Concentration-Dependent Coulombic Effects in TWIMS CCS Calibration.

Rationale Travelling wave ion mobility spectrometry (TWIMS) is increasingly being used as a method for calculating the collision cross sections (CCSs) of protein ions. To calculate the CCS values of unknown ions, however, the TWIMS device needs to be calibrated using calibrant proteins of known CCS values. The effect of calibrant protein concentration on the accuracy of the resulting calibration curve has not been explicitly studied so far. We hypothesised that at high protein concentrations the ion density within the TWIMS device will be such that ions will experience space charge effects resulting in deviations, as well as broadening, of ion arrival time distributions (ATDs). Calibration curves using these altered ATDs would therefore result in incorrect CCS values being calculated for the protein ions of interest. Methods Three protein CCS calibrants, avidin, bovine serum albumin and beta-lactgobulin, were prepared at different concentrations and used to calculate the CCS of a non-calibrant protein. Data were collected on a Synapt G1 ion mobility mass spectrometer with a nano-electrospray ionisation (nESI) source using capillaries prepared in house. Results Increasing the concentration of CCS calibrants caused ATD broadening and shifted the ATD peak tops, leading to a significant increase in calculated CCS values. Conclusions The concentration of protein calibrants can directly affect the quality of the CCS calibration in TWIMS experiments.