Study of biologically active forms of metalloproteins by electrospray ionization fourier transform ion cyclotron resonance mass spectrometry
Taylor, Peggy Kristina
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Applications of high resolution mass spectrometry to the characterization of metalloproteins are developed. Metalloproteins in nondenaturing solutions are analyzed by electrospray ionization (ESI) mass spectrometry. ESI places a number of charges on the ion, reducing the mass-to-charge ratio of the ion and making it more easily studied by mass spectrometry. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is known for its high mass accuracy and high resolution, making it an ideal tool for the study of metalloproteins. By ESI-FTICR mass spectrometry, it is possible to determine the identity of the metal at the active site of a protein. ESI-FTICR was used to examine iron- and zinc-containing rubredoxins in a time-course study, examining the rate of incorporation of iron versus zinc into this protein.|Mass accuracy in ESI-FTICR depends on accurate measurement of the cyclotron frequency. However, the frequency is affected by space charge effects, caused by the coulombic interaction of the ions in the cell, which systematically reduce the observed frequency with increasing ion density in the FTICR cell. Prior research has characterized space charge-induced frequency shifts for singly-charged ions. ESI was used to characterize frequency shifts for multiply-charged ions.|The high resolution of FTICR was utilized in the study of bovine hemoglobin dimer and tetramer. In addition to peaks arising from sodium-proton exchange, low intensity peaks were observed in the mass spectrum. Overnight dialysis in oxygensaturated buffer revealed the presence of two molecules of dioxygen bound to the dimer and four molecules of dioxygen bound to the substrate, the first mass spectral evidence of oxygen binding to hemoglobin.|Noncovalent interactions play an important role in biology. Often the active site of the complex lies at the interface between the subunits. For this and a number of other reasons, it is important to apply the mass resolution and mass accuracy of FTICR to noncovalent complexes. However, these complexes are typically high mass molecules, stretching the limits of the technique. ESI-FTICR was used to characterize a range of biologically relevant noncovalent complexes, in order to determine the parameters necessary to achieve isotopic resolution of these molecules.