posted on 2021-10-18, 19:15authored byAlexis
N. Edwards, Hien M. Tran, Elyssia S. Gallagher
Native mass spectrometry
(MS) is used to elucidate the stoichiometry
of protein complexes and quantify binding interactions by maintaining
native-like, noncovalent interactions in the gas phase. However, ionization
forces proteins into specific conformations, losing the solution-phase
dynamics associated with solvated protein structures. Comparison of
gas-phase structures to those in solution, or to other gas-phase ion
populations, has many biological implications. For one, analyzing
the variety of conformations that are maintained in the gas-phase
can provide insight into a protein’s solution-phase energy
landscape. The gas-phase conformations of proteins and complexes can
be investigated using ion mobility (IM) spectrometry. Specifically,
drift tube (DT)-IM utilizes uniform electric fields to propel a population
of gas-phase ions through a region containing a neutral gas. By measuring
the mobility (K) of gas-phase ions, users are able
to calculate an average momentum transfer cross section (DTCCS), which provides structural information on the ion. Conversely,
in traveling-wave ion mobility spectrometry (TWIMS), TWCCS values cannot be derived directly from an ion’s mobility
but must be determined following calibration. Though the required
calibration adds uncertainty, it is common to report only an average
and standard deviation of the calculated TWCCS, accounting
for uncertainty associated with replicate measurements, which is
a fraction of the overall uncertainty. Herein, we calibrate a TWIMS
instrument and derive TWCCSN2 and TWCCSN2→He values for four proteins: cytochrome c, ubiquitin, apo-myoglobin, and holo-myoglobin. We show
that compared to reporting only the standard deviation of TWCCS, propagating error through the calibration results in a significant
increase in the number of calculated TWCCS values that
agree within experimental error with literature values (DTCCS). Incorporating this additional uncertainty provides a more thorough
assessment of a protein ion’s gas-phase conformations, enabling
the structures sampled by native IM-MS to be compared against other
reported structures, both experimental and computational.