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Electrospray ionization (ESI) mass spectrometry
Mass spectrometry Advanced Methods_Elviri
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Electrospray ionization (ESI)
Electrosprayed ‘aerosol’ Liquid sample Mass spectrometer 1-3 kV needle potential ++ + ++ + + + + + + + Gas-phase ions + + + ++ + + + ++ + + + +
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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
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Charge state distribution obtained by ESI MS reflects protein conformation
Native (water) Denatured (weak acid)
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Denaturation of Azurin by acid as observed by ESI MS
Spectrum deconvolution 14001 Da 13945 Da 13945 Da
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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)
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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
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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!
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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]
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Electrospray Spray of charged microdroplets
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Electrospray ion source
Pressure and Electrical potential gradient coaxial nebulization gas flow Countercurrent gas flow to aid desolvation
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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]
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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
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A charged droplet undergoing coulombic fission
Parent droplet Offspring droplets Gomez et al., Phys. Fluids 6 (1994)
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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
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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.
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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)
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Equimolar mixture of 6 tripeptides with different C-terminal residues
Ref. [2]
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Hydrophilic substituents
Hydrophilic substituents such as phosphorylation or glycosylation reduce the ionization efficiency of proteins and peptides (in complex mixtures).
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Flow rate and ionization efficiency
Ref. [3]
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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)
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ESI spectra of disulfide-intact og disulfide-reduced lysozyme
Konermann et al. J. Am. Soc. Mass Spectrom. 1998, 9,
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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
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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)
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Gold-coated borosilicate glass
Nano-Electrospray Gold-coated borosilicate glass
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Nanoelectrospray Flow rates 50 to 200 nL/min
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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
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NanoES vs. conventional ES (2)
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NanoES vs. conventional ES (3)
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The formation of heterodimers
3+ 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,
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ESI-MS of intact virus Bacteriophage MS2
+121 Molecular mass ( 25000) Da Virus maintains its infectivity! (Ref. [6]) Ref [5]
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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)
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Noncovalent complexes in the gas phase
Pressures Standard Noncovalents p mbar mbar p mbar mbar p mbar mbar
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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
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The ribosome 810,208 +/-963 Da 1,516,052 30S +/-1986 Da ??? 2,325,463
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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
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