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 u sing electrospray ionization (ESI) is reported for the first time. High-res olution mass analysis using Fourier transform ion cyclotron resonance mass spectrometry allows the oxidized and reduced forms of the proteins to be di stinguished. The metalloproteins are reduced both chemically and electroche mically. Under normal sample handling conditions, the proteins that are red uced in solution appear in their oxidized form in their ESI mass spectra. R igorous exclusion of oxygen from the solution of the reduced protein allows the observation of the reduced form in the gas phase. The metal centers in vestigated include heme and non-heme iron proteins, copper, and a manganese -substituted iron-sulfur cluster of the form [3FeMn-4S]. The electrochemica l method is shown to provide several advantages over chemical reduction. Th e oxidation state of the metal center is stable with respect to electrospra y ionization in both positive and negative ionization modes.