A Simple Method to Determine Electrospray Response Factors of Noncovalent Complexes

The quantitative study of noncovalent complexes by electrospray mass spectrometry requires the determination of the relative response of each species. The method proposed here to determine the electrospray response factors is based on the use of (1) an internal standard and (2) the mass balance equation applied to one binding partner M, for which different complexes MxLy are detected in the electrospray mass spectra. A set of experiments providing various ratios between the complexes (e.g. different ligand concentrations in a titration experiment or different time points in a kinetics experiment) is used to generate a set of independent linear equations that can be solved using simple matrix algebra to find the response factors of each MxLy complex relative to that of the internal standard. The response factors can then be used to determine equilibrium dissociation constants or for the quantitative monitoring of reaction kinetics. The first is illustrated with a study of DNA-ligand complexes, where we show that neither minor groove binding nor intercalation dramatically affects the DNA response factor. The second is illustrated with a study of the association kinetics of the telomeric G-quadruplex dGGG(TTAGGG)3 with its complementary strand, where the response factors allow correcting for the relative response of the quadruplex and the long duplex and obtaining reproducible association rate constants independently of the source tuning potentials

Probing hemoglobin structure by means of traveling-wave ion mobility mass spectrometry

Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two alpha- and two beta-globin chains each associated with a heme group. Its exact assembly pathway is a matter of debate. Disorders of hemoglobin are the most common inherited disorders and subsequently the molecule has been extensively studied. This work attempts to further elucidate the structural properties of the hemoglobin tetramer and its components. Gas-phase conformations of hemoglobin tetramers and their constituents were investigated by means of traveling-wave ion mobility mass spectrometry. Sickle (HbS) and normal (HbA) hemoglobin molecules were analyzed to determine whether conformational differences in their quaternary structure could be observed. Rotationally averaged collision cross sections were estimated for tetramer, dimer, apo-, and holo-monomers with reference to a protein standard with known cross sections. Estimates of cross section obtained for the tetramers were compared to values calculated from X-ray crystallographic structures. HbS was consistently estimated to have a larger cross section than that of HbA, comparable with values obtained from X-ray crystallographic structures. Nontetrameric species observed included apo- and holo- forms of alpha- and beta-monomers and heterodimers: alpha- and beta-monomers in both apo- and holo- forms were found to have similar cross sections, suggesting they maintain a similar fold in the gas phase in both the presence and the absence of heme. Heme-deficient dimer, observed in the spectrum when analyzing commercially prepared Hb, was not observed when analyzing fresh blood. This implies that holo-alpha-apo-beta is not an essential intermediate within the Hb assembly pathway, as previously proposed. (J Am Soc Mass Spectrom 2009, 20, 625-631) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometr