In this report, we describe a dual ionization source ion mobility-mass spectrometer (IM-MS) instrument platform for investigations that critically compare ion mobility collision cross section (CCS) measurements obtained from different ionization methods. The instrument incorporates both matrix-assisted laser desorption ionization (MALDI) and nanoelectrospray ionization (nESI) sources. The nESI source incorporates a keyhole geometry ion funnel design which facilitates axial ion focusing, accumulation, and generation of short duration (10-30 mus) ion pulses for use with the IM-MS. The IM-MS instrument operation is independent of which ionization source is used. This allows comparisons of collision cross section measurements to be made between both ion sources with minimal differences in the instrumental arrangement. The performance of the nESI ion source is evaluated by measuring the collision cross section values of the charge states of equine cytochrome c (z = 9-16), and values are in good agreement (<2% deviation) with those previously reported in the literature. Several charge states (z = 8-11) of cytochrome c exhibit multiple cross sectional features in the ion mobility analysis. An analysis of the tryptic peptides of cytochrome c formed by both ESI and MALDI demonstrate that, on average, +1 MALDI ions are similar in CCS to +1 ESI ions and are smaller than +2 ESI ions. The ion mobility resolving power with ESI (30-35) is comparable to that obtained using MALDI (35-40), which suggests that both sources produce sufficiently narrow ion pulses for the measurement to be predominately diffusion rather than gate pulse width limited.