Propagating Error through Traveling-Wave Ion Mobility Calibration

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 ^TWCCS_N2 and ^TWCCS_N2→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.