Mass Spectrometry of Proteinsand PeptidesElectrospray Ionization(ESI) andMatrix Assisted Laser DesorptionIonization(MALDI)...
Outline• Brief introduction to Proteomics• How ESI and MALDI work• Advantages/setbacks to each method• ESI and MALDI in ac...
Proteomics• “To really understand biological processes, we  need to understand how proteins function in and  around cells ...
Advances in Protein and PeptideAnalysis• Such an enormous task requires every  conceivable technique to analyze proteins• ...
Electrospray IonizationSteps to Ionization• Mix liquid sample with polar, volatile solvent• Sample is put through a capill...
Electrospray Ionization
Electrospray IonizationEvaporation Chamber• The mixture starts out in “large” droplets. The addition  of nitrogen gas and ...
Matrix Assisted Laser Desorption    Ionization (MALDI)•Liquid sample first mixed with an excess of  matrix on a MALDI plat...
MALDI• MALDI plate is put into a high vacuum chamber and the laser is fired in bursts atcrystals on the spots on the plate...
Advantages/Disadvantages to ESIAdvantages                     Disadvantages• High accuracy                • Complicated sp...
Advantages/Disadvantages to MALDIAdvantages                         Disadvantages• Preferable for large molecules   • Fine...
Intens.                   x105ESI Spectra           3CPV                   2Bromelin_30minMax. Entropy          1Deconvolu...
Intens.                  x10 5ESI Spectra                     3CPV                  2Bromelin_150                     1min...
ESI Spectra                 Intens.                   x10 5                    2.5CPV                 2.0Bromelin_23 hr   ...
2007_05_31MALDI analysis ofCPV reacted withTrypsin @ 45°C    5min
Sources• C.Nelson, E.Minkkinen, M. Bergkvist, K.Hoelzer, M. Fisher, B. Bothner, and C.Parrish  (2008). “Detecting Small Ch...
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Mass spec of proteins

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Mass spec of proteins

  1. 1. Mass Spectrometry of Proteinsand PeptidesElectrospray Ionization(ESI) andMatrix Assisted Laser DesorptionIonization(MALDI)by Matt Fisher
  2. 2. Outline• Brief introduction to Proteomics• How ESI and MALDI work• Advantages/setbacks to each method• ESI and MALDI in action
  3. 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 Childrens 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. 4. Advances in Protein and PeptideAnalysis• 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. 5. Electrospray IonizationSteps 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. 6. Electrospray Ionization
  7. 7. Electrospray IonizationEvaporation 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. 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. 9. MALDI• MALDI plate is put into a high vacuum chamber and the laser is fired in bursts atcrystals on the spots on the plate• At the right wavelength the crystals are irradiated and sublime. Energy is transferredto the analytes which are now in the gas phase• These protonated ions are accelerated into a mass analyzer such as a TOF tube
  10. 10. Advantages/Disadvantages to ESIAdvantages Disadvantages• High accuracy • Complicated spectra• Large mass range • salts drown signal and take• Can be coupled with liquid time to remove from the chromatography to separate machine samples further • A high intensity peak can• Fast eclipse smaller intensity peaks• Auto run with sampler or • Fine tuning work: flow rate, direct injection solvent/sample ratios, etc to• Soft ionization get the analytes to ionize
  11. 11. Advantages/Disadvantages to MALDIAdvantages Disadvantages• Preferable for large molecules • Fine tuning: spotting plate,• Quick, quick, quick! getting good crystals, adjusting• Sensitive to small amounts of intensity of laser, finding sample crystals on plate with sample• Easy spectra • Low shot to shot• Accurate reproducibility• Not affected by salts • Short sample life• Soft ionization
  12. 12. Intens. x105ESI Spectra 3CPV 2Bromelin_30minMax. Entropy 1Deconvolution 0 0 5 10 15 20 25 Time [min] CPV_B_30min_41_01_2213.d: TIC +All MS Intens. +MS, 12.0-12.9min #(1013-1091) 300 831.8 250 624.1 979.3 864.9 200 943.5 150 100 50 0 200 400 600 800 1000 1200 1400 1600 m/z Intens. +MS, 12.0-12.9min #(1013-1091), Deconvoluted (maximum entropy) x104 Intact protein(VP2): 64,567.2 Da 1.0 64567.2 0.8 Digested protein(VP3): 62,315.8 Da 62615.8 0.6 0.4 65664.7 0.2 67369.8 69381.7 51835.7 57351.0 60727.7 53151.8 55110.0 0.0 50000 52000 54000 56000 58000 60000 62000 64000 66000 68000 m/z
  13. 13. Intens. x10 5ESI Spectra 3CPV 2Bromelin_150 1min MaxEntropy 0Deconvolution 0 5 10 15 20 25 Time [min] CPV_B_120min_42_01_2217.d: TIC +All MS Intens. +MS, 12.0-12.6min #(1019-1073) 831.8 300 624.1 979.3 864.9 200 100 0 200 400 600 800 1000 1200 1400 1600 m/z Intens. +MS, 12.0-12.6min #(1019-1073), Deconvoluted (maximum entropy) x10 4 1.25 62614.4 Intact protein(VP2): 64,567.6 Da 64567.6 1.00 Digested protein(VP3): 62,614.4 Da 0.75 0.50 65666.0 0.25 61510.5 67372.1 69805.9 51836.7 57344.5 59841.6 53151.7 54833.0 0.00 50000 52000 54000 56000 58000 60000 62000 64000 66000 68000 m/z
  14. 14. ESI Spectra Intens. x10 5 2.5CPV 2.0Bromelin_23 hr 1.5 1.0Max. Entropy 0.5Deconvolution 0.0 0 5 10 15 20 25 Time [min] CPV_B_23hrs_43_01_2221.d: T IC +All MS Intens. +MS, 12.0-12.6min #(1024-1070) 831.8 300 624.1 864.8 979.3 200 1099.6 100 0 200 400 600 800 1000 1200 1400 1600 m/z Intens. +MS, 12.0-12.6min #(1024-1070), Deconvoluted (maximum entropy) x10 4 1.25 Intact Protein(VP2): 64,568.8 Da 62615.8 1.00 Digested Protein(VP3): 62,615.8 Da 64568.8 0.75 0.50 65663.8 0.25 61504.5 67375.2 69389.9 51835.0 57353.8 59839.4 53148.6 54840.0 0.00 50000 52000 54000 56000 58000 60000 62000 64000 66000 68000 m/z
  15. 15. 2007_05_31MALDI analysis ofCPV reacted withTrypsin @ 45°C 5min
  16. 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 Abc’s (and xyz’s) of Peptide Sequencing”. Nature Reviews Molecular Cell Biology 5, 699-711• http://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.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

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