Chemical and on-line electrochemical reduction of metalloproteins with high-resolution electrospray ionization mass spectrometry detection.

The observation of the reduced forms of several metal-containing proteins using electrospray ionization (ESI) is reported for the first time. High-resolution mass analysis using Fourier transform ion cyclotron resonance mass spectrometry allows the oxidized and reduced forms of the proteins to be distinguished. The metalloproteins are reduced both chemically and electrochemically. Under normal sample handling conditions, the proteins that are reduced in solution appear in their oxidized form in their ESI mass spectra. Rigorous exclusion of oxygen from the solution of the reduced protein allows the observation of the reduced form in the gas phase. The metal centers investigated include heme and non-heme iron proteins, copper, and a manganese-substituted iron-sulfur cluster of the form [3FeMn-4S]. The electrochemical method is shown to provide several advantages over chemical reduction. The oxidation state of the metal center is stable with respect to electrospray ionization in both positive and negative ionization modes.