plaques, which is of crucial importance for understanding the pathological mechanisms of AD and for drug design. According to the accumulated knowledge, the path of protein/peptide self-assembly is Abbreviations AD, Alzheimer’s disease; Ab, amyloid beta; DEPC, diethyl pyrocarbonate; ESI Q-TOF MS/MS, electrospray ionization quadrupole time-of- flight tandem mass spectrometry; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; ThT, thioflavin T; a-CHCA, a-cyano-4- hydroxycinnamic acid. 1072 FEBS Open Bio 10 (2020) 1072–1081 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.