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- 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
- 2. Abstract <ul><li>The dissociation kinetics of a small biological molecule, leucine </li></ul><ul><li>enkephalin (LE), are examined using a Quadruple Ion Trap Mass </li></ul><ul><li>Spectrometer in order to determine the effect of activation waveform on </li></ul><ul><li>ion effective temperature (T eff ). The effective temperature is found to </li></ul><ul><li>have a linear relationship with the applied activation amplitude. The </li></ul><ul><li>dissociation kinetics of LE are found to be greatly affected by pressure </li></ul><ul><li>in the mass spectrometer, showing faster dissociation at lower </li></ul><ul><li>pressures. The effects of other experimental parameters, including the </li></ul><ul><li>temperature of the inlet capillary and sensitivity to the frequency of the </li></ul><ul><li>activation waveform, are also explored. Calibration of T eff as a function </li></ul><ul><li>of activation waveform will provide a way to obtain Arrhenius activation </li></ul><ul><li>parameters (activation energy and frequency factor) for other biological </li></ul><ul><li>molecules and lead to better understand of their intrinsic properties. </li></ul>
- 3. Why Mass Spectrometry? <ul><li>Many biological molecules have been studied previously in solvents, which makes it difficult to differentiate between the intrinsic properties of the molecules and how their properties are affected by the interactions with water. Mass spectrometry offers a unique opportunity to study the properties of biological molecules in the absence of solvent - in the gas phase. </li></ul>Esquire3000 1 Ubiquitin 2 1 Bruker Daltonics. “Esquire Series.” Retrieved from http://www.bdal.com/ 2 Jaremko, L., Jaremko M. “Ubiquitin.” Retrirved from http://stud.chem.uni.wroc.pl/users/lucek/JAREMKO/ubiquitin.htm Introduction
- 4. <ul><li>4 basic steps: </li></ul><ul><li>1. create gas-phrase ions (electrospray ionization) </li></ul><ul><li>2. isolate ions of desired m/z ratio </li></ul><ul><li>3. dissociate isolated ions </li></ul><ul><li>4. detect and measure the quantity of separated ions </li></ul><ul><li>m/z ratio: mass/charge </li></ul>Quadruple Ion Trap Mass Spectrometry Materials and Method
- 5. <ul><li>Entrance to Instrument: </li></ul><ul><li>Electrospray Ionization </li></ul>3 Adapted from Max-Planck-Institute Nanoscale Science Department. “Electrospray Ionization Deposition Source.” Retrived from http://www.fkf.mpg.de/kern/facilities/esi/esi.html Spray needle containing solvent and molecules or clusters Hot N 2 counter current gas capillary to first vacuum stage Charged and uncharged droplets formed by (electro-) spray Entrance plate at – kV pulls positive molecules Size reduction due to solvent evaporation and electrostatic repulsion Protonated ions
- 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.
- 7. Manipulation and Isolation of Ions <ul><li>Retention or ejection of ion with </li></ul><ul><li>different m/z by varying : 2 </li></ul><ul><li>- the amplitude of rf applied on the ring electrode </li></ul><ul><li>- a supplemental rf can be applied to end caps to resonantly excite ions </li></ul><ul><li>… which affect trapping parameter, q z (Figure 1) </li></ul><ul><li>if q z > q eject = 0.908, ions are ejected through end-cap electrode </li></ul>Figure 1. Region of stability within the ion trap. 3 4 Jonscher, K., Yates, J.”The Whys and Wherefores of Quadruple Ion Trap Mass Spectrometry.” Retrieved from http://www.abrf.org/ABRFNews/1996/September1996/sep96iontrap.html
- 8. Isolation of Ions <ul><li>Isolation of Precursor Ion of interest: </li></ul><ul><li>-apply specific frequency to excite and ejected all ions except ion of interest </li></ul><ul><li>-can leave a m/z “window” in which ions are not ejected </li></ul>
- 9. <ul><li>Theoretically, the “window” of frequencies applied isolate the ions are of the same amount for all m/z value(blue) </li></ul><ul><li>but… </li></ul><ul><li>empirically, the amount of frequency applied is not the same for all m/z within the window (red) </li></ul>Fragmentation of Ions
- 10. <ul><li>The excitation “window” (pink) must be centred so that the maximum amplitude of the excitation waveform is at the correct frequency to excite the ions (black). </li></ul>
- 11. Collision Induced Dissociation (CID) <ul><li>Molecules accelerate inside ion trap due to excitation waveform, collide with helium gas fragmentation </li></ul>Le ·H + Le ·Na + Le ·K +
- 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.
- 14. Leucine Enkephalin <ul><li>Tyr-Gly-Gly-Phe-Ala-Leu </li></ul><ul><li>Pentapeptide with morphine-like activities </li></ul>5RSC, “Leucine Enkephalin.’ Retrieved from http://www.chemsoc.org/chembytes/ezine/images/1998/bodfig2.gif 6 NIAID . “Leucine Enkephalin.” Retrieved from http://chemdb2.niaid.nih.gov/struct_search/images/structures/030267.gif
- 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
- 17. <ul><li>What could have affected the dissociation rates between the two days? </li></ul>Major Factors that affect dissociation or kinetics of ions: 1. method of analysis 2. temperature of counter-current gas 3. pressure of the helium gas 4. fragmentation waveform Method of Analysis Incorrect Method Correct Method Different number of avg. spectra for each activation time Same number of avg. spectra for each activation time Linearity improved
- 18. <ul><li>Changes within 100 °C have little effect on the linearity or the slope of the dissociation plot sp the random fluctuations of temperature during experiment (±1°C) should have insignificant effect. </li></ul>Temperature of Drying Gas
- 19. Excitation “Window” Variation in excitation window (which changes the waveform for fragmentation of ions) result in changes in dissociation kinetics (different slopes).
- 20. <ul><li>Very small changes in pressure affects the slope of the dissociation plots significantly. </li></ul><ul><li>The method of analysis and the pressure affect the dissociation kinetics of leucine enkephalin, so these parameters needs to be controlled for. </li></ul><ul><li>The centre of excitation window needs to adjusted for each experimental run to ensure maximum dissociation. </li></ul>
- 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).
- 24. <ul><li>The effective temperature of other peptides and proteins needs to be </li></ul><ul><li>determined using the same method to construct a calibration plot </li></ul><ul><li>(Bradykinin+ H) + and (Bradykinin + 2H) 2+ are currently being examined]. </li></ul><ul><li>The effective temperature of other proteins subject to collision activation in a </li></ul><ul><li>QIT can then be estimated using this plot. Thus, this can be further used to </li></ul><ul><li>estimate activation energies and Arrhenius A-factor for proteins and peptides </li></ul><ul><li>whose dissociation parameters are currently unknown. </li></ul>Implications enkephalin (Bradykinin+2H) 2 + Acknowledgements: Professor Jockusch, Geng Li, Matthew Forbes, Dr. Qunzhou Bian Reference Gabelica, V., Karas, M., De Pauw, E. (2003). Calibration of Ion Effective Temperature Achieved by Resonant Activation in a Quadrupole Ion Tap. Anal. Chem., 75 , 5152-5159. Goeringer, D.E., Asano, K.G., McLuckey, S.A. (1999). Ion Internal temperature and ion trap collisional activation: protonated leucine enkephalin. International Journal of Mass Spectrometry. 182/183, 275-288.

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