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