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by Matt Fisher
Outline
Brief introduction to Proteomics
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!
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
Electrospray Ionization
Electrospray Ionization
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
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
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
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
Intens. x10 5 3
0 0 5 CPV_B_30min_41_01_2213.d: TIC +All MS Intens. 300 250 624.1 200 150 100 50 0 200 Intens. x10 4 1.0 400 600 800 1000 1200 1400 1600 m/z 864.9 979.3 943.5 831.8 +MS, 12.0-12.9min #(1013-1091) 10 15 20 25 Time [min]
64567.2
0.8
62615.8
0.6
0.4
65664.7 0.2 67369.8 51835.7 0.0 50000 52000 54000 56000 58000 60000 62000 64000 66000 68000 m/z 53151.8 55110.0 57351.0 60727.7 69381.7
Intens. x10 5
ESI Spectra
CPV Bromelin_150 min Max Entropy Deconvolution
0 0 5 CPV_B_120min_42_01_2217.d: TIC +All MS Intens. 831.8 300 +MS, 12.0-12.6min #(1019-1073) 10 15 20 25 Time [min]
200
100
0 200 Intens. x10 4 1.25 400 600 800 1000 1200 1400 1600 m/z
62614.4 64567.6
65666.0 0.25 51836.7 0.00 50000 52000 54000 56000 58000 60000 62000 64000 66000 68000 m/z 57344.5 59841.6 61510.5 67372.1 53151.7 54833.0 69805.9
ESI Spectra
CPV Bromelin_23 hr Max. Entropy Deconvolution
Intens. x10 5
2.5
2.0
1.5 1.0
0.5
0.0 0 5 CPV_B_23hrs_43_01_2221.d: TIC +All MS Intens. 831.8 300 624.1 864.8 200 979.3 +MS, 12.0-12.6min #(1024-1070) 10 15 20 25 Time [min]
1099.6
100
0 200 Intens. x10 4 1.25 400 600 800 1000 1200 1400 1600 m/z
62615.8
1.00
64568.8
0.75
0.50
0.25 51835.0 0.00 50000 52000 54000 56000 58000 60000 57353.8 59839.4
61504.5
53148.6
54840.0
62000
64000
66000
68000
m/z
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.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