Determining Copper and Zinc Content in Superoxide Dismutase Using Electron Capture Dissociation Under Native Spray Conditions

Abstract

Localizing metal binding to specific sites in proteins remains a challenging analytical problem in vitro and in vivo. Although metal binding can be maintained by “native” electrospray ionization with intact proteins for quantitation by mass spectrometry, subsequent fragmentation of proteins with slow-heating methods like collision-induced dissociation (CID) can scramble and detach metals. In contrast, electron capture dissociation (ECD) fragmentation produces highly localized bond cleavage that is well known to preserve posttranslational modifications. We show how a newly available ECD tool that can be retrofitted on standard QTOF mass spectrometers allows the sites of copper and zinc binding to be localized in the antioxidant enzyme Cu, Zn superoxide dismutase (SOD1). The loss of zinc from Cu, Zn SOD1 has been shown to induce motor neuron death and could have a causal role in the fatal neurodegenerative disease, amyotrophic lateral sclerosis (ALS). The methods described enable copper loss to be distinguished from zinc using distinct ECD fragments of SOD1 and are broadly applicable to other metalloproteins.