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Mass Spectrometry of Proteins and Peptides

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Mass Spectrometry of Proteins and Peptides Powered By Docstoc
					Mass Spectrometry of Proteins
and Peptides
Electrospray Ionization(ESI) and
Matrix Assisted Laser Desorption
Ionization(MALDI)


by Matt Fisher
Outline
• Brief introduction to Proteomics

• How ESI and MALDI work

• Advantages/setbacks to each method

• ESI and MALDI in action
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!
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
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
    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
    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                     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
Advantages/Disadvantages to MALDI
Advantages                         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
                 Intens.
                   x10 5

ESI Spectra           3




CPV                   2


Bromelin_30min
Max. Entropy          1



Deconvolution
                      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)
                   x10 4

                    1.0
                                Intact protein(VP2): 64,567.2 Da                                                                                      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
                Intens.
                  x10 5


ESI Spectra
                     3




CPV                  2



Bromelin_150
min Max
                     1




Entropy              0

Deconvolution             0                           5

                                 CPV_B_120min_42_01_2217.d: TIC +All MS
                                                                                     10                             15                        20                            25             Time [min]



                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
ESI Spectra
                 Intens.
                   x10 5


                    2.5

CPV                 2.0

Bromelin_23 hr      1.5




Max. Entropy
                    1.0




Deconvolution
                    0.5


                    0.0
                           0                           5                              10                             15                        20                            25             Time [min]

                                  CPV_B_23hrs_43_01_2221.d: TIC +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



                            Intact Protein(VP2): 64,568.8 Da
                  1.25                                                                                                              62615.8




                            Digested Protein(VP3): 62,615.8 Da
                  1.00

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

				
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