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Niels Lion
Laboratoire dElectrochimie Physique et Analytique
Ecole Polytechnique Fdrale de Lausanne
niels.lion@epfl.ch
Outline
What is a mass spectrometer?
Analytical features
Ion sources: ESI and MALDI
Basic instruments
Quadrupolar instruments
Ion trap instruments
Time-of-flight mass analyzers
Fourier Transform-Ion Cyclotron Resonance-MS
Hybrid instruments
Examples from proteomic research
3
What is a mass
spectrometer?
Ion
source
Ion
optics
Mass
analyzer
Detector
How to evaluate MS
performances?
m/z range or mass range
How to evaluate MS
performances?
m/z range or mass range
Mass resolving power
How to evaluate MS
performances?
m/z range or mass range
Mass resolving power
10x10
m/m50%
Intensity
8
6
4
2
0
1746
1748
1750
m/z
1752
1754
How to evaluate MS
performances?
m/z range or mass range
Mass resolving power
Mass resolution
How to evaluate MS
performances?
m/z range or mass range
Mass resolving power
Mass resolution
10x10
(m1-m2)/
m1
Intensity
8
6
4
2
0
1748
1752
m/z
1756
1760
How to evaluate MS
performances?
m/z range or mass range
Mass resolving power
Mass resolution
Mass precision (repeatability)
How to evaluate MS
performances?
m/z range or mass range
Mass resolving power
Mass resolution
Mass precision (repeatability)
Mass accuracy: difference between measured
and true mass)
Scaling MS plateaus
10
Bibliography
11
Outline
What is a mass spectrometer?
Analytical features
Ion sources: ESI and MALDI
Basic instruments
Quadrupolar instruments
Hybrid instruments
Examples from proteomic research
12
Electrospray ionization
13
500
1000 m/z
1500
2000
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
15
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
1.
Charge state
determination:
2.
Molecular weight
evaluation
m/z=(M+zH)/z
16
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
m/z=(M+zH)/z
M=z*m/z-zH
17
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
m/z=(M+zH)/z
M=z*m/z-zH
if z=10:
M=14126.328
M=13869.4797
M=13561.2624
17
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
m/z=(M+zH)/z
M=z*m/z-zH
17
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
m/z=(M+zH)/z
M=z*m/z-zH
if z=11:
M= 15538.960
M= 15410.533
M= 15256.4202
17
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
m/z=(M+zH)/z
M=z*m/z-zH
17
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
m/z=(M+zH)/z
M=z*m/z-zH
If z=12:
M=16951.5936
M=16951.5863
M=16951.578
17
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
m/z=(M+zH)/z
M=z*m/z-zH
17
28
27
26
25
24
23
22
21
20
19
18
998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
17
16
15
m/z=(M+zH)/z
M=z*m/z-zH
14
13
12
11
10
9
8
z=12:
M=16951.5936
M=16951.5863
M=16951.578
M=16951.63730.0513
Da
7
18
ESI features
+ Multiply charged analytes
+ Good for on-line coupling with liquid phase
separations
Multiply charged analytes (need for
deconvolution)
Ion suppression
Requires good desolvation (presence of organic
solvent)
Intolerant to salts
19
Outline
Analytical features
Basic instruments
Quadrupolar instruments
Hybrid instruments
21
Delayed extraction
Matrix and analyte
ions are mixed;
expansion is
necessary before
acceleration to limit
collision and
fragmentations
22
MALDI features
+ Mostly singly charged ions
+ Good throughput
+ Relative tolerance to salts
Mostly singly charged ions
Off-line analysis
Sample preparation
23
ESI bibliography
Rohner TC, Lion N, Girault HH, Theoretical and electrochemical aspects of electrospray ionization, PCCP, in press.
Fenn, J. B.; Rosell, J.; Meng, C. K., In electrospray ionization, how much pull does an ion need to escape its droplet
prison?, Journal of the American Society for Mass Spectrometry, 1997, 8, 1147-1157.
Labowsky, M.; Fenn, J. B.; de la Mora, J. F., A continuum model for ion evaporation from a drop: effect of curvature
and charge on ion solvation energy, Analytica Chimica Acta, 2000, 406, 105-118.
Kebarle, P.; Peschke, M., On the mechanisms by which the charged droplets produced by electrospray lead to gas
phase ions, Analytica Chimica Acta, 2000, 406, 11-35.
Kebarle, P., A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry,
Journal of Mass Spectrometry, 2000, 35, 804-817.
Peschke, M.; Blades, A.; Kebarle, P., Charged states of proteins. Reactions of doubly protonated alkyldiamines with
NH3: Solvation or deprotonation. Extension of two proton cases to multiply protonated globular proteins observed
in the gas phase, Journal of the American Chemical Society, 2002, 124, 11519-11530.
[Felitsyn, N.; Peschke, M.; Kebarle, P., Origin and number of charges observed on multiply-protonated native
proteins produced by ESI, International Journal of Mass Spectrometry, 2002, 219, 39-62.
deJuan, L.; delaMora, J. F., Charge and size distributions of electrospray drops, Journal of Colloid and Interface
Science, 1997, 186, 280-293.
24
MALDI bibliography
Karas, M.; Bahr, U.; Giessmann, U., Matrix-Assisted Laser Desorption Ionization MassSpectrometry, Mass Spectrometry Reviews, 1991, 10, 335-357.
Karas, M.; Kruger, R., Ion formation in MALDI: The cluster ionization mechanism,
Chemical Reviews, 2003, 103, 427-439.
Knochenmuss, R.; Zenobi, R., MALDI ionization: The role of in-plume processes,
Chemical Reviews, 2003, 103, 441-452.
Zenobi, R.; Knochenmuss, R., Ion formation in MALDI mass spectrometry, Mass
Spectrometry Reviews, 1998, 17, 337-366.
25
Outline
What is a mass spectrometer?
Ion sources: ESI and MALDI
Analytical features
Basic instruments
Quadrupolar instruments
Hybrid instruments
Examples from proteomic research
26
U
V
27
Trajectories in a quadrupole
d2 x
m dt 2 = ze x
d2 y
m 2 = ze
y
dt
= U V cost
u = x or y
t
=
2
8zeU
au =
2 2
mr0
4zeV
qu =
mr02 2
d u
2 + (au 2qu cos2) u = 0
d
28
Stable trajectories
au U
qu V
= U V cost
29
Features of quadrupolar MS
m/z<4000
Low resolution (3000~5000)
Fast scans
Low cost
In the RF mode, quadrupoles, hexapoles and
octopoles are used as ion beam focusing
devices
30
Q1
Q2
Q3
31
32
Collision induced
dissociation of peptides
33
Resulting fragments
etc........
34
Ordered fragments
dm1
dm1
dm2
dm2
dm3
dm3
dm4
dm4
35
Collision induced
dissociation of peptides
36
37
G G
37
G G
V G G
37
collision cell
MS 2
m/z selection
CID
scanning
scanning
collision cell
MS 2
CID
m/z selection
scanning
m/z=x
collision cell
CID
MS 2
scanning
m/z=x-a
Outline
What is a mass spectrometer?
Ion sources: ESI and MALDI
Analytical features
Basic instruments
Quadrupolar instruments
Ion trap instruments
Time-of-flight mass analyzers
Fourier Transform-Ion Cyclotron Resonance-MS
Hybrid instruments
Examples from proteomic research
41
42
Ion trajectories
Ion destabilizationEjectionDetection
43
100
50
0
250
300
350
m/z
400
450
1.5
inj= 50 ms
1.0
0.5
0.0
200
250
300
350
m/z
400
450
inj= 100 ms
2.0
1.5
1.0
0.5
0.0
150
200
250
300
350
m/z
400
450
500
inj= 250 ms
2
1
0
150
200
250
300
350
m/z
400
450
500
5x10
4
inj= 500 ms
3
2
1
0
500
2.5x10
4x10
500
2.0x10
150
200
150
inj= 5 ms
150x10
150
200
250
300
350
m/z
400
450
500
5x10
4
inj= 1000 ms
3
2
1
150
200
250
300
350
m/z
400
450
500
44
What to optimize?
35
250
SNR / [dB]
300
200
150
30
'SNR mean'
'SNR stdev'
25
100
50
20
200
400
600
800
1000
200
400
600
800
1000
45
Ion isolation
= all ions are ejected except the one of interest
47
48
MS/MS bibliography
http://www.matrixscience.com/help/fragmentation_help.html
49
Outline
What is a mass spectrometer?
Ion sources: ESI and MALDI
Analytical features
Basic instruments
Quadrupolar instruments
Ion trap instruments
Time-of-flight mass analyzers
Fourier Transform-Ion Cyclotron Resonance-MS
Hybrid instruments
Examples from proteomic research
50
Time-of-flight MS
Field-free drift
tube
+Eacc
l
0V
dv
zeE = m
dt
t flight
2l
L m
=
+
z
eE
2eEl
51
+Eacc
l
0V
2eEl
v= m
z
52
53
TOF features
54
Outline
What is a mass spectrometer?
Ion sources: ESI and MALDI
Analytical features
Basic instruments
Quadrupolar instruments
Ion trap instruments
Time-of-flight mass analyzers
Fourier Transform-Ion Cyclotron Resonance-MS
Hybrid instruments
Examples from proteomic research
55
Fourier Transform-Ion
Cyclotron (FT-ICR) MS
56
dv
m
= zev B
dt
2
xy
v
m
= zev xy B0
r
m r = zeB0r
2
zeB0
c =
m
57
Direct consequences
B of few tesla:
Ion excitation
E = E 0 cos(t )
E0
E
cos(t) j + 0 sin(t)i
2
2
E
E
E l = 0 cos(t ) j 0 sin(t)i
2
2
Er =
E 0Texc V0 Texc
r=
=
2B0
2dB0
Kinetic energy in the keV range
59
Ion detection
Synchronized ion
motion induces a
current between the
two detector plates
60
Spectrum computation
61
FT-ICR-MS features
+ Incomparable mass resolving power, resolution,
and accuracy
+ Resolving power: 105-106
+ Mass accuracy: 1-20 ppm
+ Compatible with ESI and MALDI
- Cost
- More or less difficult to operate
62
FT-ICR-MS bibliography
Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S., Fourier transform ion cyclotron resonance
mass spectrometry: A primer, Mass Spectrometry Reviews, 1998, 17, 1-35.
Marshall, A. G., Milestones in Fourier transform ion cyclotron resonance mass spectrometry
technique development, International Journal of Mass Spectrometry, 2000, 200, 331-356.
Marshall, A. G., Ion cyclotron resonance mass spectrometry: a brief history, Actualite Chimique,
2001, 18-22.
Hughey, C. A.; Rodgers, R. P.; Marshall, A. G., Resolution of 11 000 compositionally distinct
components in a single Electrospray ionization Fourier transform ion cyclotron resonance mass
spectrum of crude oil, Analytical Chemistry, 2002, 74, 4145-4149.
Marshall, A. G.; Hendrickson, C. L.; Shi, S. D. H., Scaling MS plateaus with high-resolution FTICRMS, Analytical Chemistry, 2002, 74, 253A-259A.
Marshall, A. G.; Hendrickson, C. L., Fourier transform ion cyclotron resonance detection:
principles and experimental configurations, International Journal of Mass Spectrometry, 2002,
215, 59-75.
63
Summary of mass
spectrometer performances
Mass
range
Accuracy
Resolution
(ppm)
cost
Quad or
<2000 or
ion
3000-5000 200-300
4000
traps
low
TOF
unlimited
104-105
20-100
medium
<2000 or
FT-ICR
4000
105-107
1-50
high
64
Conclusions
Hybrid instruments
Miniaturization (micro ion traps)
MS/MS: IRMPD, ECD, CID, SID
New analyzers (ion mobility)
Cost
Sample delivery
65
Take-home message
Mass spectrometers are wonderful instruments
66