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Electrospray ionization (ESI)mass spectrometry
Mass spectrometry Advanced Methods_Elviri
Electrospray ionization (ESI)
Liquid sample
1-3 kVneedle potential
++
++
+
+
+++
+
+++
+
+++
Electrosprayed‘aerosol’
+
++
++
+
+
++ Gas-phase ions
Mass spectrometer
ES SPECTRUM OF HORSE MYOGLOBIN MW 16.951 A) multiply charged ion distribution from +12-24 shown at low resolution B) the 17+ charge state at a resolution of about 15000 showing the resolved isotope peaks
Charge state distribution obtained by ESI MS reflects protein conformation
rekombinant CytC4 HC151 i vand pos cone 20
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000m/z0
100
%
m06341ams 365 (7.124) Cm (18:403) TOF MS ES+ 1.02e41902.08
1743.61
1145.04
1030.571288.02 1609.51
2092.08
1907.72C10
2095.35
HC151 i 5% FA pos cone 20
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000m/z0
100
%
m06345ams 206 (4.233) Cm (2:469) TOF MS ES+ 3.93e3839.23
805.23
775.46
747.80
722.05
697.99
874.18912.14
953.56
1048.82 1233.74
1398.10
2096.641497.81 1906.13
1613.09 1901.93 2105.74
Native(water)
Denatured (weak acid)
Denaturation of Azurin by acidas observed by ESI MS
Azurin positiv
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000m/z0
100
%
m06105ams 67 (1.437) Cm (33:134) TOF MS ES+ 2.34e4A9
1556.49
A101400.89
A81751.11
1560.48
2001.401753.67
Azurin i 1% FA
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000m/z0
100
%
m06169ams 176 (3.525) Cm (128:178) TOF MS ES+ 3.78e31557.26
A15930.54
A16872.50
A17821.23
A18775.63
A14996.93
A131073.63 1550.37
1163.001395.40
1751.81
1744.111756.09
Azurin i 4,5% FA
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000m/z0
100
%
m06166ams 1 (0.020) Cm (1:111) TOF MS ES+ 8.13e3A15
930.54
A16872.46
A17821.16
A18775.66
A19734.86
458.18
A14996.93
A131073.51
1751.71A9
1550.28A12
1162.92A11
1268.53
1557.131756.14
Azurin positiv
13000 13200 13400 13600 13800 14000 14200 14400 14600 14800 15000mass0
100
%
m06105ams 67 (1.437) M1 [Ev-45152,It21] (Gs,0.750,1384:2369,1.00,L33,R33); Cm (33:134) TOF MS ES+ 1.10e5A
14001.00
13938.00
14005.00
14035.00
Azurin i 1% FA
13000 13200 13400 13600 13800 14000 14200 14400 14600 14800 15000mass0
100
%
m06169ams 176 (3.525) M1 [Ev-40243,It25] (Gs,0.750,745:1770,1.00,L33,R33); Cm (128:178) TOF MS ES+ 8.73e4A
13945.00
14006.00
Azurin i 4,5% FA
13000 13200 13400 13600 13800 14000 14200 14400 14600 14800 15000mass0
100
%
m06166ams 1 (0.020) M1 [Ev-56363,It29] (Gs,0.750,625:1869,1.00,L33,R33); Cm (1:111) TOF MS ES+ 2.30e5A
13945.00
13976.00
Spectrum deconvolution
13945 Da
13945 Da
14001 Da
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)
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
From Liquid to Gas Phase
Dispersion of liquid into highly charged droplets
Formation of smaller off-spring droplets
Formation of highly solvated pseudo-ionsIon 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!
Electrical nebulization of liquid and electrochemical oxidation
Electrochemical oxidation in the metal capillary (needle) at the positive (+) high voltage terminal
Electrons
Reduction at (-)
Electrons
High voltage power supplyRef [1]
Electrospray
Spray of charged microdroplets
Electrospray ion source
Pressure and Electrical potential gradient
coaxial nebulization gas flow
Countercurrent gas flow to aid desolvation
The onset voltage (Von) for electrospray is a function of capillary diameter and surface tension
()
CH3OH CH3CN (CH3)2SO H2O
Von (kV) 2.2 2.5 3.0 4.0
(N/m) 0.0226 0.030 0.043 0.073
Taylor cone
ccon r
drV
4ln102 5
d: capillary - electrode distance rc: inner diameter of capillaryFor rc = 0.1 mm og d = 40 mm:
Ref [1]
Electrospray: From solution to gas phase(I)
I. Electrical nebulization of liquid results in the formation of charged micro droplets.
II. Vaporization increases the charge density on the surface of the droplets. Electrostatic repulsion increases.
III. When the electrostatic repulsion exceeds the surface tension the droplet undergoes coulombic fission.
IV. The formation of charged ions in the gas phase
A charged droplet undergoing coulombic fission
Parent dropletOffspring droplets
Gomez et al., Phys. Fluids 6 (1994) 404-414
Parent droplet after 1 fissionVol. = 3.5 m3
Area = 11 m2
Solvent evaporation causes sequential fissions of charged droplets
N=51250R=1.5µm
t=462µs 512500.945
435600.939
3840.09
435600.848
t=74µs
t=70µs
t=39µs
370260.844
3260.08
370260.761
314720.756
2780.07
2780.03
20.003
Kebarle et al. Anal. Chem. 65 (1993) 972A
Asymmetrical fission process: 20 offspring droplets are formed carrying ~2% of the total mass and ~15% of the net charge.
~20 offspring droplets:Total volumen = 0.06 m3
Total surface area = 2 m3
The formation of smaller droplets increases the total surface area and this relieves the coulombic repulsion
N: No. of chargesR: droplet radius
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.
Surface activity (hydrofobicity + charge) determines ionization efficiency
IA : Abundance of A in the mass spectrumCA: conc. of AkA: A’s responsefactor ~ ionization efficiency
Kebarle P., J. Mass Spectrom. 35, 804-817 (2000)
Equimolar mixture of 6 tripeptides with different C-terminal residues
Ref. [2]
Hydrophilic substituents
• Hydrophilic substituents such as phosphorylation or glycosylation reduce the ionization efficiency of proteins and peptides (in complex mixtures).
Flow rate and ionization efficiency
Ref. [3]
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)
ESI spectra of disulfide-intact og disulfide-reduced lysozyme
Konermann et al.J. Am. Soc. Mass Spectrom. 1998, 9, 1248-1254
ES SPECTRUM OF HORSE MYOGLOBIN MW 16.951 A) multiply charged ion distribution from +12-24 shown at low resolution B) the 17+ charge state at a resolution of about 15000 showing the resolved isotope peaks
Mass (M) determination of proteins (II)
800 1000 1200 1400 1600 1800 2000 2200 2400m/z0
100
%
m19371tj 22 (0.808) Sm (SG, 3x8.00); Cm (12:43) TOF MS ES+ 1.88e31060.58
942.86
893.29 1211.95
1305.09
1413.77
1542.221884.65
2120.11
2422.81
nz
m
n
nM
65.18841
nz
m
11.2120
nz
m
11
1
nz
m
n
nM
(1) og (2): two equations with two unknowns: M og n (n : No. Of protons)
865.188411.2120
165.18841
1
1
nn
n
zm
zm
zm
n
(1) (2)
9.169528)11.21208(
nn
z
mM
n
Apomyoglobin (16951.50 Da)
Nano-Electrospray
Gold-coated borosilicate
glass
Nanoelectrospray
Flow rates 50 to 200 nL/min
Features of NanoES
Flow rates of 10-40 nL/min (25-100 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
NanoES vs. conventional ES (2)
Electrosprayparameters
NanoES Conventional ES
Capillary
Flow
ES voltages
1-3µm
10-40nL/min.
300-700V
>100µm
>500nL/min.
>2500V
NanoES vs. conventional ES (3)
Droplets NanoES Conventional ES
Radius
Volume
Analyte moleculesper droplet
(at 1pmol/µL)
50 - 200nm
5·10-13 - 4·10-12µL
0.3 - 2.5
1000 - 2000nm
4·10-9 - 3·10-8µL
2.5·103 - 1.9·104
AAAAAA
AAAAAAAAAAAAAAAAAA AAAAAAAAAAAA
AAAAAA
AAAAAA
AAAAAAAAAAAA
AAAAAA
AAAAAA
AAAAAA
3+
3+
3+
3+
2+
2+
2+2+3+3+3+
3+
3+
The formation of heterodimers
Eremomycin
Cl-eremomycin
Staroske, T.; O’Brien, D.P.; Jørgensen, T.J.D.; Roepstorff, P.; Williams, D.H.; Heck, A.J.R. Chem. Eur. J. 2000, 6, 504-509
ESI-MS of intact virusBacteriophage MS2
Molecular mass 2 484 700 ( 25000) Da
Virus maintains its infectivity! (Ref. [6])
+121
Ref [5]
Noncovalent complexes in the gas phase
Mcalc.= 800,770 Da
Mexp. = 803,700 ±100
14mer
Nano-ESI, 10µM in aqueousamm. acetate (100mM, pH7)
E.coli GroEL under non-denaturing conditions
Noncovalent complexes in the gas phase
Pressures StandardNoncovalents
p2 10-4 mbar 10-2-10-3 mbar
p3 10-6 mbar 10-4-10-5
mbar
p4 10-7 mbar 10-6-10-7 mbar
Noncovalent complexes in the gas phase
E.coli GroEL 14mer (≈800 kDa)
p2= 0.4·10-2 mbar
p2= 1.3·10-2 mbar
p2= 1.6·10-2 mbar
p2= 1.0·10-2 mbar
p2= 0.7·10-2 mbar
Colli
sion
al co
olin
g
???
810,208 +/-963 Da
30S
1,516,052 +/-1986 Da
50S
2,325,463+/-2003 Da
70S
The ribosome
Referencer
1. “Electrospray Ionization Mass Spectrometry” Ed. R.B. Cole, John Wiley & Sons, 1997
2. Cech et al. “Practical implications of some recent studies in electrospray ionization fundamentals” Mass Spectrometry Reviews, 2001, 20, 362-387
3. Covey et al. “Nanospray Electrospray Ionization Development” i Applied Electrospray Mass Spectrometry, Ed. N. Birendra et al., Marcel Dekker, 2002
4. Tito et al. J. Am. Chem. Soc. 2000, 122, 3550-35515. Siuzdak, G. et al. Chem Biol. 1996, 3, 45-48