1. Mass Spectrometry of Proteins
and Peptides
Electrospray Ionization(ESI) and
Matrix Assisted Laser Desorption
Ionization(MALDI)
by Matt Fisher

2. Outline
• Brief introduction to Proteomics
• How ESI and MALDI work
• Advantages/setbacks to each method
• ESI and MALDI in action

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 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!

4. 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

5. 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

6. Electrospray Ionization

7. 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

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. 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

10. 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

11. 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

12. Intens.
x105
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)
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. Intens.
x10 5
ESI Spectra
3
CPV 2
Bromelin_150
1
min Max
Entropy 0
Deconvolution
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. ESI Spectra
Intens.
x10 5
2.5
CPV 2.0
Bromelin_23 hr 1.5
1.0
Max. Entropy 0.5
Deconvolution 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. 2007_05_31
MALDI analysis of
CPV reacted with
Trypsin @ 45°C 5
min

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