Microelectrospray ionization analysis of noncovalent interactions within the electron transferring flavoprotein

Cofactor associations within the electron transferring flavoprotein (ETF) w ere studied in real time using microelectrospray ionization-mass spectromet ry (mu ESI-MS). Initial analysis of porcine (pETF) and human ETF (hETF) rev ealed only the holoprotein. When mu ESI-MS source energies were increased, both pETF and hETF readily lost AMP. Analysis of hETF and pETF in methanol revealed intact alpha- and beta -subunits, and beta -subunit with AMP. The pETF also contained beta -subunit with FAD and beta -subunit with both cofa ctors. In contrast to crystal structure predictions, AMP dissociates more r eadily than FAD, and the pETF beta -subunit has an intimate association wit h FAD. This work demonstrates the complementarity of mu ESI-MS with NMR X-r ay and optical spectroscopy in the analysis of noncovalent complexes. (C) 2 001 Academic Press.