Recently reported results (Konn et al. [14]) on the collisional cooling of atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) and nano-electrospray ionization (nano-ESI) generated ions in a quadrupole ion trap mass spectrometer (QITMS) are inconsistent with measured collisional cooling rates. The work reported here presents a re-examination of those previous results. Collision induced dissociation (CID) has been used to probe various properties of ions contained in a QITMS. It is shown experimentally that when trapping large numbers of ions, an effective dc trapping voltage is induced that varies with changes in the size of the ion cloud. A decrease in the resonant frequency for maximum CID efficiency is observed as the cool time between parent ion isolation and CID is increased. Ion trajectories in a QITMS are simulated to demonstrate how ion density changes over the course of parent ion isolation. The effect of space charge on ion motion is simulated, and Fourier transformations of ion axial motion plus simple calculations corroborate the experimentally observed transient frequency shifts. The relative stability of ions formed by AP-MALDI and nano-ESI is compared under low charge density conditions. These data show that the ions have reached equilibrium internal energy and, thus, that differences in dissociation onsets and "50% fragmentation efficiency points" between the ionization mechanisms are due to the formation of distinct ion conformations as previously shown in reference [28] A n understanding of ion internal energy is of primary interest to researchers studying the structures of gas-phase ions. How fast an ion will dissociate, which mechanisms will be involved, and how many products will be observed all have a large dependence on the amount of internal energy in the ion and the experimental time window for dissociation [1][2][3][4]. If the internal energy of an ion is distributed statistically among its bonds, RRKM theory can determine the dissociation kinetics of the various dissociation pathways, and mass spectra or tandem mass spectra (MS/MS) could be predicted [5][6][7]. MS/MS spectra are related to ion structure on a fundamental level because the vibrational bond frequencies, the ground state energy, and the critical energy of dissociation are all determined on some level by the three dimensional conformation of the ion.Because MS/MS spectra have a dependence on structure and internal energy, it is important to understand the various factors that determine ion internal energy and how its magnitude might change over the course of an experiment. A typical experiment using a quadrupole ion trap mass spectrometer (QITMS) lasts several hundred ms, during which time a large number of variables can affect an ion population's distribution of internal energy. The theoretical calculation of ion internal energy in a QITMS is a complex quantum chemical calculation in which many factors must be considered, such as the kinetic energy of the ion, the vibrational energy states of the io...