Probing Hemoglobin Structure by Means of Traveling-Wave Ion Mobility Mass Spectrometry

Charlotte A. Scarff, Vibhuti J. Patel, Konstantinos Thalassinos, James Scrivens

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    55 Citations (Scopus)


    Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two α- and two β-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 α- and β-monomers and heterodimers; α- and β-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-α-apo-β is not an essential intermediate within the Hb assembly pathway, as previously proposed.

    Original languageEnglish
    Pages (from-to)625-631
    Number of pages7
    JournalJournal of the American Society for Mass Spectrometry
    Issue number4
    Publication statusPublished - 1 Apr 2009


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