Mass Spec of Proteins.ppt

7/29/2019 Mass Spec of Proteins.ppt

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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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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


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


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

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Mass Spec of Proteins.ppt