1. Electrospray ionization (ESI) mass spectrometry
Mass spectrometry Advanced Methods_Elviri

2. Electrospray ionization (ESI)
Electrosprayed
‘aerosol’
Liquid sample
Mass spectrometer
1-3 kV
needle potential ++ + ++ + + + + + + +
Gas-phase ions + + + ++ + + + ++ + + + +

3. ES SPECTRUM OF HORSE MYOGLOBIN MW 16
ES SPECTRUM OF HORSE MYOGLOBIN MW A) multiply charged ion distribution from shown at low resolution B) the 17+ charge state at a resolution of about showing the resolved isotope peaks

4. Charge state distribution obtained by ESI MS reflects protein conformation
Native
(water)
Denatured
(weak acid)

5. Denaturation of Azurin by acid as observed by ESI MS
Spectrum
deconvolution
14001 Da
13945 Da
13945 Da

6. The Nobel Prize in Chemistry 2002
"for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules”
John B. Fenn (USA): Elektrospray ionization
Koichi Tanaka (Japan): Soft laser desorption
”Electrospray ion source. Another variation on the free-jet theme” M. Yamashita and J.B. Fenn J. Phys. Chem. 4451, 88 (1984)
"Electrospray Ionization for Mass Spectrometry of Large Biomolecules" J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong and C. M. Whitehouse, Science 246, 64 (1989)

7. Operational definitions
Electrospray:
An electrical nebulization of liquid that results in the formation of charged micro droplets
Electrospray ionization:
The transfer and ionization of molecules from solution to gas phase by electrospray

8. From Liquid to Gas Phase
Dispersion of liquid into highly charged droplets
Formation of smaller off-spring droplets
Formation of highly solvated pseudo-ions
Ion desorption model (desorption of charged
ions from the surface of the droplet (active)
Charged residue model (passive)
Formation of desolvated ions
ESI produces a stable, continuous ion beam!

9. Electrical nebulization of liquid and electrochemical oxidation
Electrochemical oxidation in the metal capillary (needle) at the positive (+) high voltage terminal
Reduction at (-)
Electrons
Electrons
High voltage power supply
Ref [1]

10. Electrospray
Spray of charged microdroplets

11. Electrospray ion source
Pressure and
Electrical potential gradient
coaxial nebulization gas flow
Countercurrent gas flow to aid desolvation

12. The onset voltage (Von) for electrospray is a function of capillary diameter and surface tension ()
Taylor cone
d: capillary - electrode distance
rc: inner diameter of capillary
For rc = 0.1 mm og d = 40 mm:
CH3OH
CH3CN
(CH3)2SO
H2O
Von (kV)
2.2
2.5
3.0
4.0
g (N/m)
0.0226
0.030
0.043
0.073
Ref [1]

13. Electrospray: From solution to gas phase(I)
Electrical nebulization of liquid results in the formation of charged micro droplets.
Vaporization increases the charge density on the surface of the droplets. Electrostatic repulsion increases.
When the electrostatic repulsion exceeds the surface tension the droplet undergoes coulombic fission.
The formation of charged ions in the gas phase

14. A charged droplet undergoing coulombic fission
Parent droplet
Offspring droplets
Gomez et al., Phys. Fluids 6 (1994)

15. Solvent evaporation causes sequential fissions of charged droplets
The formation of smaller droplets increases the total surface area and this relieves the coulombic repulsion
t=462µs
N=51250
R=1.5µm
51250
0.945
Parent droplet after 1 fission
Vol. = 3.5 m3
Area = 11 m2
43560
0.939
t=74µs
43560
0.848
N: No. of charges
R: droplet radius
384
0.09
37026
0.844
t=70µs
~20 offspring droplets:
Total volumen = 0.06 m3
Total surface area = 2 m3
37026
0.761
326
0.08
31472
0.756
Asymmetrical fission process:
20 offspring droplets are formed carrying ~2% of the total mass and ~15% of the net charge.
t=39µs
278
0.07
278
0.03 2
0.003
Kebarle et al. Anal. Chem. 65 (1993) 972A

16. Ionization mechanisms
Two models for the formation of gas phase ions:
Ion Evaporation Theory (IET)
The most likely mechanism for the formation of low molecular gas phase ions (<200 Da).
Single Ion in Droplet Theory (SIDT) also known as Charged Residue Model (CRM)
The most likely mechanism for the formation of macromolecular gas phase ions.

17. Surface activity (hydrofobicity + charge) determines ionization efficiency
IA : Abundance of A in the mass spectrum
CA: conc. of A
kA: A’s responsefactor ~ ionization efficiency
Kebarle P., J. Mass Spectrom. 35, (2000)

18. Equimolar mixture of 6 tripeptides with different C-terminal residues
Ref. [2]

19. Hydrophilic substituents
Hydrophilic substituents such as phosphorylation or glycosylation reduce the ionization efficiency of proteins and peptides (in complex mixtures).

20. Flow rate and ionization efficiency
Ref. [3]

21. Mass determination of intact proteins (I)
When a protein is ionized with ESI a Gauss-like distribution of charge states is observed.
Positive ions are usually formed by protonation
Negative ions are usually formed by deprotonation
The conformation of the protein affects the width and mean value of the charge state distribution
Each peak in the charge state distribution in the mass spectrum corresponds to one charge state of the protein.
Assumptions:
Adjacent peaks differ by a net charge of one
The charge results from attachment or detachment of cations (usually protons)

22. ESI spectra of disulfide-intact og disulfide-reduced lysozyme
Konermann et al.
J. Am. Soc. Mass Spectrom.
1998, 9,

23. ES SPECTRUM OF HORSE MYOGLOBIN MW 16
ES SPECTRUM OF HORSE MYOGLOBIN MW A) multiply charged ion distribution from shown at low resolution B) the 17+ charge state at a resolution of about showing the resolved isotope peaks

24. Mass (M) determination of proteins (II)
(1)
(2)
(1) og (2): two equations with two unknowns: M og n (n : No. Of protons)
Apomyoglobin ( Da)

25. Gold-coated borosilicate glass
Nano-Electrospray
Gold-coated borosilicate glass

26. Nanoelectrospray
Flow rates 50 to 200 nL/min

27. Features of NanoES
Flow rates of nL/min ( min analysis time)
Sample volumes down to 300 nL
Near 100 % sample utilization
Minimal instrument contamination
Zero sample cross contamination
Spray from 0% to 100% aqueous solvents

28. NanoES vs. conventional ES (2)

29. NanoES vs. conventional ES (3)

30. The formation of heterodimers3+
AAAAAA 3+ 3+ 3+
AAAAAA 3+
AAAAAA
AAAAAA 2+ 2+
AAAAAA
AAAAAA
AAAAAA
AAAAAA 3+
AAAAAA
AAAAAA 3+ 2+
Cl-eremomycin 3+
AAAAAA 3+ 2+
Eremomycin
AAAAAA
AAAAAA
Staroske, T.; O’Brien, D.P.; Jørgensen, T.J.D.; Roepstorff, P.; Williams, D.H.; Heck, A.J.R. Chem. Eur. J. 2000, 6,

31. ESI-MS of intact virus Bacteriophage MS2
+121
Molecular mass ( 25000) Da
Virus maintains its infectivity! (Ref. [6])
Ref [5]

32. Noncovalent complexes in the gas phase
E.coli GroEL under non-denaturing conditions
Mcalc.= 800,770 Da
Mexp. = 803,700 ±100
14mer
Nano-ESI, 10µM in aqueous
amm. acetate (100mM, pH7)

33. Noncovalent complexes in the gas phase
Pressures Standard Noncovalents
p mbar mbar
p mbar mbar
p mbar mbar

34. Noncovalent complexes in the gas phase
p2= 1.6·10-2 mbar
E.coli GroEL mer (≈800 kDa)
Collisional cooling
p2= 1.3·10-2 mbar
p2= 1.0·10-2 mbar
p2= 0.7·10-2 mbar
p2= 0.4·10-2 mbar

35. The ribosome 810,208 +/-963 Da 1,516,052 30S +/-1986 Da ??? 2,325,463

36. Referencer
“Electrospray Ionization Mass Spectrometry” Ed. R.B. Cole, John Wiley & Sons, 1997
Cech et al. “Practical implications of some recent studies in electrospray ionization fundamentals” Mass Spectrometry Reviews, 2001, 20,
Covey et al. “Nanospray Electrospray Ionization Development” i Applied Electrospray Mass Spectrometry, Ed. N. Birendra et al., Marcel Dekker, 2002
Tito et al. J. Am. Chem. Soc. 2000, 122,
Siuzdak, G. et al. Chem Biol. 1996, 3, 45-48