Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 956 High Rate Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry BY YOURI TSYBIN ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2004 !" !##$ %#&%' ( ) ( ( *) )+ ,) - . )+ , /+ !##$+ ) 0 . 1 , ( 2 1 0 3 4 + 54 ( 6 (7 ( 89 6 8 6 :+ + ;'<+ << + + 24=> ;%8''$8';%?8@ ) ) ) ) ) ( ) ( A + = ) ( ) ) B + - ( ) B ( ) + 2 ) ) ) ( ) C ) ) B 8 8 ) C ( + ,) ( ) ( ) ) ) ( ( 3 (( )) (( ) B+ ,) ( ) ) B - ( -8 9 ) ) + ) ( ) - 9 ( - ( ) B )) ( ) + 4 ( - ) ) ) ( D ) ) C + ) )) - 8 ) B ) )A ) 8 + ,) ( ) ) B ) 8 - B ( !; 6 B ( - ) ( ( 5 ( : - ) C ( B ) ) ) + ) ( ( C ( ( - ) 8 ( + ( 8 ( ) B ) ) ) ! " # $ # % & # '( )*+# # ,-)./. # E / , !##$ 244> %%#$8!"!@ 24=> ;%8''$8';%?8@ & &&& 8$%"< 5) &DD +6+D F G & &&& 8$%"<: To my parents Publications included in this thesis This thesis is a summary of the following publications, referred to in the text by their Roman numerals. I. Peptide and Protein Characterization by High Rate Electron Cap- ture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Tsybin Y.O., Ramström M., Witt M., Baykut G., Håkansson P. Journal of Mass Spectrometry, submitted II. Improved Low-Energy Electron Injection Systems for High Rate Electron Capture Dissociation in Fourier Transform Ion Cyclo- tron Resonance Mass Spectrometry Tsybin Y.O., Håkansson P., Budnik B.A., Haselmann K.F., Kjeldsen F., Gorshkov M., Zubarev R.A. Rapid Communications in Mass Spectrometry 15, 2001, 1849 III. Combined Infrared Multiphoton Dissociation and Electron Cap- ture Dissociation with a Hollow Electron Beam in Fourier Trans- form Ion Cyclotron Resonance Mass Spectrometry Tsybin Y.O., Witt M., Kjeldsen F., Baykut G., Håkansson P. Rapid Communications in Mass Spectrometry 17, 2003, 1759 IV. Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry in the Electron Energy Range 0- 50 eV Tsybin Y.O., Witt M., Baykut G., Håkansson P. Rapid Communications in Mass Spectrometry, submitted V. Liquid Chromatography and Electron Capture Dissociation in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Palmblad M., Tsybin Y.O., Ramström M., Bergquist J., Håkansson P. Rapid Communications in Mass Spectrometry 16, 2002, 988 VI. Capillary Electrophoresis and Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Peptide Mixture and Protein Digest Analysis Tsybin Y.O., Wetterhall M., Håkansson P., Markides K.E., Bergquist J. European Journal of Mass Spectrometry 8, 2002, 389 © John Wiley & Sons Inc. (II, III, V) © IM Publications (VI) My contribution to papers I-VI: I have been responsible for planning and carrying out the experimental work, analysis of results and writing of papers I-IV, and VI. Paper II is a joint re- port with the Mass Spectrometry Laboratory, Odense University, Denmark. The experiments on which papers III and IV are based were partly made in collaboration with Bruker Daltonics, Bremen, Germany. Paper V was mostly written by M. Palmblad, but I took an active part in experiments, data analy- sis, and discussions. Paper VI was made in a collaboration with the Analyti- cal Chemistry Department, Uppsala University, Sweden. Publications not included in this thesis: 1. Shifted-basis technique improves accuracy of peak position determina- tion in Fourier transform mass spectrometry. Savitski M.M., Ivonin I.A., Nielsen M.L., Tsybin Y.O., Håkansson P., and Zubarev R.A. Journal of the American Society for Mass Spectrometry 2004, in press 2. A novel mass spectrometric approach to the analysis of hormonal pep- tides in extracts of mouse pancreatic islets. Ramström M., Hagman C., Tsybin Y.O., Markides K., Håkansson P., Salehi A., Lundquist I., Håkanson R., Bergquist J. European Journal of Biochemistry 270 (15), 2003, 3146 3. Micro-liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry - a useful tool for peptide identification. Ramström M., Palmblad M., Amirkhani A., Tsybin Y.O., Markides K.E., Håkansson P., and Bergquist J. Acta Biochim. Pol. 48, 2001, 1101 4. Ionization energies of singly and multiply protonated polypeptides ob- tained by tandem ionization in Fourier transform mass spectrometers Budnik B.A., Tsybin Y.O., Håkansson P., Zubarev R.A. Journal of Mass Spectrometry 37, 2002, 1141. 5. A theoretical investigation of the kinetic energy of ions trapped in a ra- dio-frequency hexapole ion trap McDonnell L.A., Giannakopulos A.E., Tsybin Y.O., Håkansson P., Der- rick P.J. European Journal of Mass Spectrometry 8, 2002, 181. 6. Electron Capture Dissociation with a Hollow Electron Beam in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Tsybin Y.O., Håkansson P., Weiss G., Witt M., Baykut G. Manuscript 7. Ion desorption under impact of surface current on metallic and semicon- ductor samples in vacuum. Tsybin O.Y., Tsybin Y.O., Kravetz N.M. Izvestija RAS, serija fizicheskaja 66, 2002, 1204. Conference presentations (invited talks) that included parts of the work presented in the thesis: x Optimization of the electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry. The 7th European FT ICR MS Workshop. Konstanz, Germany, 2004 x Electron injection systems in ECD FT ICR MS. The 1st International Symposium on ECD of Biomolecules and Related Phenomena. Stock- holm, Sweden, 2003 x Peptide and protein characterization by high rate electron capture disso- ciation mass spectrometry. The 16th International Mass Spectrometry Conference, Edinburgh, Scotland, 2003 x Development and application of combined IRMPD and ECD with a hollow electron beam in FT ICR MS. The 51st ASMS Conference on Mass Spectrometry and Allied Topics, Montreal, Canada, 2003 x Electron capture dissociation combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry for protein and protein mixture characterization. The 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, USA, 2002 x Large emitting area electron gun for electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry. The 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, USA, 2001 Contents 1. Introduction …………………………………………………….. 9 2. Peptide and protein mass spectrometry …………..…………..... 12 2.1. Peptides and proteins…………………………………......... 12 2.2. Principles of mass spectrometry……………….................... 13 2.3. Tandem mass spectrometry………………………………… 16 2.4. Top-down and bottom-up protein characterization…............ 21 2.5. Challenges in tandem mass spectrometry…………….......... 22 3. Experimental methods …………………….…………................ 24 3.1 Overview…………………………………………................ 24 3.2 Electrospray ionization…………………………………….. 25 3.3 Fourier transform mass spectrometry……………..……….. 26 3.4 Ion collisions with neutral molecules……………................ 32 3.5 Infrared laser beam injection system ……………................ 33 3.6 Coupling of separation techniques………………………..... 33 4. Optimization of electron injection systems for electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry ……………………………………………………. 35 4.1. Analysis of electron beam parameters…………………..... 35 4.2. Pencil electron beam injection system…………………..... 38 4.3. Hollow electron beam injection system…………………... 38 4.4. Properties of improved electron beams…………………… 40 4.5. Application in time-limited experiments………………..... 43 5. Development of high rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry………..... 44 5.1 High rate electron capture dissociation.…………………... 44 5.2 Top-down peptide sequencing…………………………..... 45 5.3 Top-down protein sequencing…………………………..... 48 5.4 Bottom-up protein analysis………………….…………..... 50 5.5 Post-translational modification analysis………….............. 53 5.6 Secondary fragmentation………………………………..... 54 5.7 Analysis of peptides with disulfide bonds………………... 55 6. Concluding remarks .................................................................... 57 7. Acknowledgements ..................................................................... 59 8. Snabb fragmenteringsmetod genom elektroninfångning i Fouriertransform-joncyklotronresonans-masspektrometri ……. 60 9. References ................................................................................... 62 Abbreviations AI ECD Activated ion electron capture dissociation BIRD Blackbody infrared radiative dissociation CE Capillary electrophoresis CE/MS Capillary electrophoresis/mass spectrometry CID Collision induced dissociation Da Dalton (molecular mass unit) DR Dissociative recombination ECD Electron capture dissociation EDD Electron detachment dissociation EED Electronic excitation dissociation EI Electron (impact) ionization ESI Electrospray ionization