Collisionally activated dissociation and tandem mass spectrometry of intact hemoglobin β-chain variant proteins with electrospray ionization

Authors

  • Karen J. Light-Wahl,

    1. Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352, USA
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  • Joseph A. Loo,

    1. Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352, USA
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  • Charles G. Edmonds,

    1. Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352, USA
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  • Richard D. Smith,

    Corresponding author
    1. Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352, USA
    • Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352, USA
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  • H. Ewa Witkowska,

    1. Clinical Mass Spectrometry Facility, Children's Hospital, Oakland Research Institute Oakland, California 94609, USA
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  • Cedric H. L. Shackleton,

    Corresponding author
    1. Clinical Mass Spectrometry Facility, Children's Hospital, Oakland Research Institute Oakland, California 94609, USA
    • Clinical Mass Spectrometry Facility, Children's Hospital, Oakland Research Institute Oakland, California 94609, USA
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  • Chuen-Shang C. Wu

    1. Cardiovascular Research Institute, University of California, San Francisco, California 94143, USA
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Abstract

Electrospray ionization collisionally activated dissociation (CAD) mass spectra of multiply charged human hemoglobin β-chain variant proteins (146 amino acid residues, 15.9 kDa), generated in the atmospheric pressure/vacuum interface and in the collision quadrupole of a triple-quadrupole mass spectrometer, are shown and compared. Several series of structurally informative singly and multiply charged b- and y-mode product ions are observed, with cleavage of the Thr 50–Pro 51 CO[BOND]NH bond to produce the complementary y96 and b50 sequence ions as the most favored fragmentation pathway. The eight different β-globin variants studied differ by a single amino acid substitution and can be differentiated from the observed m/z shifts of the assigned product ions. The overall fragmentation patterns for the variant polypeptides are very similar, with the exception of the Willamette form, in which Arg is substituted for Pro- 51, and multiply charged y96 product ions are not observed. Circular dichroism spectra of normal βA and βWillamette show very little difference under a variety of solvent conditions, indicating that fragmentation differences in their respective CAD mass spectra are substantially governed by primary rather than secondary structure.

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