mass spec of proteins.ppt
TRANSCRIPT
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Mass Spectrometry of Proteins
and PeptidesElectrospray Ionization(ESI) andMatrix Assisted Laser Desorption
Ionization(MALDI)
by Matt Fisher
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Outline
Brief introduction to Proteomics
How ESI and MALDI work
Advantages/setbacks to each method
ESI and MALDI in action
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Proteomics
To really understand biological processes, weneed to understand how proteins function in andaround cells since they are the functioningunits. - Hanno Steen, director of the ProteomicsCenter at Children's Hospital Boston
30,000 genes code for 100,000 functionalproteins in humans
Extreme cases of a single gene coding for 1,000proteins!
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Advances in Protein and PeptideAnalysis
Such an enormous task requires everyconceivable technique to analyze proteins
The 2002 Nobel Prize for Chemistry was shared
between John Fenn, and Koichi Tanaka for theirdevelopment of ESI and MALDI, respectively
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Electrospray Ionization
Steps to Ionization
Mix liquid sample with polar, volatile solvent
Sample is put through a capillary with a fine tip on theend
A high voltage(~2000 V) is applied to the tip of the
capillary, charging the proteins and peptides in thesolvent. (Multiply-charged species common in ESI)
Mixture is pushed through to an evaporation chamber
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Electrospray Ionization
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Electrospray Ionization
Evaporation Chamber
The mixture starts out in large droplets. The additionof nitrogen gas and heat begins to evaporate the solventin the droplets
The droplets get smaller and the charged molecules getcloser together and repel, splitting into smallerdroplets(Coulombic fission)
The process continues until each droplet consists of asingle molecule that is charged
Molecules then enter a mass analyzer such as a time of
flight(TOF) tube to measure m/z
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Matrix Assisted Laser DesorptionIonization (MALDI)
Liquid sample first mixed with an excess ofmatrix on a MALDI plate
The liquid in the mixture evaporates in open air,with some of the sample incorporated into finecrystals of the matrix
The matrix is a UV-absorbing species, usually of
low molecular weight
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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
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Advantages/Disadvantages to ESIAdvantages
High accuracy Large mass range
Can be coupled with liquidchromatography to separatesamples further
Fast Auto run with sampler or
direct injection
Soft ionization
Disadvantages
Complicated spectra salts drown signal and take
time to remove from themachine
A high intensity peak can
eclipse smaller intensity peaks Fine tuning work: flow rate,
solvent/sample ratios, etc toget the analytes to ionize
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Advantages/Disadvantages to MALDI
Advantages
Preferable for large molecules Quick, quick, quick!
Sensitive to small amounts ofsample
Easy spectra
Accurate Not affected by salts
Soft ionization
Disadvantages
Fine tuning: spotting plate,getting good crystals, adjustingintensity of laser, findingcrystals on plate with sample
Low shot to shot
reproducibility Short sample life
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0 5 10 15 20 25 Time [min]
0
1
2
3
5x10
Intens.
CPV_B_30min_41_01_2213.d: T IC +All MS
624.1
831.8
864.9943.5
979.3
+MS, 12.0-12.9min #(1013-1091)
0
50
100
150
200
250
300
Intens.
200 400 600 800 1000 1200 1400 1600 m/z
51835.7 53151.8 55110.057351.0 60727.7
62615.8
64567.2
65664.7
67369.8
69381.7
+MS, 12.0-12.9min #(1013-1091), Deconvoluted (maximum entropy)
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.
50000 52000 54000 56000 58000 60000 62000 64000 66000 68000 m/z
ESI Spectra
CPV
Bromelin_30minMax. EntropyDeconvolution
Intact protein(VP2): 64,567.2 Da
Digested protein(VP3): 62,315.8 Da
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0 5 10 15 20 25 Time [min]
0
1
2
3
5x10
Intens.
CPV_B_120min_42_01_2217.d: TIC +All MS
624.1
831.8
864.9
979.3
+MS, 12.0-12.6min #(1019-1073)
0
100
200
300
Intens.
200 400 600 800 1000 1200 1400 1600 m/z
51836.7 53151.7 54833.057344.5 59841.6
61510.5
62614.4
64567.6
65666.0
67372.1 69805.9
+MS, 12.0-12.6min #(1019-1073), Deconvoluted (maximum entropy)
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.
50000 52000 54000 56000 58000 60000 62000 64000 66000 68000 m/z
ESI Spectra
CPVBromelin_150min MaxEntropyDeconvolution
Intact protein(VP2): 64,567.6 Da
Digested protein(VP3): 62,614.4 Da
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0 5 10 15 20 25 Time [min]
0.0
0.5
1.0
1.5
2.0
2.5
5x10
Intens.
CPV_B_23hrs_43_01_2221.d: TIC +All MS
624.1
831.8
864.8 979.3
1099.6
+MS, 12.0-12.6min #(1024-1070)
0
100
200
300
Intens.
200 400 600 800 1000 1200 1400 1600 m/z
51835.053148.6
54840.0
57353.8 59839.4
61504.5
62615.8
64568.8
65663.8
67375.269389.9
+MS, 12.0-12.6min #(1024-1070), Deconvoluted (maximum entropy)
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.
50000 52000 54000 56000 58000 60000 62000 64000 66000 68000 m/z
ESI Spectra
CPVBromelin_23 hr
Max. EntropyDeconvolution
Intact Protein(VP2): 64,568.8 DaDigested Protein(VP3): 62,615.8 Da
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2007_05_31
MALDI analysis ofCPV reacted withTrypsin @ 45C 5
min
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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 ParvovirusCapsid 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 ReviewsMolecular 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 inPhysiology. American Journal of Physiology Gastrointestinal and Liver Physiology.10.1152
http://www.innovadyne.com/Assets%20Doc/MALDI%20spots%20Biomek%20plate.jpg
http://www.magnet.fsu.edu/education/tutorials/tools/ionization_esi.htmlhttp://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.htmlhttp://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.htmlhttp://www.innovadyne.com/Assets%20Doc/MALDI%20spots%20Biomek%20plate.jpghttp://www.innovadyne.com/Assets%20Doc/MALDI%20spots%20Biomek%20plate.jpghttp://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.htmlhttp://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.htmlhttp://www.magnet.fsu.edu/education/tutorials/tools/ionization_esi.html