1. Mass Spectrometry of Proteins and Peptides Electrospray Ionization(ESI) and Matrix Assisted Laser Desorption Ionization(MALDI) by Matt Fisher

2. Outline Brief introduction to Proteomics How ESI and MALDI work Advantages/setbacks to each method ESI and MALDI in action

3. Proteomics To really understand biological processes, we need to understand how proteins function in and around cells since they are the functioning units. - Hanno Steen, director of the Proteomics Center at Children's Hospital Boston 30,000 genes code for 100,000 functional proteins in humans Extreme cases of a single gene coding for 1,000 proteins!

4. Advances in Protein and Peptide Analysis Such an enormous task requires every conceivable technique to analyze proteins The 2002 Nobel Prize for Chemistry was shared between John Fenn, and Koichi Tanaka for their development of ESI and MALDI, respectively

5. Electrospray Ionization Steps to Ionization Mix liquid sample with polar, volatile solvent Sample is put through a capillary with a fine tip on the end A high voltage(~2000 V) is applied to the tip of the capillary, charging the proteins and peptides in the solvent. (Multiply-charged species common in ESI) Mixture is pushed through to an evaporation chamber

6. Electrospray Ionization

7. Evaporation Chamber The mixture starts out in large droplets. The addition of nitrogen gas and heat begins to evaporate the solvent in the droplets The droplets get smaller and the charged molecules get closer together and repel, splitting into smaller droplets(Coulombic fission) The process continues until each droplet consists of a single molecule that is charged Molecules then enter a mass analyzer such as a time of flight(TOF) tube to measure m/z

8. Matrix Assisted Laser Desorption Ionization (MALDI) Liquid sample first mixed with an excess of matrix on a MALDI plate The liquid in the mixture evaporates in open air, with some of the sample incorporated into fine crystals of the matrix The matrix is a UV-absorbing species, usually of low molecular weight

9. MALDI MALDI plate is put into a high vacuum chamber and the laser is fired in bursts at crystals on the spots on the plate At the right wavelength the crystals are irradiated and sublime. Energy is transferred to the analytes which are now in the gas phase These protonated ions are accelerated into a mass analyzer such as a TOF tube

10. Advantages/Disadvantages to ESI Advantages High accuracy Large mass range Can be coupled with liquid chromatography to separate samples further Fast Auto run with sampler or direct injection Soft ionization Disadvantages Complicated spectra salts drown signal and take time to remove from the machine A high intensity peak can eclipse smaller intensity peaks Fine tuning work: flow rate, solvent/sample ratios, etc to get the analytes to ionize

11. Advantages/Disadvantages to MALDI Advantages Preferable for large molecules Quick, quick, quick! Sensitive to small amounts of sample Easy spectra Accurate Not affected by salts Soft ionization Disadvantages Fine tuning: spotting plate, getting good crystals, adjusting intensity of laser, finding crystals on plate with sample Low shot to shot reproducibility Short sample life

12. ESI Spectra CPV Bromelin_30min Max. Entropy Deconvolution Intact protein(VP2): 64,567.2 Da Digested protein(VP3): 62,315.8 Da

13. ESI Spectra CPV Bromelin_150 min Max Entropy Deconvolution Intact protein(VP2): 64,567.6 Da Digested protein(VP3): 62,614.4 Da

14. ESI Spectra CPV Bromelin_23 hr Max. Entropy Deconvolution Intact Protein(VP2): 64,568.8 Da Digested Protein(VP3): 62,615.8 Da

15. 2007_05_31 MALDI analysis of CPV reacted with Trypsin @ 45°C 5 min

16. Sources C.Nelson, E.Minkkinen, M. Bergkvist, K.Hoelzer, M. Fisher, B. Bothner, and C.Parrish (2008). Detecting Small Changes and Additional Peptides in the Canine Parvovirus Capsid Structure. J. Virol. 82: 10397-10407 http://www.magnet.fsu.edu/education/tutorials/tools/ionization_esi.html H. Steen, M. Mann (2004). The Abcs (and xyzs) of Peptide Sequencing. Nature Reviews Molecular Cell Biology 5, 699-711 http://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602 P0.htmlhttp://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602 P0.html F.Witzmann, J. Li (2002). Proteomics: Core Technologies and Applications in Physiology. American Journal of Physiology – Gastrointestinal and Liver Physiology. 10.1152 http://www.innovadyne.com/Assets%20Doc/MALDI%20spots%20Biomek%20plate.jpg