Mass Spectroscopy
Alireza Ghassempour (PhD)
Medicinal Plants and Drugs Research Institute
Shahid Beheshti University
Evin, Tehran
Operational sequence
introduce
sample
ionize
separate by
mass/charge
detect ions
Sample introduction
Ionization methods
EI – direct interaction of electrons with sample
CI – electrons ionize reagent gases
DI – uses a pulse of energy to produce ions
SI – converts solvated molecules into ions
electron ionization
There will be different degrees of fragmentation
depending on the stability of the sample molecule
For a stable aromatic compound the primary peak is the parent ion
For a less stable cyclic compound fragmentation is predominant
Chemical ionization
Chemical ionization is a more controlled method
of ionization than electron ionization
In CI a neutral analyte (M) reacts with a reagent
ion that is generated by EI to form a variety of
molecular ions
*
Desorption ionization
1. Energetic primary ions
secondary ion mass spectrometry (SIMS)
2. Energetic atoms
fast atom bombardment (FAB)
3. Nuclear fission fragments
plasma desorption (PD)
4. Photons
laser desorption (LD)
5. Very rapid heating
desorption chemical ionization (DCI)
FAB ionization matrices
Optimal matrix properties
Strongly absorbs the energy provided
Contributes few ions to the spectrum
Interacts with the analyte to produce ions
Effectively transfers energy to the analyte ions
Diagram of an FAB gun. 1, Ionization of argon; the resulting
ions are accelerated and focused by the lenses 2. In 3, the
argon ions exchange their charge with neutral atoms, thus
becoming rapid neutral atoms. As the beam path passes
between the electrodes 4, all ionic species are deflected.
Only rapid neutral atoms reach the sample dissolved in a
drop of glycerol, 5. The ions ejected from the drop are
accelerated by the pusher, 6, and focused by the electrodes,
7, towards the analyser, 8.
Depending on the nature of the matrix we can obtain different molecular ions
Why are there two peaks?
107Ag
protonated
species
deprotonated
species
Ag salt
and 109Ag
Spray ionization methods
Spray ionization achieves the direct conversion of non-volatile,
solvated molecules into gas phase ions
Electrospray (ESI)
Electrospray ionization
ESI-MS of cytochrome c (mw = 12,360)
peak separation is 1/15
The isotope distribution also allows charge assignment, since each
isotopic peak is separated from the next by 1/n where n is the charge
Principles of MALDI






The sample is dispersed in a large excess of matrix material which will
strongly absorb the incident light.
The matrix contains chromophore for the laser light and since the matrix
is in a large molar excess it will absorb essentially all of the laser
radiation
The matrix isolates sample molecules in a chemical environment which
enhances the probability of ionization without fragmentation
Short pulses of laser light (UV, 337 nm) focused on to the sample spot
cause the sample and matrix to volatilize
The ions formed are accelerated by a high voltage supply and then
allowed to drift down a flight tube where they separate according to
mass
Arrival at the end of the flight tube is detected and recorded by a high
speed recording device
Matrix Assisted Laser Desorption
Matrices
Matrix
1,8,9-Trihydroxyanthracen
(Dithranol)
OH
OH
OH
polymers
COOH
2,5-Dihydroxybenzoic acid
(DHB)
proteins, peptides, polymers
OH
HO
-Cyano-4-hydroxycinnamic acid
N C C CH COOH
peptides, (polymers)
OH
4-Hydroxypicolinic acid
OH
oligonucleotides
N
COOH
Trans-Indol-3-acrylacid
(IAA)
COOH
N
H
polymers
Sample Preparation: Dried Droplet
solved Matrix
solved sample
Mixing and Drying
Sample Preparation: Thin Layer
solved Matrix
fast
drying
solved sample
Drying
thin homogenuous
layer of crytslas
MALDI spectra of a monoclonal
antibody (above) and of a
polymer PMMA 7100
(below).
Secondary Ion Mass
Spectrometry (SIMS)
Secondary ion
generation



The sample is prepared
in an ultra high vacuum.
A beam of primary ions
or neutral particles
impacts the surface with
energies of 3-20 keV.
A primary ion or particle causes a collision cascade
amongst surface atoms and between .1 and 10
atoms are usually ejected. This process is termed
sputtering. The sputter yield depends on the nature of
the analyte.
Static SIMS




Low ion flux is used. This means a small amount of
primary ions is used to bombard the sample per area
per unit time. Sputters away approximately only a
tenth of an atomic monolayer.
Ar+, Xe+, Ar, and Xe are the commonly used particles
present in the primary particle beam, which has a
diameter of 2-3 mm.
The analysis typically requires more than 15 minutes.
This technique generates mass spectra data well
suited for the detection of organic molecules.
Imaging SIMS







The mass spectrometer is
set to
only detect one mass.
The particle beam traces
a
raster pattern over the
sample
with a low ion flux beam,
much like
Static SIMS.
Typical beam particles consists of Ga+ or In+ and the beam
diameter is approximately 100 nm.
The analysis takes usually less than 15 min.
The intensity of the signal detected for the particular mass is
plotted against the location that generated this signal.
Absolute quantity is difficult to measure, but for a relatively
homogeneous sample, the relative concentration differences are
measurable and evident on an image.
Images or maps of both elements and organics can be
generated.
Images created using the Imaging SIMS mode.
Scanning ion image of granite from the Isle of Skye.
-University of Arizona SIMS 75 x 100 micrometers.
Mass Analyzers
Mass Analyzer divided into:
1. Scanning analysers transmit:
1.1 only the ions of a given mass-to-charge
ratio to go through at a given time (magnetic, qudrupole)
1.2. allow the simultaneous transmission of all ions (ion trap, TOF)
2. ion beam versus ion trapping types,
3. continuous versus pulsed analysis,
4. low versus high kinetic energies
The five main characteristics for mass analyser:
1. The mass range limit (Th)
2. The analysis speed (u s−1)
3. The transmission (the ratio of the number of ions reaching
the detector and the number of ions entering the mass analyser, a
quadrupole MS used in SIM mode has a duty cycle of 100 % but a
quadrupole MS scanning over 1000 amu, the duty cycle is
1/1000=0.1%. )
4. The mass accuracy (ppm)
5. The resolution.
Two peaks are considered to be resolved if the valley
between them is equal to 10% of the weaker peak
intensity when using magnetic or ion cyclotron
resonance (ICR) instruments and 50% when using
quadrupoles, ion trap, TOF, and so on.
R=m/∆m
Low resolution or high resolution is usually used to describe analysers with a
resolving power that is less or greater than about 10 000 (FWHM), respectively
The first example is human insulin, a protein having the
molecular formula C257H383N65O77S6.
The nominal mass of insulin is 5801 u using the integer
mass of the most abundant isotope of each element,
such as 12 u for carbon, 1u for hydrogen, 14 u for
nitrogen, 16 u for oxygen and 32 u for sulfur.
Its monoisotopic mass of 5803.6375 u is calculated using
the exact masses of the predominant isotope of each
element such as C=12.0000 u, H=1.0079 u, N=14.0031
u, O=15.9949 u and S=31.9721 u.
Monoisotopic mass / Average Mass
Average mass
Monoisotopic mass
Magnetic-Sector
THEORY:
The ion source accelerates ions to a kinetic energy given by:
KE = ½ mv2 = qV
Where m is the mass of the ion, v is its velocity, q is the charge on
the ion, and V is the applied voltage of the ion optics.
Magnetic-Sector
•The ions enter the flight tube and are deflected by the magnetic
field, B.
•Only ions of mass-to-charge ratio that have equal centripetal and
centrifugal forces pass through the flight tube:
mv2 /r = BqV, where r is the radius of curvature
Magnetic-Sector
mv2 /r = BqV
•By rearranging the equation and eliminating the velocity term using
the previous equations, r = mv/qB = 1/B(2Vm/q)1/2
•Therefore, m/q = B2r2/(2V)
•This equation shows that the m/q ratio of the ions that reach the
detector can be varied by changing either the magnetic field (B) or
the applied voltage of the ion optics (V).
Basis of Quadrupole Mass Filter





consists of 4 parallel metal
rods, or electrodes
opposite electrodes have
potentials of the same sign
one set of opposite electrodes
has applied potential of
[U+Vcos(ωt)]
other set has potential of
- [U+Vcosωt]
U= DC voltage, V=AC voltage,
ω= angular velocity of
alternating voltage
The trajectory of an ion will be stable if the values of x and y never reach r0, thus if it never hits
the rods. To obtain the values of either x or y during the time, these equations need to be
integrated. The following equation was established in 1866 by the physicist Mathieu :
Mass analyzers
An ion trap is a device that uses an oscillating electric
field to store ions.
The ion trap works by using an RF quadrupolar field that
traps ions in two or three dimensions (2D and 3D).
Ion Trap MS



Ions are trapped by applying
rf frequencies on the ring
electrode and endcaps
Then ions are scanned out
of the trap by m/z as the
base mass voltage is
increased over time
This is accomplished by
maintaining in the trap a
pressure of helium gas
which removes excess
energy from the ions by
collision.
Time of Flight Schematic
• The Back Plate and Grid are used to accelerate the ions
• The Ion source is used to ionize the Sample
• The Sample Inlet introduces the sample to the source
• The vacuum is used to maintain a low pressure
• The Drift Region separates the ions according to their mass
• The Detector outputs current as each ion strikes it
• The Oscilloscope displays the detector output
Time-of-Flight Converted to Mass

An accelerating potential (V) will give an ion of charge z an energy of zV. This can be
equated to the kinetic energy of motion and the mass (m) and the velocity (v) of the ion

zV = 1/2mv2

Since velocity is length (L) divided by time (t) then

m/Z = [2Vt2]/L2

V and L cannot be measured with sufficient accuracy but the equation can be rewritten

m/Z = B(t-A)2
where A and B are calibration constants that can be determined by calibrating to a known
m/Z
Reflectron TOF-MS Improved mass resolution in MALDI TOF-MS has been obtained by the utilization of a
single-stage or a dual-stage reflectron (RETOF-MS). The reflectron, located at the end of the flight tube, is
used to compensate for the difference in flight times of the same m/z ions of slightly different kinetic
energies by means of an ion reflector. This results in focusing the ion packets in space and time at the
detector
A typical MALDI mass spectrum of substance P in CHCA (see Table 1) employing both linear and
reflectron TOF-MS in the continuous ion extraction mode with a 500 MS/s transient digitizer is
shown. The maximum mass resolution observed in the linear mass spectrum of substance P
employing continuous ion extraction is about 600 which is typical for a peptide of this size. Only the
average chemical mass can be determined from this mass spectrum. In the reflectron mass
spectrum, the isotopic multiplet is well resolved producing a full width half maximum (FWHM) mass
resolution of about 3400
FT-ICR-MS instrument general scheme
FTICR: New Dimensions of High
Performance Mass Spectrometry
Ions are trapped and oscillate with low,
incoherent, thermal amplitude
Excitation sweeps resonant ions into a large,
coherent cyclotron orbit
Preamplifier and digitizer pick up the induced
potentials on the cell.
FTICR: New Dimensions of High
Performance Mass Spectrometry
The frequency of the cyclotron gyration of an ion
is inversely proportional to its mass-to-charge
ratio (m/q) and directly proportional to the
strength of the applied magnetic field B.
Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Detection electrodes
100
80
60
Intensity [%]
Intensity [%]
40
0
Time
Time
+
20
0
-20
-40
-60

v
+

B

-80
-100
Excitation
electrodes
Fourier
Transform
Fourier Transform
 
FL  qv  B
Frequency
qB0
f 
2m
High Resolution of FTICR MS
Ubiquitin (14+)
Mass resolution: 170,000
(up to 5,000,000)
Finnigan LTQ FT Ultra
51
Finnigan LTQ FT Ultra
52
Biological samples
Ceribrospinal fluid (CSF), 1µL Injection volume
200 nL/min, Nanospray,
D:\Data\...\HUPO\CSF79_120-30_SIM_LC154
18.08.2005 06:06:46
RT: 0.00 - 150.00
95.17
100
NL:
2.70E8
TIC F:
MS
CSF79_120
30_SIM_LC
154
TIC  Separation of peptides
90
80
56.02
70
69.62
60
104.48
80.90
50.39
22.48
50
80.54
56.58
20.47
70.07
141.41
86.39
40
96.92
90.53
30
38.31
20
26.79
10
35.99
20.11
9.68 14.28 18.48
0
0
10
143.18
73.74
110.76 114.57
117.43
65.74
44.45
33.35
20
30
40
50
MS  Peptide mass
CSF79_120-30_SIM_LC154 #7925 RT: 49.57 AV: 1 NL: 1.40E6
T: FTMS + p NSI Full ms [ 300.00-2000.00]
660.91
100
60
70
80
Time (min)
90
100
110
130.52
134.06
123.06
120
130
Retention Time, min
MS/MS  Peptide sequence
140
CSF79_120-30_SIM_LC154 #7929 RT: 49.60 AV: 1 NL: 2.01E5
T: ITMS + c NSI d Full ms2 661.51@23.00 [ 170.00-2000.00]
838.67
100
660.91
90
90
499.75
80
80
551.09
60
50
451.27
40
825.89
30
394.99
70
Relative Abundance
70
60
767.20
50
40
30
702.55
613.77
20
10
20
435.77
542.31
330.46
737.35
319.86
850.90
10
936.72
1080.97
337.49
1207.85
0
743.35
458.38
1028.77 1138.85
1214.29 1396.21
876.52
0
400
600
m/z
m/z
800
1000
1200
200
400
600
800
m/z
1000
m/z
1200
1400
Finnigan LTQ FT Ultra – MS/MS
54
Biological samples
Ceribrospinal fluid (CSF), 1µL Injection volume
200 nL/min, Nanospray,
D:\Data\...\HUPO\CSF79_120-30_SIM_LC154
18.08.2005 06:06:46
RT: 0.00 - 150.00
95.17
100
NL:
2.70E8
TIC F:
MS
CSF79_120
30_SIM_LC
154
TIC  Separation of peptides
90
80
56.02
70
69.62
60
104.48
80.90
50.39
22.48
50
80.54
56.58
20.47
70.07
141.41
86.39
40
96.92
90.53
30
38.31
20
26.79
10
35.99
20.11
9.68 14.28 18.48
0
0
10
143.18
73.74
110.76 114.57
117.43
65.74
44.45
33.35
20
30
40
50
MS  Peptide mass
CSF79_120-30_SIM_LC154 #7925 RT: 49.57 AV: 1 NL: 1.40E6
T: FTMS + p NSI Full ms [ 300.00-2000.00]
660.91
100
60
70
80
Time (min)
90
100
110
130.52
134.06
123.06
120
130
Retention Time, min
MS/MS  Peptide sequence
140
CSF79_120-30_SIM_LC154 #7929 RT: 49.60 AV: 1 NL: 2.01E5
T: ITMS + c NSI d Full ms2 661.51@23.00 [ 170.00-2000.00]
838.67
100
660.91
90
90
499.75
80
80
551.09
60
50
451.27
40
825.89
30
394.99
70
Relative Abundance
70
60
767.20
50
40
30
702.55
613.77
20
10
20
435.77
542.31
330.46
737.35
319.86
850.90
10
936.72
1080.97
337.49
1207.85
0
743.35
458.38
1028.77 1138.85
1214.29 1396.21
876.52
0
400
600
m/z
m/z
800
1000
1200
200
400
600
800
m/z
1000
m/z
1200
1400
LC-MS/MS
56
Orbitrap
- a new type of FTMS
Principle
of Trapping
the Orbitrap
Click
to editinMaster
title
style
 The Orbitrap is an ion trap – but there are no RF or
magnet fields!
• Moving ions are trapped around an electrode
- Electrostatic attraction is compensated by centrifugal
force arising from the initial tangential velocity.
Analogon: a satellite is “trapped” around the Earth;
gravitational attraction is compensated by centrifugal
force arising from initial tangential velocity.
• Potential barriers created by end-electrodes confine
the ions axially
• The frequencies of oscillations (especially the axial
ones) could be controlled by shaping the electrodes
appropriately
• Thus we arrive at …
Orbital traps
Kingdon (1923)
58
LTQ Orbitrap™ Hybrid Mass Spectrometer
Launched in Summer 2005
Finnigan LTQ™ Linear Ion Trap
API Ion source
Linear Ion Trap
Differential pumping
C-Trap
Orbitrap
Differential pumping
Inventor: Dr. Alexander Makarov, Thermo Fisher Scientific (Bremen, Germany)
LTQ Orbitrap Operation Principle
1. Ions are stored in the Linear Trap
2. …. are axially ejected
3. …. and trapped in the C-trap
4. …. they are squeezed into a small cloud and injected into the Orbitrap
5. …. where they are electrostatically trapped, while rotating around the central electrode
and performing axial oscillation
The oscillating ions induce an image current into the two
outer halves of the Orbitrap, which can be detected using
a differential amplifier
Ions of only one mass generate a sine
wave signal
Frequencies and Masses
The axial oscillation frequency follows the formula w 
Where
w = oscillation frequency
k
= instrumental constant
m/z = …. well, we have seen this before
Many ions in the Orbitrap generate a complex
signal whose frequencies are determined using a
Fourier Transformation
k
m/ z
What‘s the size of the Orbitrap?
LTQ Orbitrap™ Hybrid Mass
Spectrometer
Tandem mass spectrometers
Uses of tandem mass spectrometry:
(1) Characterize individual compounds in complex mixtures
(2) Completely identify the molecular structure of a compound
To accomplish either of these goals mass analysis
must be carried out twice in a tandem instrument
This can be achieved either by separating the
mass analysis operations in space or in time
Tandem mass spectrometers
Information obtained from MS experiments
GC-MS
LC-MS
-TOF Product (185.1): 1.267 to 2.317 min from Sample 5 (ESI-: pinic P185 CE=-6, CAD=2) of 0...
a=3.56228408459780370e-004, t0=4.72207389233553840e+001
Max. 170.6 counts.
185
170
Chemical Ionization
(CI)
160
150
MS
140
130
In te n s ity , c o u n ts
120
110
Molecular weight
100
90
80
70
60
50
40
30
20
10
-TOF Product
(185.0): 1.750 to 3.300 min from pinic_P185.wiff
0
40
50
60 t0=4.61223072761931690e+001
70
80
90
100
a=3.56215314471457500e-004,
Max. 46.2 counts.
110
120
m/z, amu
130
140
150
160
170
180
141.09268
Electron impact Ionization
(EI)
45
40
MS/MS
COOH
I n t e n s it y , c o u n t s
35
30
Structural information
COOH
25
20
15
10
185.08193
123.08251
5
0
115
120
125
167.07195
130
135
140
145
150
155
160
m/z, amu
165
170
175
180
185
190
195
190
Separation in space can be achieved by coupling two mass analyzers
For example, a sector magnet (MS 1) can be coupled to a
quadrupole mass filter (MS 2)
A parent ion is selected by the sector magnet and
separated from the other ions in the sample
Each selected ion is activated by a collision process
The resulting set of product ions are analyzed by the
quadrupole mass filter
Tandem mass spectrometers
The most common tandem mass spectrometer
is a triple quadrupole mass spectrometer
Mass Spec Ion Detectors




Faraday Cup
Electron Multiplier and Channel Electron
Multiplier
Microchannel Plate
Daly Detector (Scintillation Counter or
Photomultiplier)
Electron Multipliers
Continuous Electron Multiplier
Photomultiplier (Daly detector)
Photomultiplier





Also called daly detector or scintillation counter
Metal dynode emits secondary electrons
Secondary electrons hit phosphorus screen and
trigger photon emission; photon abundance
measured by photomultiplier
Advantage: keep detector in vacuum -- no
contamination = low noise and long lifetime
Disadvantage: cannot be exposed to light
Data Collection

Typically, the computer controls:




Scanning of mass spec
Data acquisition
Data processing
Interpretation of data (generation of spectra;
possible comparison to spectra in database)
Sequencing Using MALDI
When the molecular mass of a peptide is known, for example Gly2Tyr = 296, we may use a
digested, or fragmented effect of MALDI to learn the sequence of the peptide.
Backbone (peptide bond) cleavages in the mass spectrometer generate two types of ions.
Acylium ions are produced when the charge is retained on the N-terminal side of the
peptide, while protonated peptides are produced when the charge is retained on the Cterminal side of the peptide.
These ion fragments are known as bn and yn fragments respectively. In straightforward
cases, all possible fragments are produced using prior peptide digestion. This means that
for a given peptide sequence, two unique sets of fragment ions are produced (the bn and yn
sets).
In the following table, the “predicted” fragments for a given peptide are listed. Comparison
of these predicted fragments with experimentally observed peptide fragments allows for a
given peptide mass to be assigned to a unique peptide sequence.
Formulas for prediction of mass spectrometer fragments during
the analysis of peptides.
Two series (bn and yn) are produced, depending on the peptide
end on which cleavage occurs
b1
Mresidue + H
y1
Mresidue + 19
b2
Mresidue + b1
y2
Mresidue + y1
bn-1
MH+ - 18 - Mresidue
Yn-1
MH+ - Mresidue
Example
Consider the peptide GlyTyr. Two possible sequences are possible:
H-Gly-Tyr-OH
H-Tyr-Gly-OH
The predicted fragments for these two sequences would be:
H-Tyr-Gly-OH
H-Gly-Tyr-OH
b1 = 164
b1 = 58
b2 = 221
b2 = 221
y1 = 76
y1 = 182
y2 = 239
y2 = 239
Suppose experimentally, we detected fragments at mass 164, 221 but none at 239.
This missing mass is sufficient to identify the sequence as H-Tyr-Gly-OH.
Peptide Fingerprinting

MALDI may be used to determine an exact
“peptide fingerprint” of a section of a protein
that carries a “disease” or differs from the
normal protein in some way.
Image Analysis for Differential Protein Expression
C-1
Digitized Image
Tox-1
Standard Control
2D Gel
C-2
Tox-2
C -3
Tox-3
Results: 196 features detected; 40 ( ) at 2-fold change.
Digitized PDQuest image
following matchset comparison,
which allows comparison of all
control versus all toxicant
images. Each black spot
represents a protein; spots
circled in yellow indicate prot.
eins with at least a 2-fold change
based on spot intensity or
statistical test
Protein Identification by MALDI-MS
• Differentially expressed proteins
identified by image analysis are
excised from 2D gels and trypsin
digested. The resulting peptide
fragments are analyzed on a MALDI
mass spectrometer (MS).
• The MALDI spectra displays a
“peptide fingerprint” of the protein
using corresponding peptide masses.
Database Searching using MALDI-MS Ions for
Protein Identification
• Proteins are identified by entering the
masses (ions from MALDI spectrum) of
the peptides into a peptide mapping
database such as ProFound.
• Search parameters are refined by
including experimental mass and
isoelectric point (pI) determined by 2D
PAGE, as well as by taxonomic category
and any modifications.
http://prowl.rockefeller.edu/cgi-bin/ProFound