Protein conformations can be probed in top-down HDX MS experiments utilizing electron transfer dissociation of protein ions without hydrogen scrambling |
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Authors: | Rinat R Abzalimov Desmond A Kaplan Michael L Easterling Igor A Kaltashov |
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Institution: | aDepartment of Chemistry, University of Massachusetts-Amherst, Amherst, Massachusetts, USA;bBruker Daltonics, Billerica, Massachusetts, USA |
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Abstract: | Electron-transfer dissociation (ETD) is evaluated as a technique to provide local information on higher order structure and
dynamics of a whole protein molecule. Isotopic labeling of highly flexible segments of a model 18 kDa protein is carried out
in solution under mildly denaturing conditions by means of hydrogen/deuterium exchange (HDX), followed by transfer of intact
protein ions to the gas phase by means of electrospray ionization, and mass-selection of a precursor ion for subsequent reactions
with fluoranthene radical anions. The ETD process gives rise to abundant fragment ions, whose deuterium content can be measured
as a function of duration of the HDX reaction in solution. No backbone protection is detected for all protein segments spanning
the 25-residue long N-terminal part of the protein, which is known to lack structure in solution. At the same time, noticeable
protection is evident for segments representing the structured regions of the protein. The results of this work suggest that
ETD of intact protein ions is not accompanied by detectable hydrogen scrambling and can be used in tandem with HDX to probe
protein conformation in solution. |
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