Microelectrospray ionization analysis of noncovalent interactions within the electron transferring flavoprotein

Cofactor associations within the electron transferring flavoprotein (ETF) were studied in real time using microelectrospray ionization-mass spectrometry (muESI-MS). Initial analysis of porcine (pETF) and human ETF (hETF) revealed only the holoprotein. When muESI-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 cofactors. In contrast to crystal structure predictions, AMP dissociates more readily than FAD, and the pETF beta-subunit has an intimate association with FAD. This work demonstrates the complementarity of muESI-MS with NMR X-ray and optical spectroscopy in the analysis of noncovalent complexes.