The dissociation kinetics of a small biological molecule, leucine enkephalin (LE), are examined using a Quadruple Ion Trap Mass Spectrometer in order to determine the effect of activation waveform on ion effective temperature (Teff). The effective temperature is found to have a linear relationship with the applied activation amplitude. The dissociation kinetics of LE are found to be greatly affected by pressure in the mass spectrometer, showing faster dissociation at lower pressures. The effects of other experimental parameters, including the temperature of the inlet capillary and sensitivity to the frequency of the activation waveform, are also explored. Calibration of Teff as a function of activation waveform will provide a way to obtain Arrhenius activation parameters (activation energy and frequency factor) for other biological molecules and lead to better understand of their intrinsic properties.

1. Understanding Intrinsic Properties of Biological Molecules in Absence of Solvent: Effective Temperature of Ions in a QIT Mass Spectrometer Jenny Pui Shan Wong Supervisor: Professor R. Jockusch

6. Quadruple Ion Trap The Lissajous or “figure-eight” trajectory of a single ion (blue) and the projections of the trajectory (red) at the centre of the ion trap.

12. Dissociation of Leucine Enkephalin LE = leucine enkephalin LE* = excited leucine enkephalin = rate constant Pseudo First-order dissociation : activation depends on collisions with He, P He is held constant ( 1 , -1 >> 2 ) Rate constant, = Ae -Ea/R T (can be rearranged to) T eff Arrhenius (A), Activation energy (Ea), and k known from previous experiments From the Arrhenius Equation: The Distribution of ion internal energy is approximately Boltzmann.

13. Effective Temperature “ Temperature” is the statistical distribution of molecular kinetic energy (the Boltzmann Distribution). The population of the LE and LE* is not exactly at a “temperature” but the distribution of ion internal energy is similar to Boltzmann distribution, hence, effective temperature.

15. Experiment 1. Solution: 5ug/ml of leucine enkephalin (50/50 acetonitrile/0.1% acetic acid). 2. Isolate Parent ion (556.2Da) and fragment using various voltages (0.18V – 0.23V). 3. Plot intensity ratio of parent ion: parent ion+fragments vs. activation time A line of best fit is generated with the resulting regression analysis r 2 -value to determine linearity and the slope of the line gives dissociation constant. Day “1”

16. Day “2” Comparison of the dissociation plots between the two days -improved linearity (want r 2 ~ 0.99) Problem: -the slope of the plots are very different (should be within a narrow range) Results and Discussion

19. Excitation “Window” Variation in excitation window (which changes the waveform for fragmentation of ions) result in changes in dissociation kinetics (different slopes).

21. Similar slope and good linearity for the dissociation on separate days after improved experimental method.

22. Dissociation Kinetics of Leucine Enkephalin Well-fit by 1 st order dissociation kinetics. Leucine enkephalin dissociates faster at higher voltages (k= -slope)

23. Effective Temperature Plot Data from dissociation plots at different voltages plotted as an effective temperature plot where slope is –Ea/R T eff depends linearly on activation amplitude. (This result is in contrast To a model which predicted a quadratic relationship (Goeringer et al). This result agrees with other experimental results obtained using different kinds of ion traps (Gabelica, et al).