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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
Abstract  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Why Mass Spectrometry? ,[object Object],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
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Quadruple Ion Trap  Mass Spectrometry Materials and Method
[object Object],[object Object],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
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.
Manipulation and Isolation of Ions ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],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
Isolation of Ions ,[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],Fragmentation of Ions
[object Object]
Collision Induced Dissociation (CID) ,[object Object],Le ·H + Le ·Na + Le ·K +
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.
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.
Leucine Enkephalin ,[object Object],[object Object],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
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”
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
[object Object],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
[object Object],Temperature of Drying Gas
Excitation “Window” Variation in excitation window (which changes the waveform for fragmentation of ions) result in changes in dissociation kinetics (different slopes).
[object Object],[object Object],[object Object]
Similar slope and good linearity for the dissociation on separate days after improved experimental method.
Dissociation Kinetics of  Leucine Enkephalin  Well-fit by 1 st  order dissociation kinetics. Leucine  enkephalin dissociates faster at higher voltages (k= -slope)
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).
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],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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Understanding Intrinsic Properties of Biological Molecules in Absence of Solvent: Effective Temperature of Ions in a QIT Mass Spectrometer

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