CH 908: Mass Spectrometry
Lecture 5
Ionization sources, EI, MALDI,
and ESI
Prof. Peter B. O’Connor
Objectives for this lecture
• Ionizing molecules
• The many attempts to ionize biomolecules
– Direct Chemical Ionization
– Plasma desorption
– FAB
– LDI
• ESI and MALDI
MS Block Diagram
Sample
Cleanup
Fragmentation
Method
Chromatography
Inlet
Ion
Source
Mass
Separator
Detector
Computer
Data
Reduction
Mass Spectrometers do not measure mass, they
measure mass/charge ratio.
Bioinformatics
A simple, laser desorption ion source.
Laser
Sample
Ionization
Because most molecules prefer to be
electrically neutral, ionization requires
addition or removal of one or more
charges particles (usually electrons or
protons). This can be done by hitting
the sample with one of the following.
Exceptions: samples that naturally
prefer to be ions or are somehow
stabilized as ions.
• Photons
• Electrons
• Fast Neutral Molecules
• Other Ions.
• Heat
• Combination methods
EI Mass Spectrum of an
acetylated and reduced
peptide
Field ionization/ field desorption MS
(FI/FDMS) H. D. Beckey, IJMSIP, 1969
H. R. Schulten, Int. J. Mass Spectrom. Ion Phys., 32, 97-283, 1979
Emitters for field ionization/ field
desorption MS (FI/FDMS)
C. E. Costello, electron microscopy courtesy of JEOL , 1979
Field desorption MS of bradykinin
S. Asante-Poku, W. G. Wood, and D. E. Schmidt, Jr.
Biomed. Mass Spectrom, 2, 121, 1975
Field desorption MS of Vitamin B12
H.-R. Schulten and H.
M. Schiebel
Naturwissenschaften,
65, 223, 1978
Methods for “soft” ionization
•
•
•
•
•
•
•
•
•
Direct chemical ionization MS (DCIMS)
Field ionization/ field desorption MS (FI/FDMS)
Secondary ionization MS (SIMS)
252Cf Plasma desorption (PDMS)
Fast atom bombardment (FABMS)
Liquid secondary ionization MS(LSIMS)
Laser desorption MS (LDMS)
Electrospray ionization MS (ESIMS)
Matrix-assisted laser desorption/ionization MS
(MALDI MS)
[Direct] chemical ionization MS (DCIMS)
Frank H. Field and Burnaby Munson
ASMS Distinguished Contribution, 1996
252Cf
Plasma desorption (PDMS)
Ronald D.
Macfarlane
ASMS Distinguished
Contribution, 1990
R. D. Macfarlane, Anal. Chem., 55,
1247A-1264A, 1983
252Cf
Plasma desorption (PDMS) of
porcine trypsin (23,463 Da)
Peter
Roepstorff
Per
Håkansson
B. Sundquist, P. Roepstorff, et al., Science, 226, 696 – 698, 1984
Fast atom bombardment (FABMS)
Michael Barber
ASMS Distinguished
Contribution, 1991
M. Barber, R. S. Bordoli, G. J. Elliott,
R. D. Sedgwick and A. N. Tyler, Anal.
Chem., 54, 645A-657A, 1982
Fast atom bombardment mass spectra of (a)
vitamin B12 and (b) the coenzyme of vitamin B12
(a)
(b)
M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Nature, 293, 270-275, 1981
Fast atom bombardment mass spectra:
(a) human insulin and (b) proinsulin
(a)
A. Dell and H. R. Morris, Biochem.
Biophys. Res. Commun, 106,
1456-1462, 1982
(b)
M. Barber, R. S. Bordoli, G. J. Elliott, N. J.
Horoch and B. N. Green, Biochem. Biophys.
Res. Commun, 110, 753-757, 1983
+
Anode
-
e- e e-
+
Sample
+
+
+
+
+
Target
cathode
Figure 2. Laser Desorption plus Electron Impact Ionization
Two Special Cases
• Matrix Assisted Laser Desorption/Ionization (MALDI)1,2
• Analyte is mixed with a "Matrix" and dried down on a surface
• Laser hits the spot and desorbs matrix and analyte together
• some of the analyte ends up charged
• Electrospray Ionization (ESI)3,4
•Solution containing the sample is sprayed out a needle into a large
electric field. Droplets are formed and are sucked down into the
mass spectrometer. They evaporate and leave charges on the
analytes.
1. Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T., Matrix-Assisted Laser Desorption/Ionization Mass
Spectrometry of Biopolymers Anal. Chem. 1991, 63, 1193A-1203A.
2. Karas, M. I.; Bachmann, D.; Bahr, U.; Hillenkamp, F., Matrix-Assisted Ultraviolet Laser Desorption of Non-Volatile
Compounds Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68.
3. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray ionization for mass spectrometry
of large biomolecules Science 1989, 246, 64-71.
4. Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B., Electrospray interface for LCMS Anal. Chem. 1985,
57, 675-679.
ESI and MALDI
Developed Electrospray
Prof. John Fenn
Nobel prize, 2002
Developed MALDI
Prof. Dr. Ing. Franz Hillenkamp
Prof. Dr. Michael Karas
+ + ++
++ + +
+ + + +
+
++++ + +++ + ++ +++ + ++
+
Oil Drop Experiment
Robert A. Millikan (Nobel prize in physics, 1923)
A simple ESI ion source.
Electrospray Ionization
• Electrospray Ionization (ESI)
•Solution containing the sample is sprayed out a needle into a
large electric field. Droplets are formed and are sucked down
into the mass spectrometer. They evaporate and leave
charges on the analytes.
•Sample is a flowing liquid - compatible with LC
•flow rates typically in the <1 μl/min range
•sample cleanup is required!
•sensitivity (Best case <1 amol, normal case 10-1000 fmol)
•Multiple charging
•upper mass range not really limited, but big masses get
confusing if you don't have sufficient mass resolution
•extremely soft ionization method - allows ionization of noncovalently bound species (e.g. protein complexes)
Generic Atmospheric Pressure Ionization
Source/Mass Spectrometer System
Vacuum system with multiple stages:
1st stage - free-jet expansion, molecular-beam stage
2nd stage - ion optics
3rd stage - mass analyzer
Electrospray
Major Components and Processes in ES-MS
Electrospray
Emitter
 production of
charged droplets
at emitter tip
Atmospheric
Pressure Region
(The Gap)
Atmosphere
to Vacuum
Interface
 shrinkage/subdivison
of droplets
 gas-phase ion
production
 final
desolvation
 fragmentation
 gas-phase
reactions
 charge
- ionization
permutation
- charge permutation
Mass
Analyzer
Taylor cone
Spray ~1 microliter/min
In “nanospray”, flow rates of ~1 nl/min are used. The
taylor cone and plume become invisible because the
droplets are in the 100 nm diameter range, and sensitivity
goes up due to greatly reduced space charge and
improved capture efficiency.
A few examples of
electrospray mass spectra…
~80 Da difference
~25 Da wide
Bad mass
spectrometer?
p21Ras
Normal distribution
MI=21284.4032 (21284.5511) MI=21284.4080 (21284.5511)
21291
21303
1185
16+
21291
17+
21307
Mass/Charge (m/z) 1230
15+
Peroxynitrite
14+
18+
1000
1200
1400
1600
1800
1000
1200
Mass/Charge (m/z)
1400
1600
Mass/Charge (m/z)
DTT treated distribution
MI=21282.3422 (21282.53543)
MI=21282.3880 (21282.53543)
21290
21291
21303
24+
23+
22+
21+
21304
20+
19+
18+
17+
25+
800
16+
900
1000
1100
1200
1300
1400
1500
Mass/Charge (m/z)
Zhao, C.; Sethuraman, M.; Clavreul, N.; Kaur, P.; Cohen, R. A.; O'Connor, P. B. A Detailed Map of Oxidative Post-translational Modifications of Human p21ras using Fourier
Transform Mass Spectrometry Anal. Chem. 2006, 78, 5134-5142.
1800
Intens.
x104
+MS, 0.3-1.5min #(16-91)
Calmodulin (Entire protein)
1120.3351
Micro-TOF
1200.2786
1.5
1050.3689
1292.5220
1.0
1400.1262
0.5
988.6485
1527.3098
933.7842
1680.0067
884.7075
0.0
800
900
1000
1100
1200
1300
1400
1500
1600
1700
m/z
Intens.
+MS, 0.4-1.2min #(23-70)
880.5196
6000
908.0058
854.6555
Carbonic anhydrase (Entire protein
Micro-TOF
830.2614
937.2675
5000
807.2280
785.4459
968.4701
1001.8412
4000
1037.5745
764.7936
1075.9647
714.6845
3000
1117.3119
1161.9558
1210.3295
2000
1262.9047
659.8101
1320.2672
1000
1383.1014
1452.1785
429.2474
593.1336
543.4134
1528.5965
1613.4771
1747.1915
0
400
600
800
1000
1200
1400
1600
1800
m/z
Intens.
x104
+MS, 0.0-0.3min #(3-16)
1147.5278
Insulin (Entire protein)
Micro-TOF
6
956.4497
4
2
819.8634
1433.7548
0
600
800
1000
1200
1400
1600
1800
m/z
Intens.
x105
+MS, 0.1-1.3min #(7-79)
714.7
Ubiquitin (Entire protein)
Micro-TOF
1.0
0.8
779.5
0.6
659.8
0.4
857.4
0.2
952.6
1071.5
0.0
500
600
700
800
900
1000
1100
1200
1300
1400
m/z
MALDI
• Matrix Assisted Laser Desorption/Ionization (MALDI)
• Analyte is mixed with a "Matrix" and dried down on a surface
• Laser hits the spot and desorbs matrix and analyte together
• some of the analyte ends up charged "Lucky Survivor Model"
• pulsed beam - nicely couples with pulsed mass analyzers
• sensitivity (best case ~1 amol, normal case 10-1000 fmol)
• more tolerant of salts than ESI, less cleanup required
• upper mass limit defined by
1) metastable decay (PSD)
2) detector
• relatively soft, but generally not soft enough for non-covalent
complexes
MALDI mass spectrometry
+
+
+
Laser
Most commonly, Laser is a <10 nsec pulse at 337
nm (N2 laser) or 355 (frequency tripled Nd:YAG).
50 nsec pulse at 2.94 (Er:YAG) is also used.
ESI and MALDI
Developed Electrospray
Prof. John Fenn
Developed MALDI
Prof. Dr. Ing. Franz Hillenkamp
Prof. Dr. Michael Karas
Nobel prize, 2002
Nobel prize, 2002
Excimer lasers
ArF
193 nm
ND:YAG laser
KrF
248 nm
XeCl
308 nm
4xf
266 nm
ER:YAG laser
2.94 µm
3xf
353 nm
Nitrogen laser
337 nm
193 nm
200 nm
300 nm
400 nm
6.3 eV
6,1 eV
4 eV
3 eV
ultraviolet
OPO-lasers
1xf
1.06 µm
800 nm
1.5 eV
visible
1 µm 3µm
0.4 eV
CO2 laser
9.1-10.6 µm
10µm wavelength 
0.01 eV
photon
energy h
infrared
Figure 1. Wavelength and photon energy of frequently used desorption lasers
Camera
Ocular
system
2D
Sample
stage.
Laser
Ion Optics
TOF Analyzer
Figure 3. Laser Microprobe for Mass Analysis (LaMMA) configuration
d2
d1
Target

1
2
Laser
d2
d1
f1
f2
f3
Infinite
Conjugation
Figure 2. Laser focusing optics for adjustment of the spot size on the target
A.
B.
Figure 4. Two of the first MALDI spectra. A)
the matrix effect, desorbing Alanine mixed
with Tryptophan allowed observation of the
Alanine peaks at 10x lower laser fluence
than possible with pure Alanine. B) the first
MALDI of a protein, showing that
desorption/ionization could occur with no
observable fragmentation.
Karas, M.; Bachmann, D.; Hillenkamp, F.
Influence of the wavelength in highirradiance ultraviolet laser desorption
mass spectrometry of organic molecules
Anal Chem 1985, 57, 2935-2939.
Karas, M. I.; Bachmann, D.; Bahr, U.;
Hillenkamp, F. Matrix-assisted ultraviolet
laser desorption of non-volatile
compounds Int J Mass Spectrom Ion
Processes 1987, 78, 53-68.
Peptide digests
+
Sample
+
+
+
+
+
Target
Temperature
Figure 1. Laser Desorption/Ionization
MALDI of a 0.6 kDa Polymer
PEG (entire compound)
MALDI
Intens. [a.u.]
MALDI of a 1.6 kDa Polymer
1581.914
1405.812
1758.019
1361.785
8000
1802.049
1317.760
6000
1846.074
1273.735
1890.098
1229.709
1934.124
4000
1978.143
1185.686
2022.169
1141.659
2000
2066.187
1097.626
2110.212
1053.586
2198.245
0
1000
1200
1400
1600
1800
2000
2200
m/z
Intens. [a.u.]
MALDI of a 13 kDa polymer
1250
1000
750
500
250
0
10000 11000
12000 13000 14000 15000 16000
m/z
Intens. [a.u.]
16794.350
2000
Calmodulin (Entire protein)
MALDI
1500
1000
500
0
10000
12000
14000
16000
18000
20000
22000
24000
26000
m/z
Other laser wavelengths
In-gel digest experiment
In-gel digest experiment
Self Assessment
1. list 3 methods that people used to try to ionize
proteins before 1985.
2. What two methods were discovered in 19851987 that revolutionized the study of
biomolecules via mass spectrometry? How do
they work?
3. What’s a good matrix for proteins/peptides in
MALDI? What’s the structure? What laser
wavelengths are used for MALDI?
4. How do you determine the mass of a protein
from an ESI mass spectrum? 2 methods (at
least).
CH908: Mass spectrometry
Lecture 5 – Ionization sources.
Fini…
Typical MALDI-TOF mass spectra of small proteins
First MALDI-TOF mass spectrum of a glycoprotein.
Electrons
The oldest, most controllable method of ionizing most species is to
smash electrons into them.
A few important ionization techniques using this method
• Electron Impact
• >10 eV electrons
• Gas phase samples only
• Typically used for GC/MS
• Deposits lots of energy into the molecule - causing extensive
fragmentation
• Electron capture ionization
•< 5 eV electrons get captured by the molecule
• relatively gentle, but only works for some species with high
electron affinity (like PCB's)
Photons
Using light is the next most reliable method. Some commonly used
techniques are:
• Photoionization
•Uses high energy photons (UV typically) to knock an electron
off the molecule.
• Very wavelength dependent - every molecule has particular
wavelengths for ionization
• Resonance Enhanced Multiphoton Ionization (REMPI)
• Only used for gas phase samples
• Very controllable energy deposition
• Laser Desorption/Ionization (LD/I)
• Absorbs laser energy into electronic and vibrational modes of
the molecule blasting it off of a surface (typically steel)
• Not very controllable in energy deposition
• Large suppression effects
• Very small fraction of the desorbed molecules are ionized.
C60 IR-LDI
Buda v1.2 C:\IONSPEC\FTDATA\PO010226.002
Buda v1.2 C:\IONSPEC\FTDATA\PO010226.009
MALDI : C60 spectrum, IR laser
MALDI : ir laser, c60
110
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ScaleFactor : 25.64
100
Intensity (arbitrary units)
90
80
70
60
50
40
30
20
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15:15:42.13
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100
90
Intensity (arbitrary units)
110
80
70
60
50
40
30
20
10
10
0
0
500
750
1000
1250
Mass/Charge (m/z)
1500
1750
2000
500
Buda v1.2 C:\IONSPEC\FTDATA\PO010226.004
750
1000
1500
1750
2000
Buda v1.2 C:\IONSPEC\FTDATA\PO010226.009
MALDI : C60 spectrum, IR laser, adjusting timing
MALDI : ir laser, c60
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ScaleFactor : 37.55
100
90
80
70
60
50
40
30
20
10
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ScaleFactor : 1.39
100
90
Intensity (arbitrary units)
110
Intensity (arbitrary units)
1250
Mass/Charge (m/z)
80
70
60
50
40
30
20
10
0
0
500
750
1000
1250
Mass/Charge (m/z)
1500
1750
2000
718
719
720
721
722
Mass/Charge (m/z)
723
724
725
Neutral Molecules
High velocity neutral molecules can be used to generate ions.
• Fast Atom Bombarment (FAB)
• Cesium ion gun (with deflector)
• Used heavily in the early 80's
• high energy deposition, very good for lipids
Other Ions
• Secondary Ion Mass Spectrometry (SIMS)
• Cesium ion gun (with no deflector)
• Not generally used for liquid/gaseous samples
• Commonly used for imaging of solids
• Deposits lots of energy into the molecule - causing extensive
fragmentation
• Massive Cluster Bombardment
• clusters (droplets) of glycerol ions
• lower energy deposition
• not used much, mostly in fundamentals work
Heat
• Thermal Desorption
•Solid samples placed directly on a surface that is rapidly ramped
up in temperature until the samples leap off the surface.
•Mostly an experimental technique that got dropped with the advent
of ESI/MALDI methods.
• Pyrolysis
•determining species desorbed as a function of temperature.
Combined Methods
• Chemical Ionization (CI)
•EI is used to ionize a background gas, and the background gas then
undergoes a charge-transfer reaction to the analyte.
•e.g. CH4  CH4+·  CH5+ + CH3·  A+H+ + CH4
• "somekind of" Desorption with "somekind of" postionization
• for example, laser desorption with EI post ionization.
• these are mostly fundamental methods used in physical chemistry
experiments.
• ICP-MS, plasma desorption,
Electrospray is a soft
ionization method.
Protein folding and protein
complexes are accessible
Myoglobin natural theoretical
isotope distribution
C769 H1215 N209 O221 S4
100
13C
= 1.1%
2H=0.015%
15N = 0.36%
17O=0.04%
18O=0.2%
33S=0.76%
34S=4.2%
90
80
70
60
Monoisotopic
peak
50
40
30
20
10
0
17,040
17,050
17,060
17,070
ESI-FTMS of a tryptic digest
Buda v1.2Electrospray
C:\IONSPEC\FTDATA\PO000808.002
: aldolase 107 eap digested with trypsin
110
08-AUG-2000
14:22:33.98
# Scans : 20
# Zero Fills : 1
ScaleFactor : 9.66
635
100
Intensity (arbitrary units)
90
80
70
60
670
829
50
680
690
700
1137
40
30
1332
20
1556
2274
10
0
500
750
1000
1250
1500
Mass/Charge (m/z)
1750
2000
2250
2500
ESI-FTMS of a tryptic digest can give extremely
high mass accuracy