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Mass spec basics:

main instruments
Niels Lion
Laboratoire dElectrochimie Physique et Analytique
Ecole Polytechnique Fdrale de Lausanne
niels.lion@epfl.ch

Mass spectrometry history

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

Transfer of ions from liquid or solid phase


to gas phase, and separation of ions
according to their m/z ratio

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

Marshall A. G., Hendrickson C. L., Shi S. D. H., Scaling MS plateaus with


high-resolution FT-ICR-MS, Analytical Chemistry, 2002, 74(9):
253A-259A

11

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
12

Electrospray ionization

13

Multiply charged spectra


(M+nH)n+

500

1000 m/z

1500

2000

Each peak corresponds to one protonation state (n


protons)
calculation of the molecular weight
14

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

998.1549
1060.4766
1131.1078
1211.8293
1304.9694
1413.6328
1542.0533
1696.1578
1884.5078
2119.9453
2422.6506
15

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

Molecular weight calculation


606.4186
628.8412
652.9886
679.0679
707.3204
738.0296
771.5306
808.2221
848.5828
893.1921
942.7577

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

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


20

Matrix assisted laser


desorption ionization
Analyte co-deposition with an acid matrix

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

Ion trap instruments

Time-of-flight mass analyzers

Fourier Transform-Ion Cyclotron Resonance-MS

Hybrid instruments
Examples from proteomic research
26

The basic quadrupole

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

Triple quadrupole instruments

Q1

Q2

Q3

Q1 and Q3: mass analyzers


Q2: collision cell (inert gas)

31

Peptides tandem mass


spectrometry
One can fragment peptides in the gas phase by increasing
their energy
Energy uptake = breakage of the peptide backbone (one
peptidic bond=two breakage positions)

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

Peptide sequencing by CID

37

Peptide sequencing by CID

G G

37

Peptide sequencing by CID

G G

V G G

37

Triple quad scanning modes:


daughter ions scan
MS 1

collision cell

MS 2

m/z selection

CID

scanning

Identification of a molecule through its


fragmentation pattern (peptide
sequencing)
38

Triple quad scanning modes:


parent ions scan
MS 1

scanning

collision cell

MS 2

CID

m/z selection

Identification of all molecules giving a


particular ion
39

Triple quad scanning modes:


neutral loss scan
MS 1

scanning
m/z=x

collision cell

CID

MS 2

scanning
m/z=x-a

Identification of all molecules losing a particular


neutral group under collision (phosphorylation search)
40

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

Quadrupole ion traps

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

Intensity / [ion counts]

200

Intensity / [ion counts]

Intensity / [ion counts]

150

Intensity / [ion counts]

inj= 5 ms

150x10

150

Intensity / [ion counts]

Intensity / [ion counts]

Effect of injection time

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]

Mass resolving power / [-]

300

200
150

30
'SNR mean'
'SNR stdev'
25

100
50

20

200

400

600

800

Injection time / [ms]

1000

200

400

600

800

Injection time / [ms]

1000

45

Ion isolation
= all ions are ejected except the one of interest

Tandem mass spec


= collision-induced-dissociation with inert gas (He,
Ar) in the trap

Gas phase chemistry


= reaction of trapped ions with a reactive gas (control of
gas pressure and reaction time)
46

Ion trap features


m/z < 4000
Resolution 3000-5000
Mass accuracy 200-300 ppm
Low cost
MSn with n<3,4
Versatile, fast scans

47

Quad and ion trap


bibliography

March RE, Quadrupole ion trap mass spectrometer, Encyclopedia of


Analytical Chemistry, 2000.

48

MS/MS bibliography

http://www.matrixscience.com/help/fragmentation_help.html

Roepstorff, P and Fohlman, J, Proposal for a common nomenclature for


sequence ions in mass spectra of peptides. Biomed Mass Spectrom, 11(11) 601
(1984).

Papayannopoulos, IA, The interpretation of collision-induced dissociation


tandem mass spectra of peptides. Mass Spectrom. Rev., 14(1) 49-73 (1995).

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

Reflectron mode (1)


Goal: to compensate for kinetic energy dispersion

Field-free drift tube

+Eacc
l

0V

2eEl
v= m
z

52

Reflectron mode (2)


Ions are repelled by an
electrostatic field. Energetic
ions penetrate the reflectron
more than slow ions
Focalization of ions of
same m/z in the same plane

53

TOF features

Virtually unlimited mass range


Excellent resolution: 104-5.104
Mass accuracy: 20-100 ppm
Need internal calibration
Limited MS/MS
Especially adapted to off-line, pulsed
ionization

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

Ion cyclotron motion


An ion of mass m and charge ze in a magnetic
field is subjected to:

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:

c from kHz to Mhz

For an ion in temperature equilibrium with its


surroundings:
v xy2
m
kT
2
Ion radius < 1cm at room temperature
Ion of mass 100 u in 7 Te: 30 km/s
58

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

An oscillating current is induced in the


detector plates by rotating, synchronized ions
of given m/z

For each m/z, current frequency is acquired,


and the resulting frequency spectrum is
converted into mass spectrum by inverse FFT.

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

Mass spectrometry is the central tool for peptide and


protein identification (see next talk)

A few instrument types are available (quadrupoles, ion


traps, TOF, FT-ICR) that cover most of needs

But the technology is evolving quickly:

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

But new developments are always


needed (and going on).

66