1
Mass spectrometry of biomolecules 2010 1
C7895 Mass Spectrometry of Biomolecules
Jan Preisler
Analytical chemistry division, Chemistry dept. A14-312, tel.: 549496629
preisler@chemi.muni.cz
The course is focused on mass spectrometry of biomolecules, i.e. ionization
techniques MALDI and ESI, modern mass analyzers, such as time-of-flight
MS or ion traps and bioanalytical applications. However, the course covers
much broader area, including inorganic ionization techniques, virtually all
types of mass analyzers and hardware in mass spectrometry.
Schedule of lectures for 2010
The lectures will take place in A14-207 every Wednesday 14:00 – 15:50. The
changes will be announced in advance.
Consultations
Please contact me in advance to make an appointment.
Mass spectrometry of biomolecules 2010 2
Content
I. Introduction
II. Ionization methods and sample introduction
III. Mass analyzers
IV. Biological applications of MS
V. Example problems
Mass spectrometry of biomolecules 2010 3
Preliminary Schedule of the Lectures for 2004
I. 22. 9. A Brief Historical Perspective: Overview of Techniques and
Technology. Basic Concepts of MS (resolution, sensitivity). Isotope
patterns of organic molecules. Ionization techniques and sample
introduction. Electron impact ionization (EI).
II. 29. 9. Chemical Ionization (CI). Glow Discharge. Inductively Coupled
Plasma (ICP). Field Ionization/Desorption. Fast Atom Bombardment
(FAB). Secondary Ion Mass Spectrometry (SIMS). Photoionization (PI).
Plasma Desorption (PD)
III. 6. 10. Laser Desorption (LD). Matrix-Assisted Laser Desorption/
Ionization (MALDI).
IV. 13. 10. Thermospray (TSI). Ionspray (IS). Electrospray (ESI). Mass
Spectrometers: Ion Optics. Wein Filter. Energy Analyzer (E).
V. 20. 10. Magnetic Sector (B). Quadrupole Filter (Q). Ion Trap (IT).
VI. 27. 10. Linear Trap (LT). Ion Cyclotron Resonance Fourier Transform
Mass Spectrometer (FT-ICR-MS). Orbitrap. Electrostatic Trap.
Simulation of Ion Movement (Simion), examples.
Mass spectrometry of biomolecules 2010 4
VII. 3. 11. Time-of-Flight Mass Spectrometer (TOFMS). Techniques for
Enhanced Resolution in TOF MS (Reflector, Delayed Extraction and
Orthogonal Extraction).
VIII. 10. 11. Ion Dissociation (CID, SID, ECD, ETD, IRMPD). Tandem
Mass Spectrometry (MS/MS). In-Source Decay (ISD), Post-Source
Decay (PSD). New Techniques (TOF-TOF, LIFT). Ion mobility
spectrometry (IMS).
IX. 17. 11. Vacuum: Principles & Techniques. Detectors. Data
Acquisition. Coupling of Separation and MS (on-line, off-line, chips)
X. 24. 11. Applications: Proteins and Peptides. Protein Identification:
Peptide Mapping, Sequence Tag, Accurate Mass Tag.
XI. 3. 12. Proteins and peptides. Isotope Labelling. ICAT. Sequence
Determination. Post-translational Modification.
XII. 10. 12. Disulfide Bridge Analysis. Proteins. MS databases. DNA,
Saccharides, Synthetic Polymers.
XIII. 17.12. Christmas consultation session
Preliminary Schedule of the Lectures for 2004
Mass spectrometry of biomolecules 2010 5
Introduction to Mass spectrometry. Brief History of MS. A Survey
of Methods and Instrumentation. Basic Concepts in MS:
Resolution, Sensitivity. Isotope patterns of organic molecules.
Ionization Techniques and Sample Introductin. Electron Impact
Ionization (EI)
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Mass spectrometry of biomolecules 2010 6
I. Introduction
• Information sources about mass spectrometry
• Brief history of mass spectrometry, a survey of methods and
instrumentation
• Basic concepts of mass spectrometry
• Isotope patterns of organic molecules.
2
Mass spectrometry of biomolecules 2010 7
Study Material
Lecture notes
Advice: please take notes, but do not copy the slides; the English slides will
be provided at the end of the semester. The Czech slides can be found
on the internet: http:\\bart.chemi.muni.cz
Additional literature
• Robert J. Cotter: Time-of-Flight Mass Spectrometry - Instrumentation and
Applications in Biological Research, American Chemical Society, 1997.
• Richard B. Cole et al.: Electrospray Ionization Mass Spectrometry Fundamentals,
Instrumentation & Applications, John Wiley & Sons, Inc.,
1997.
Mass spectrometry of biomolecules 2010 8
Additional Sources of Information
Internet
Textbook http://www.ms-textbook.com/
Comprehensive information source www.spectroscopynow.com
Laboratories, e.g. www.mpi-muelheim.mpg.de/stoecki/mass_server.html
Protein Prospector prospector.ucsf.edu
Proteometrics - PROWL www.proteometrics.com
Etc etc.
Specialized journals
International Journal of Mass Spectrometry
Journal of Mass Spectrometry
Journal of the American Society for Mass Spectrometry
Mass Spectrometry Reviews
Rapid Communications in Mass Spectrometry
Mass spectrometry of biomolecules 2010 9
Ionization Techniques and Sample Introduction
Glow discharge (GD)
Electron impact ionization (EI)
Chemical ionization (CI)
Field ionization (FI)
Inductively coupled plasma (ICP)
Fast atom bombardment (FAB)
Secondary ion mass spectrometry (SIMS)
Thermospray (TSI)
Ionspray (IS)
Elektrospray (ESI)
Plasma Desorption (PD)
Laser Desorption (LD)
Matrix-assisted laser desorption/ionization (MALDI)
Coupling of separation and mass spectrometry (on-line, off-line,
microdevices)
Mass spectrometry of biomolecules 2010 10
Mass Spectrometers
Ion optics. Simulation of ion movement, Simion
Energy analyzers
Magnetic sectors
Quadrupole filter
Ion cyclotron with Fourier transformation (ICR-FT-MS)
Ion trap (IT), linear trap (LT)
Time-of-flight mass spectrometer (TOFMS)
New mass spectrometers: Orbitrap, TOF-TOF, LIFT-TOF
Tandem mass spectrometry (MS/MS, MSn)
Collision induced dissociation (CID)
Surface induced dissociation (SID)
In source and post source fragmentation (ISF and PSD)
Principles of vacuum instrumentation
Detectors a detection electronics
Chromatography - MS (on-line, off-line, in-line, microdevices)
Mass spectrometry of biomolecules 2010 11
MS Applications
Analysis of biological compounds:
• Proteins, peptide mapping, protein databases, new methods (ICAT)
• Peptide analysis (disulfide bonds, post-translational modifications)
• Nucleic acids
• Saccharides
Analysis of synthetic polymers
and more…
Mass spectrometry of biomolecules 2010 12
Brief History of Mass Spectrometry
1803 Dalton atomic theory
―mass consists of atoms; all atoms
of a kind have the same mass‖
… not really: isotopes…
Proof of existence of isotopes:
- optical spectroscopy: a slight shift
of spectral lines
... requires a very high quality
instrument
- MS: easy determination
3
Mass spectrometry of biomolecules 2010 13
Glow Discharge Ionization
1880’s Crookes: glow discharge
H2 + H2
+  (H2
+)* + H2
(H2)*  H2 + 4 hn
vrstva iontu
------
++
++++p ~ 0.2 Pa
viditelný negativní výboj+ U
~ 700 V DC
I ~ 1 mA
o
glow discharge
visible discharge
Mass spectrometry of biomolecules 2010 14
The First Mass Spectrometer
1911 J. J. Thompson: Parabola MS (Phil Mag. 1911, 21, 225)
―Rays of positive electricity‖ 1913
Glow discharge in Ne at 1 Torr, hollow cathode, magnet
+
Ne
dutákatoda
fotografickádeska
magnet
selektrodami
-
vakuová
pumpa
doutnavý
výboj v Ne,
1 Torr
-
+
vacuum
pump
Ne
1 Torr
glow
discharge
magnet
electrodes
hollow
cathode
photographicplate
Mass spectrometry of biomolecules 2010 15
Photographic plate as a detector: 20Ne and 22Ne lines
The First Mass Spectrometer
20Ne+22Ne+
Mass spectrometry of biomolecules 2010 16
Magnetic Sector with Energy Analyzer
1919 F. W. Aston: Mass Spectrograph (Phil. Mag. 1919, 38, 209)
Magnetic sector with electrostatic energy analyzer
Abundance of most natural isotopes determined by 1930
In Nobel prize ceremony lecture, 1934: ―MS is dead, everything's done …‖
(+)
(+)
(-)
extrakce
a fokusace
(-)
+ + +
+ + +
+ + +
B
r = f(m/z)
selekce
kinetické
energie
štèrbina
analýza
hmotnosti
zdroj
detekce
kinetic
energy
analysis
mass
analysis
ion
source
extraction,
focusing
slit
detection
Mass spectrometry of biomolecules 2010 17
But the History of MS Goes on ...
1940 C. Berry: Electron impact ionization (EI) for ionization of organic
compounds
1950-70 MS applied mostly in structural analysis of organic compounds
1980+ Analysis of heavy molecules due to new ionization techniques:
FAB, PD, ESI a MALDI
2006 MS for qualitative,structural and quantitative analysis
Wide scale of commercial mass spectrometers available
MS necessary for analysis of organic and biological molecules
Biospectrometry
Mass spectrometry of biomolecules 2010 18
Basic Concepts of Mass Spectrometry
Mass spectrometer
instrument, in which ions are formed from analytes and their mass-tocharge
ratio is analyzed
Components of mass spectrometer
1. Ion source chamber (contains device for sample introduction, ion optics)
2. Mass analyzer (ion optics, electrodes, magnets, detector)
3. Vacuum pumps (rough, high and ultrahigh vacuum)
4. Control and data processing unit, software
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Mass spectrometry of biomolecules 2010 19
Mass Spectrum
Ion signal vs. m/z
Ion signal
charge, current, often converted to voltage, arbitrary units
signal normalization: intensity of the dominant peak = 100%
0
10
20
30
40
50
60
70
80
90
100
%Int.
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500
Mass/Charge
normalization of ion signal
Mass spectrometry of biomolecules 2010 20
Mass Spectrum
Mass, m
a.m.u., u, Da (Dalton), molecular weight
number of atom mass units numerically equivalent to molar weight
m/z ... mass-to-charge ratio, Th (Thomson)
Number of charges, z:
number of elemental charges of an ion
usually ±1
exceptions, e.g. electrospray-generated ions: |z| >> 1
Pozitivní and negative ions, not cations and anions.
Mass spectrometry, not spectroscopy.
Mass spectrometry of biomolecules 2010 21
Selected Powers in Mass Spectrometry
Mass resolution
A measure of separation of two adjacent peaks.
Two definitions:
1. FWHM (full width at half maximum), R = m/m
2. Max. mass (m/z), at which two adjacent peaks with a unit mass
difference may be resolved.
Ion energy, W
Instead of Joule (J) use electronVolts (eV) and atoms or ions rather than
moles
1 eV = 1.6 x 10-19 J
simplicity: acceleration voltage = 100 V, charge = 1 ... W = 100 eV
can be easily compared with ionization energy, bond energy, photon energy...
Pressure, p
1 atm = 760 Torr = 101 325 Pa = 1.01325 bar = 14.70 PSI
Mass spectrometry of biomolecules 2010 22
Abbreviations
m mass of ion, atom, molecule (u, a.m.u., Da)
z number of charges; (-)
m/z mass-to-charge ratio (Th, Thomson)
e elemental charge (1.6x10-19C)
U voltage (V)
E intensity of electric field (V/m, N/C)
W energy, labor (eV, J)
v ion velocity (m/s)
r curvature radius (m)
L path (m)
l mean free path of a molecule
t time (s)
s collision diameter (m)
s2 collision cross-section (m2)
m reduced mass (a.m.u., Da, kg)
Mass spectrometry of biomolecules 2010 23
Abbreviations
n numerical concentration (m-3)
I current (A), flux (m-2), intensity (-)
T absolute temperature (K)
p pressure (Pa, Torr)
R resolution (-)
f frequency (Hz)
w angular frequency (rad/s, s-1)
a direct proportion
LD detection limit, also LOD, limit of detection (mol, g, M)
S/N signal-to-noise ratio
RSD relative standard deviation
Mass spectrometry of biomolecules 2010 24
Isotope Patterns of Organic Molecules
Carbon isotopes: 99% 12C, 1% 13C
Pattern as a function of number of carbon atoms in the molecule:
C: 99% 12C 1% 13C
C2: 98% 12C12C 2% 12C13C 0.01% 13C13C
C3: 97% 12C12C12C 3% 12C12C13C 0.04% 12C13C13C 10-4 % 13C13C13C
Binomic formula
Relative abundance of the light isotope, a
Relative abundance of the heavy isotope, b
Number of atoms, n
E.g. for n = 2: (a+b)2 = a2 + 2ab + b2
Monoisotopic molecule contains given atoms in form of a single isotope.
In the case of biomolecules usually carbon atoms in the form of 12C.
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Mass spectrometry of biomolecules 2010 25
Isotope Patterns of Organic Molecules
Relative abundance of molecules (%):
C60: 12C60 100
12C59
13C 66
12C58
13C2 21
12C57
13C3 4.6
C100: 12C100 100
12C99
13C 110
12C98
13C2 60
12C97
13C3 22
(normalized with respect to the monoisotopic molecule /only 12C/ = 100 %)
With increasing number of carbon atoms, n, the relative intensity of the
monoisotopic form decreases, the monoisotopic peak is not dominant any
more and intensities of other isotopic forms are comparable (wide
envelope) ... see examples below.
Mass spectrometry of biomolecules 2010 26
Practical Impact of Isotope Abundance
- Decrease of sensitivity
- High resolution R necessary at high m/z for correct determination of m/z
+ Use of isotopic internal standards. The best internal standards.
Example:
5 peptides/proteins with relative abundance of elements
C : H : N : O : S = 30 : 45 : 6 : 6 : 1
R = 20 000
C30H45N6O6S C60H90N12O12S2
C90H135N18O18S3 C180H270N36O36S6 (also shown for more R)
C270H405N54O54S9 C360H540N272 O272S12
C900H1350N180O180S30 C1800H2700N360O360S60
Mass spectrometry of biomolecules 2010 27
0
20
40
60
80
100
%Int.
616 617 618 619 620 621 622 623 624 625
Mass/Charge
Molecular formula: C30H45N6O6S Resolution: 20000 at 50%
617.3
618.3
619.3
620.3 621.3 622.3 623.3
Mass spectrometry of biomolecules 2010 28
0
20
40
60
80
100
%Int.
1234 1236 1238 1240
Mass/Charge
Molecular formula: C60H90N12O12S2 Resolution: 20000 at 50%
1234.6
1235.6
1236.6
1237.6
1239.6
Mass spectrometry of biomolecules 2010 29
0
20
40
60
80
100
%Int.
1852 1854 1856 1858
Mass/Charge
Molecular formula: C90H135N18O18S3 Resolution: 20000 at 50%
1852.9
1851.9
1853.9
1854.9
1855.9
1857.9
Mass spectrometry of biomolecules 2010 30
0
20
40
60
80
100
%Int.
3704 3706 3708 3710 3712 3714
Mass/Charge
Molecular formula: C180H270N36O36S6 Resolution: 20000 at 50%
3705.9
3704.9
3707.9
3708.9
3703.9
3709.9
3710.9
3712.9
6
Mass spectrometry of biomolecules 2010 31
0
20
40
60
80
100
%Int.
5555 5560 5565 5570
Mass/Charge
Molecular formula: C270H405N54O54S9 Resolution: 20000 at 50%
5559.8
5560.8
5557.8
5561.8
5562.85556.8
5563.8
5564.85555.8
5566.8 5569.8
Mass spectrometry of biomolecules 2010 32
0
20
40
60
80
100
%Int.
13405 13410 13415 13420 13425
Mass/Charge
Molecular formula: C360H540N272O272S12 Resolution: 20000 at 50%
13414.4
13412.4
13416.4
13411.3
13417.4
13410.3 13418.4
13419.4
13409.3
13420.4
13408.3
13422.4
Mass spectrometry of biomolecules 2010 33
0
20
40
60
80
100
%Int.
18520 18530 18540
Mass/Charge
Molecular formula: C900H1350N180O180S30 Resolution: 20000 at 50%
18533.4
18535.418530.4
18536.4
18529.4
18537.5
18528.3
18538.5
18527.3
18539.5
18526.3
18541.5
18524.2
18544.6
Mass spectrometry of biomolecules 2010 34
0
20
40
60
80
100
%Int.
37050 37060 37070 37080
Mass/Charge
Molecular formula: C1800H2700N360O360S60 Resolution: 20000 at 50%
37066.0
m/z ~ 12
Mass spectrometry of biomolecules 2010 35
0
20
40
60
80
100
%Int.
3695 3700 3705 3710 3715 3720
Mass/Charge
Molecular formula: C180H270N36O36S6 Resolution: 500 at 50%
3706.6
m/z ~ 9
Mass spectrometry of biomolecules 2010 36
0
20
40
60
80
100
%Int.
3695 3700 3705 3710 3715 3720
Mass/Charge
Molecular formula: C180H270N36O36S6 Resolution: 2500 at 50%
3706.2
m/z ~ 4.5
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Mass spectrometry of biomolecules 2010 37
0
20
40
60
80
100
%Int.
3695 3700 3705 3710 3715 3720
Mass/Charge
Molecular formula: C180H270N36O36S6 Resolution: 5000 at 50%
3705.9
3704.8
3707.9
3708.9
3703.8
3709.9
3711.0
3714.0 3718.0
Mass spectrometry of biomolecules 2010 38
0
20
40
60
80
100
%Int.
3695 3700 3705 3710 3715 3720
Mass/Charge
Molecular formula: C180H270N36O36S6 Resolution: 10000 at 50%
3705.9
3704.9
3707.9
3708.9
3703.9
3709.9
3710.9
3713.9 3717.9
Mass spectrometry of biomolecules 2010 39
Multiply-charged ions
• Typical example: multiply-charged ions [M+zH]z+ by electrospray
Bell-shaped envelope
• The gaps between peaks are not equidistant (contrary to isotope pattern).
Example:
A small protein with M.W. = 10 000 Da
z 4 5 6 7 8 9 10
m/z 2501 2001 1668 1429 1251 1112 1001
m/z
S
Mass spectrometry of biomolecules 2010 40
What Can Be Said about This Spectrum?
Note: This is a portion of mass spectrum of a single organic compound.
m/z values were determined with accuracy ~ 0.1.
m/z
S
1000.0
1000.3 1000.7
1001.0
Mass spectrometry of biomolecules 2010 41
II. Ionization Techniques and Sample Introduction
sample (atm. pressure) → sample (vacuum)
Unwanted phenomena
• pressure increase in the ion source
• cooling and freezing of solvent due to solvent evaporation
• adsorption of compound (e.g. water) on the walls of the ion source
chamber
Classification of samples acc. to the state
• Fluid samples
gaseous
liquid (liquid analyte, dissolved analyte)
• Solid samples
volatile (usually light compounds)
nonvolatile (polar, heavy, polymer compounds)
Mass spectrometry of biomolecules 2010 42
probe
vent
vent atmospheric pressure
ion source
(vacuum)
sample
seal
to pump
c
o
probe
vent
vent atmospheric pressure
ion source
(vacuum)
sample
seal
to pump
c
o
Off-line
On-line
Number of introduced samples
• 1 sample
• more samples
• in a queue, sample series (discrete samples or continual flow)
• in paralel
probe
vent
vent atmospheric pressure
ion source
(vacuum)
sample
seal
to pump
o
probe
vent
vent atmospheric pressure
ion source
(vacuum)
sample
seal
to pump
c
c
Sample Introduction
8
Mass spectrometry of biomolecules 2010 43
Electron Impact Ionization, EI
Classical ionization technique.
Electrons emitted from a heated filament are accelerated using a medium
voltage.
Electron energy, W(e-) = acceleration potential x charge (1).
Typical energy: 70 eV.
+15 V
-55 V
e-
ABC+
chamber
heated
filament
×
inlet of gaseous sample ABC
orthogonally oriented to
the electron beam (×)
Mass spectrometry of biomolecules 2010 44
Mechanism of EI
Interaction of electron with analyte molecule ABC:
e- (fast) + ABC  ABC+ + 2 e- (slow)
Total equation,
ABC  ABC+ + eis
characterized by ionization energy ABC, H(ABC).
Ions ABC+ with energy excess can undergo fragmentation:
(ABC+)*  AB+ + C, A + CB+ etc
Fragmentation extent depends on electron energy, E(e-) and on the analyte
structure:
a) W(e-) ~ ionization potential  production of molecular ions.
Ionization potential of simple organic molecules ~10 – 12 eV.
b) W(e-) >> ionization potential  fragmentation.
Type of fragmentation depends on analyte structure; compounds of similar
structure have similar fragmentation spectra.
Interpretation of spectra. Spectral libraries (> 100 000 spekter).
Mass spectrometry of biomolecules 2010 45
Mechanism of EI
Appearance energy (AE), at which the fragments AB+ appears, does not
have to be higher than H(ABC)!
ABC  ABC+ + e- Ionization energy ABC, H(ABC)
ABC  AB+ + C + e- Threshold energy AB+, AE(AB+)
AE(AB+) = H(ABC) + D(ABC+) Dissociation energy ABC+,
D(ABC+)
Absorption of electron during travel through the analyte
Reduction of electron flux, dI during the travel through infinitesimally thin
analyte layer:
dI = -acIdx,
after integration:
I = Io e-acx.
I electron flux (A)
c concentration of ABC, (cm-3) (c = p/RT)
x layer thickness (cm)
a cross-section (cm2) … analogy of e coefficient in the Lambert-Beerově law
Mass spectrometry of biomolecules 2010 46
Chemical Ionization (CI). Glow Discharge. InductivelyCoupled
Plasma (ICP). Field Ionization/Desorption. Fast
Atom Bombardment (FAB). Secondary Ion Mass
Spectrometry (SIMS). Photoionization (PI). Plasma
Desorption (PD)
2
Mass spectrometry of biomolecules 2010 47
Chemical Ionization (CI)
20th of the 20th century A. J. Dempster
The same source as in the case of the EI plus an inlet for reagent gas
Reagent gas (RH)
CH4, butane, H2, NH3 etc.
p(ABC) < 10-4 Pa
p(R) ~ 0.1 Pa (l ~ 0.05 mm, many collisions in the source)
Mechanism of ion formation
1) production of RH+
e- (fast) + RH  RH+ + 2 e- (slow)
2a) charge transfer
RH+ + ABC  RH + ABC+
Mass spectrometry of biomolecules 2010 48
Mechanism of Ion Formation at CI (cont.)
2b) proton transfer (more common)
RH+  R + H+ PA(R) proton affinity
ABC + H+  ABCH+ - PA(ABC)
RH+ + ABC  R + ABCH+ E = PA(R) - PA(ABC)
E < 0: exothermic, preferred reaction
E << 0: energy excess at ABCH+  fragmentation of ABCH+
structural analysis
E < 0, E  0  ABC+ a ABCH+ dominate
+ quantitative analysis
+ determination of molecular weight of ABC
+ high ionization efficiency (ABC)
- no structural information
9
Mass spectrometry of biomolecules 2010 49
Collisions during CI
Mean free path
Mean path, which a particle travels between 2 collisions
l = (2ps2n )-1
l(cm) = 0.66/p(Pa) (only the 1st approximation)
Number of collisions
z = ps2(8kT/(pm))1/2
m reduced mass, m = (m1
-1 + m2
-1)-1
s collision diameter
s2 collision cross-section
CI: 1015-16 collisions  high ionization efficiency (ABC)
Comparison of CI vs. EI
+ stronger signal
- higher noise
+ overall S/N higher (LOD of organic compounds ~ pg)
Mass spectrometry of biomolecules 2010 50
Negative Chemical Ionization
The same ion source as in the case of EI plus an inlet for reagent gas
Mechanism
1. Production of thermal (slow) electrons
e- (fast) + RH  RH+ + 2 e- (slow)
W(slow e-) ~ 3/2 kT
T ~ 400 K  E ~ 0.1 eV
2. Electron capture
ABC + e-  ABCPreferred
by compounds with electronegative groups (PCB, NO3
- etc.)
LD ~ pg
Mass spectrometry of biomolecules 2010 51
Sample Introduction for EI/CI
1. Gas/volatile liquid
Residual gas analysis
open ion source in chamber with analyzed gas: p(chamber) = p(gas)
Eluent from a separation column (GC-MS)
Problem: High flow rate of the carrier gas from classical GC columns
a) fraction collection or split flow (splitter may mean reduction of sensitivity)
b) continual interface (working without interruption)
i) particle beam separator
for analyte (ABC) enrichment
in the carrier gas
requires M(ABC) >> M(carrier)
... use He as the carrier gas
column MS
vacuum
pump
Mass spectrometry of biomolecules 2010 52
Sample Introduction for EI/CI (cont.)
ii) membrane interface - sample introduction through a membrane,
separation of the carrier gas using a gas-permeable membrane
c) direct introduction from a capillary GC column
lower gas load; lower flow rate of the carrier gas (He)
2. Volatile, thermally stable solid sample
Direct sample introduction on a probe (glass, ceramics, steel). After
introduction of the probe, the sample begins to evaporate and undergoes
ionization in the gaseous state.
Mass spectrometry of biomolecules 2010 53
Sample Introduction for EI/CI (cont.)
3. Nonvolatile compounds
- Large molecules
- Molecules with many polar groups
… many interesting compounds (proteins, DNA, saccharides)
a) Generation of volatile derivates and consecutive standard ionization (EI, CI).
Useful for molecules with M < 1000 Da.
Example: esterification, RCOOH + CH3OH  RCOOCH3
b) Application of classical ionization in desorption arrangement. Sample
deposited on a probe is inserted into an ion source, in which electrons
interact directly with the sample in condensed state.
c) Other ionization techniques
―Soft‖ ionization: production of molecular ions without their thermal
decomposition: FAB, electrospray, laser desorption techniques
Mass spectrometry of biomolecules 2010 54
Inorganic Ion Sources
Glow discharge
Thermal ionization
Inductively coupled plasma
Other techniques, e.g. laser desorption – also for organics, discussed later
10
Mass spectrometry of biomolecules 2010 55
Glow Discharge
The first ion source (J. J. Thompson)
Analysis of solid samples, usually conductive.
Precise and relatively sensitive analysis: RSD ~1%, LOD ~1 ppb.
Discharge in Ar (p ~100 Pa): Ar+ ions sputter metal atoms (M) from a
sample plate and ionize them later ... M+ ions are formed.
(+)
anode
(-)
cathode,
sample
plate
(-) entrance of a
mass analyzer
Ar
100 Pa
do MS,
0.1 Pa
Mass spectrometry of biomolecules 2010 56
Thermal Ionization (TI)
Sample deposited on a filament; the filament is resistively heated.
Evaporation, atomization and ionization:
MX (s)  MX (g)  M (g) + X (g)
M (g)  M+ (g) + e- (filament)
to MS
(+) (-)
heated
filament
0.1
Pa
Mass spectrometry of biomolecules 2010 57
Thermal Ionization
Saha-Langmuir equation
n(M+)/n(M)  exp[(W - IE)/(kT)]
Working function of metal (filament), W ~ 4 eV
Metal K Ca Fe Zn
IE (eV) 4.6 6.0 7.8 9.4
(W – IE) (eV) -0.5 -1.5 -3.9 -5.4
efficient weak
 Three filaments (with different temperature): Substitution of a single
filament, which evaporates and ionizes sample too fast.
- Generation of more stable ion flow (RSD only ~ 0.1 % !)
- Useful for determination of isotopic abundance.
filament
e-
M(g)
M+(g)
Mass spectrometry of biomolecules 2010 58
Inductively Coupled Plasma, ICP MS
1. Desolvation of MX (aq, aerosol)
2. Evaporation of MX (s)
3. Atomization of MX (g): dissociation to M(g) and X (g)
4. Ionization to M+ (g)
sample
aerosol
Ar, 1L/min
radial flow, Ar,
10 L/min
plasma
torch
coil
ions
atoms
101 kPa 100 Pa 0.1 Pa
series of apertures
(differential pumping)
Mass spectrometry of biomolecules 2010 59
ICP
• Usually 3 vacuum stages (differential pumping).
• Very efficient ionization, n(M+)/n(Mtotal) = 90 – 100%.
• Ions with a single charge prevails.
• Non-equilibrium system.
+ +
+
+
+
+
+
++
++
+++++
++
101 kPa 100 Pa
Plasma
Mass spectrometry of biomolecules 2010 60
Differential Pumping
101 kPa 1k Pa 100 Pa 1 Pa
pump 1 pump 2 pump 3
ion
source
MS
- frequently used concept in mass spectrometry
- used for atmospheric ionization methods
11
Mass spectrometry of biomolecules 2010 61
ICP
Supersonic jet
Hot plasma (5000 K) streams via an aperture (slit) into a chamber and
expands at supersonic velocity. Random movement of atoms at the
atmospheric side is characterized by wide kinetic energy distribution (5000
K) and relatively low translational velocity. The atoms move at supersonic
velocity with a very narrow kinetic energy dispersion … supersonic cooling
(~300 K). Distribution is later ruined by collisions with molecules of
background gas (barrel shock, Mach disc).
Efficient ionization
90 - 100 % elements are ionized (very uniform ionization).
Applicable for determination of isotopic abundance (low systematic
deviation), elemental composition.
Disadvantages
• not useful for structural characterization of analytes
• interferences
Mass spectrometry of biomolecules 2010 62
ICP
Detection limits
1 ppt (quadrupole)
10 ppq (magnetic sector)
for comparison: LD of ICP-AES and AAS ~ ppm - ppb
ppm ppb ppt ppq
million 106 billion 109 trillion 1012 quadrillion 1015
Interferences
1. Non-spectral
Shifts of ionization equlibria as a result of suppression by matrix, acids or
easily ionizable elements etc.
2. Spectral
Isotopic ions
Izobaric molecular ions
Mass spectrometry of biomolecules 2010 63
Spectral Interferences in ICP
Formation of molecular ions in ICP
1. Plasma gas and reaction products (Ar+, Ar2+, ArH+, ArO+, ArC+, ArN+ etc.)
2. Sample or solvent (hydride ions, OH+, ClO+, NO+, CaO+, LaO+ etc.)
3. Chemical ionization of background gas (H2O+, H3O+, CxHy
+ etc.)
Elimination of spectral interference
1. Mathematic corrections (e.g. using isotope distribution)
2. Desolvation of aerosol (e.g. by freezing in liquid N2)
3. Cold plasma (relative shifts of ionization degree)
4. Collision cell (thermalization of ions, shifts of reaction equillibria)
5. Mass spectrometer with high resolution
Mass spectrometry of biomolecules 2010 64
Spectral Interference in ICP
Examples of isobaric ions and required resolution
Isotope Interfering ion Resolution
39K 38Ar1H+ 5690
40Ca 40Ar+ 71700
41K 40Ar1H+ 4890
44Ca 14N14N16O+ 970
12C16O16O+ 1280
52Cr 40Ar12C+ 2380
56Fe 40Ar16O+ 2500
75As 40Ar35Cl+ 7770
80Se 40Ar40Ar+ 9690
Note: higher resolution often means lower sensitivity.
Mass spectrometry of biomolecules 2010 65
Field Ionization, FI
Very high electric field intensity between sharp spires, E > 109 V/m
Electron removal due to inner tunnel effect.
Mass spectrometry of biomolecules 2010 66
Desorption Ionization Techniques
LDI Laser Desorption/Ionization
1963 R. Honig
FD Field Desorption
1969 H. D. Beckey
PD Plasma Desorption
1974 R. D. MacFarlane
FAB Fast Atom Bombradment
1981 M. Barber
SIMS Secondary Ion Mass Spectrometry
1976 A. Benninghoven
MALDI Matrix-Assisted Laser Desorption/Ionization
1988 M. Karas & F. Hillenkamp, K. Tanaka
12
Mass spectrometry of biomolecules 2010 67
Field Desorption, FD
H. D. Beckey, Int. J. Mass Spectrom. Ion Phys., 1969, 2, 500-503
Sample is blown (g) or deposited (l, g) on an emitter, a heated metal
filament with a specially modified surface
Analytes are formed in very high electric field between sharp spires, E > 109
V/m; electrons are removed by inner tunnel effect.
Often more ionization mechanisms:
- thermal ionization
- electrospray ... during deposition of liquid samples
Applicable to analysis of organic analytes with M.W. < 2 kDa
Mass spectrometry of biomolecules 2010 68
Field Ionization. Field Desorption
Source: Bernhard Linden
probe for liquid injection, FD
surface of emitter
Mass spectrometry of biomolecules 2010 69
Secondary Ion Mass Spectrometry, SIMS
(Fast Ion Bombardment, FIB)
… Ionization by ions
• Primary ion beam may be scanned; the result is MS image of elements/compounds,
analyte topography. Imaging resolution is higher than in the case of optical
microscopy since the primary ion beam can be focused tighter, ~nm).
• Products … mostly atoms and neutral molecules, also ions. Postionization helpful,
e.g. photoionization using a laser.
• Inorganic and organic analysis, also lighter biopolymers at the presence of matrix ‖matrix-assisted
SIMS‖.
probe with
a sample,
+3 kV
to MS analyzer
primary ion beam
W ~ 6 keV
Mass spectrometry of biomolecules 2010 70
Fast Atom Bombardment, FAB
Ionization similar to CI (organic molecules, small peptides …).
Setup: off-line and on-line (flow probe; continuous flow FAB, CF-FAB).
Max. m/z ~ 10 000 Da. LOD ~ 10 pmol or even ~ 1 pmol (CF-FAB)
Usually z =  1.
Formed ions: ABCH+, [ABC+K]+, [ABC+H]-, [ABC+N2]+, fragments.
Fast Xe atoms
W ~ 5 keV
(+)
(-)
Probe with a layer of
viscous solvent and
analyte
(glycerol + ABC)
Mass spectrometry of biomolecules 2010 71
Generation of Fast Xe atoms for FAB
1. Generation of fast Xe+ ions
2. Conversion of Xe+ to Xe:
Xe+ (fast) + Xe (slow)  Xe (fast) + Xe+ (slow)
3. Elimination (deflection) of Xe+
Xe source
5 kV
Xe source
Xe+
fast
Xe, Xe+
(+)
(-)Xe+
fast Xe
Mass spectrometry of biomolecules 2010 72
HK
FABMSofapeptideinnormalandCFmode
13
Mass spectrometry of biomolecules 2010 73
CF-FAB MS/MS of Human Hemoglobin, chain A
HK
Mass spectrometry of biomolecules 2010 74
Photoionization, PI
M + n hn  M+ + eNecessary
condition: nhn > IE(M)
Photoionization types
1. Single photon ionization, SPI; n = 1
2. Multiple photon ionization, MPI; n > 1
• non-selective analysis useful for inorganic analytes
3. Resonance multiphoton ionization (REMPI) n > 1
• if hn = W (energy of electron transfer of M)
• very selective and very sensitive determination
• aromatic molecules, dyes, drugs
Mass spectrometry of biomolecules 2010 75
Photoionization Schemes
hn hn
hn
hn
hn
e- e-
eSPI
MPI REMPI
IE(M)
Mass spectrometry of biomolecules 2010 76
Photoionization
Example of arrangement: LD-PI
PI as a postionization for pro laser desorption
1. Desorption laser pulse 2. Desorption of plume (mostly neutrals)
3. Ionization laser pulse 4. Ion extraction
+ - + +
- + Mass
spectrometry of biomolecules 2010 77
Plasma Desorption (PD)
1974 R. D. Macfarlane
(R. D. Macfarlane, R. P. Skowronski, D. F. Torgerson, Biochem. Biophys.
Res. Commun. 1974, 60, 616.; R. D. Macfarlane, D. F. Torgerson
Science 1976, 191, 920.)
The first technique capable of ionization of heavy compounds (e.g. proteins).
Radioactive californium 252Cf source. Energy of fission fragments ~MeV.
Impact of a fission fragment from the radioactive source on sample deposited
on polyester foil may ionize analyte molecule.
Other fragment from decay of the same 252Cf flies in the opposite direction
and triggers signal recording device.
Mass spectrometry of biomolecules 2010 78
Experimental Setup of Plasma Desorption
(R. D. Macfarlane, R. P. Skowronski, D. F. Torgerson, "New approach to the mass
spectrometry of nonvolatile compounds", Biochem. Biophys. Res. Commun. 1974,
60, 616-621.)
14
Mass spectrometry of biomolecules 2010 79
Example of Plasma Desorption
Positive PD mass spectrum of ribonuclease A from bovine pancreas
(D. M. Bunk, R. D. Macfarlane Proc. Natl. Acad. Sci. USA 1992, 89, 6215-6219)
Mass spectrometry of biomolecules 2010 80
Plasma Desorption Characteristics
Ionization of relatively large organic molecules (~ thousands Da , e.g.
insulin, 5 735 Da).
Formation of molecular ions, ion clusters and multiply-charged ions.
Disadvanatges
- radioactive source.
- time-consuming signal accumulation.
MALDI and ESI preferred for ionization of heavy analytes nowadays.
Mass spectrometry of biomolecules 2010 81
Laser Desorption (LD)
Matrix-Assisted Laser Desorption/Ionization, (MALDI)3
Mass spectrometry of biomolecules 2010 82
Laser Desorption/Ionization, LDI
After laser discovery in 6th decade of 20th century
Initially for solid samples: R. Honig, Appl. Phys. Lett. 1963, 2, 138-139.
Later for Organic compounds: M. A. Posthumus, P. G. Kistemaker, H.
L. C. Meuzelaar, M. C. ten Neuver de Brauw, Anal. Chem 1978, 50,
985.
Matrix-Assisted Laser Desorption/Ionization, MALDI
Karas, M; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass
Spectrom. Ion Proc. 1987, 78, 53-68.
Tanaka, K.; Waki, H.; Ido, Y; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid
Commun. Mass Spectrom. 1988, 2, 151.
Laser Desorption
Matrix-Assisted Laser Desorption/Ionization
Mass spectrometry of biomolecules 2010 83
Source:
www.nobel.se
October 9, 2002
ESI
MALDI
Mass spectrometry of biomolecules 2010 84
1987 Karas & Hillenkamp
melitin
nicotinic acid
m/z = 2845
1988 Tanaka
lysozyme
Co/glycerol
m/z = 100 872 (heptamer)
15
Mass spectrometry of biomolecules 2010 85
MALDI Schematic (detail)
MALDI
Laser
pulse
Matrix
Analyte
Sample
layer
Gaseous
phase
Mass spectrometry of biomolecules 2010 86
MALDI Schematic
laser pulse
U1 U2
(U1 > U2 > 0) for +
target with
a thin layer
of sample
MS, usually
TOFMS
Mass spectrometry of biomolecules 2010 87
Principle of MALDI
1. Very short laser pulse, typical t ~ ns, max. ms.
Molecules vaporize before they decompose.
Collisional cooling: conversion of Evib to Etrans .
2. Energy is absorbed mostly by matrix (M), not by analyte.
e (matrix) >> e (analyte), c(matrix) >> c(analyte)
Matrix  MH+, M+, M*, fragments, fragment ions.
Analyte, originally ―dissolved‖ in matrix, vaporizes together with matrix.
3. Matrix actively participates on analyte (ABC) ionization.
Matrix is excited after absorption of one or more photons.
Dominant ionization mechanism is proton transfer:
MH+ + ABC  M + ABCH+.
Mass spectrometry of biomolecules 2010 88
Requirements on Matrix (MALDI)
1. Absorbtion at the wavelength of the used laser (UV, IR).
2. Formation of proper crystals with analyte (empiric rule).
3. Usually acid (efficient proton transfer to ionize na analyte).
4. Stability. Low volatility. Inert (no reaction with analyte).
Types of matrix
aromatic acids (Karas & Hillenkamp)
glycerol with addition of ultra fine cobalt powder (Tanaka)
modified surface, e.g. Si - DIOS (Siuzdak), SELDI
Mass spectrometry of biomolecules 2010 89
Common Matrices for MALDI
sinapinic acid (SA) gentisic acid (DHB)
(3,5-dimethoxy-4-hydroxycinnamic acid) (2,5-dihydroxybenzoic acid)
a-cyano-4-hydroxycinnamic acid (CHCA) 3-hydroxypicolinic acid (HPA)
Mass spectrometry of biomolecules 2010 90
Common Matrices for MALDI
ferulic acid
dithranol (DIT)
(4-hydroxy-3-methoxycinnamic acid)
2',4',6'-trihydroxyacetophenone 2'-6'-dihydroxyacetophenone (THAP)
16
Mass spectrometry of biomolecules 2010 91
Common Matrices for MALDI
nicotinic acid-N-oxide 2,-(4-hydroxy-phenlyazo)-benzoic acid
(HABA)
trans-3-indoleacrylic acid picolinic acid (PA)
(2-pyridine carboxylic acid)
Mass spectrometry of biomolecules 2010 92
Matrices - Applications
peptides < 10 000 CHCA, DHB
peptides, proteins > 10 000 SA, DHB
oligonucleotides < 3 kDa THAP
nucleic acids > 3 kDa HPA
synthetic polymers DHB, DIT, IAA
carbohydrates DHB, CHCA, THAP
Addition of ―comatrices‖ (e.g. monosaccharides) may lead to
improvements in crystallization, sample homogeneity, mass
resolution, suppression of fragmentation etc.
Mass spectrometry of biomolecules 2010 93
Properties of Matrices
CHCA: „hot― matrix
for peptides with M < 10 000 Da
useful for PSD (structure analysis)
DHB: „cold― matrix
universal use
Mass spectrometry of biomolecules 2010 94
Lasers in MALDI
UV-MALDI
337 nm nitrogen laser (inexpensive and the most common)
355 nm Nd:YAG (3xf) yttrium-aluminum garnet
266 nm Nd:YAG (4xf)
193 nm ArF ... fragmentation!
Note: YAG lasers are more expensive, but their life expectancy is
much higher. The repetition rate of YAG lasers can reach kHz vs.
Hz in the case of the nitrogen lasers.
IR MALDI
2.94 mm Er:YAG laser
10.6 mm CO2 laser
Mass spectrometry of biomolecules 2010 95
Influence of Laser Energy
Significant dependence of the quality of MALDI mass spectra on laser
energy, more exactly on power density, PD (power per area).
Ion signal
(ABCH+)
PD
0
PDT
working region
resolution
decreases
fragmentation
Mass spectrometry of biomolecules 2010 96
Influence of Laser Energy
Threshold power density, PDT … minimum value of laser power per area,
at which analyte peaks begin to appear in mass spectrum.
For PD > PDT : signal (ABCH+) = k.PDn, kde n = 4 – 6.
Small variation of power leads to a large variation in ion signal of ABCH+.
In practice, operator usually slowly increases energy during MALDI
experiment, simultaneously moves target with sample and observes
mass spectra from single laser shots. After the threshold power is
established, the power is set approximately 10–30% above it and spectra
are accumulated for 10-1000 laser pulses while the target is slowly being
moved.
17
Mass spectrometry of biomolecules 2010 97
Accumulation of Spectra
Usually average of 10 – 1000 desorptions is recorded to increase signalto-noise
ratio and reproducibility
signal, S  n
noise, N  √n
signal/noise, S/N  √n
Mass spectrometry of biomolecules 2010 98
MALDI Mass Spectrum
Model spectrum of 2 peptides, Pep1 a Pep2 with matrix M
[ABC+H]+, [ABC+2H]2+, dimer [(ABC)2+H]+
adducts with alkaline metals and matrix [ABC-Na]+, [ABC+K]+, [ABC+MH]+
fragment ions of matrix a analyte, cluster ions, e.g. [M2+Na]+
Matrix suppression – ideally only the peaks of analytes.
Ion
signal
0 500 1000 1500
m/z
Na+
K+
matrix ions,
fragments
of matrix and
analytes, adducts
cluster
ions
Pep1H+
Pep2H+
Pep2Na
+
Pep1Na
+
Mass spectrometry of biomolecules 2010 99
MALDI Sample Preparation
MALDI sample = analyte + matrix
c(analyte) = 0.1-10 mM
c(matrix) = 1-100 mM
target: steel, Al, synthetic polymers
detail
MALDI
vzorku
www.srsmaldi.com
Mass spectrometry of biomolecules 2010 100
MALDI: Sample Preparation Rules
Recrystallization of matrix.
Fresh matrix solution.
Selection of proper solvent (ACN, EtOH, MetOH, acetone, water).
pH(matrix) < 4 (adjustment with e.g. 0.1 % trifluoroacetic acid, TFA).
Analyte has to be dissolved.
Purification of analyte prior to MALDI analysis.
Unknown analyte – preparation of a series of solutions with concentration of
analyte in wide range.
Deposited samples are usually stable and can be stored (archived)
Thoroughful cleaning of the target
Mass spectrometry of biomolecules 2010 101
MALDI Sample Preparation Techniques
dry droplet – deposit of mixed solution, dry at room temperature
quick & dirty – mix solutions on target, dry at room temperature
vacuum drying – speed up drying using reduced pressure
fast evaporation – first deposit matrix layer in a volatile solvent
overlayer – matrix layer, then layer of analyte with matrix
sandwich – layers: matrix, analyte, matrix
crushed crystals – matrix crystals crushed and new sample sol’n deposited
acetone redeposition – dissolve dried sample in acetone droplet and dry
spin coating – deposition on rotating target
slow crystallization – slow growth of crystals
electrospray deposition – electrospray aerosol sprayed on target
modified targets – for concentration and more reproducible crystallization
often using hydrophilic/hydrophobic interface
Mass spectrometry of biomolecules 2010 102
Influence of Salt Content
MALDI MS of a peptide sample in the presence of Na and K salts
Sample desalting necessary (not a rule, sometimes ionization based on
cationization with Na+, K+, Ag+ ...)
Presence of salts  adduct formation , sensitivity 
0 200 400 600 800 1000 1200
0.0
0.2
0.4
0.6
Signal
m/z
PepH+
PepNa+
PepK+
18
Mass spectrometry of biomolecules 2010 103
Reduction of the influence of salts
• segregation on target ... selection of proper crystal for desorption
• addition of acid (TFA, HCOOH, HCl), NH4 salts
• target washing (salts are more soluble in water)
• catex on target
• desalting prior to deposition: separation, dialysis, ZipTip (C18) desalting
Na+ (waste)
sample solution:
peptide, Na+
H2O
peptide
(MALDI
target)
50% ACN1. 2. 3.
ZipTip
Mass spectrometry of biomolecules 2010 104
MALDI Characteristics
+ one of the most ionization techniques applied in mass spectrometry
of biopolymers (together with ESI)
+ soft ionization
+ simple spectra, usually z = +1 or z = -1 (for analytes with
electronegative groups)
+ pulse ionization (predestined for coupling with TOF mass analyzers)
+ detection limits ~ amol (for small peptides - best case)
+ fast sample preparation and analysis
Mass spectrometry of biomolecules 2010 105
MALDI Characteristics
- uneasy quantitative analysis (inner standard needed)
- search for the right spot on the target
- not useful for low-mass analytes due to intensive background in
the region of low m/z (matrix, fragments and matrix clusters)
- mutual ion signal suppression
Mass spectrometry of biomolecules 2010 106
MALDI Perspective
MS analysis of large series of biological samples
• peptide mapping for identification of proteins
(MALDI MS of products enzymatic protein digests)
• peptides, proteins, oligonucleotides, saccharides
Micro methods
Coupling with separation techniques
• advantage of sample archiving on MALDI target
• recent availability of MS/MS spectrometers for MALDI
• complementar to ESI (various ionization efficiency for various
analytes)
Mass spectrometry of biomolecules 2010 107
MALDI MS of Numerous Sample Series
1, 10, 96, 100, 384, 1536 ...
samples/target
384 samples/target
Mass spectrometry of biomolecules 2010 108
Analyte: 1 pmol of tryptic digest BSA, desalted using ZipTipC18
Matrix: 10 mg/ml aCHCA in ACN/0.1% TFA : 70/30
Sample preparation: dried-droplet.
MS: Voyager DE-STR
m/z
500.0 1200.0 1900.0 2600.0 3300.0 4000.0
0
2.2E+4
0
20
40
60
80
100
Signal
1439.47 1639.51
1178.32
927.34 1193.37
1867.50
550.60
1567.36 1750.52
1249.36764.33555.31
2854.351419.39
2612.34
1682.50
3815.521905.46 2358.28
MALDI MS of a Digest: Identification of BSA
19
Mass spectrometry of biomolecules 2010 109
Micro Methods
- microtargets, piezoelectric pipetors, deposition from a capillary outlet
- size of laser focus  size of sample  maximum sensitivity
high sample density on target
sample:
1 mm
laser:
50 mm
%25.0
1000
50
2
2

sample
laser
S
S
Mass spectrometry of biomolecules 2010 110
Micro Methods
(Ekstrom, S., Onnerfjord, P., Nilsson, J., Bengtsson, M., Laurell, T., MarkoVarga,
G. Anal. Chem. 2000, 72, 286-93)
(A) sample
processing and
injection
(B) reactor with
immobilized
enzyme
(C) micropipetor
(D) nanovials (300 x
300 x 20 mm) on
MALDI target
(E) automated
MALDI-TOF MS
Mass spectrometry of biomolecules 2010 111
Comparison of LDI and MALDI
LDI MALDI
Ionization relatively hard soft
Sample only analyte analyte in excess of
matrix
Max. m (Da) <10 000 106
Typical analyte small organic
molecules, small
peptides, synthetic
polymers
peptides,
proteins,
DNA,
saccharides, synthetic
polymers
Mass spectrometry of biomolecules 2010 112
Thermospray (TS). Ion Spray (IS). Electrospray (ES).
Isotopic Patterns of Organic Molecules4
Mass spectrometry of biomolecules 2010 113
On-Line Ionization Techniques
Atmospheric Pressure Ionization
(also spray ionization techniques)
Common attributes
• Analyte: polar, often ionized and dissolved in solution.
• Heated capillaries and other elements of ion source (hundreds ºC)
• Additional elements to increase ionization efficiency (electron beam,
electric arc)
• Differential pumping
• Often after separation  2D separation (MS as the second dimension).
• Ionization process stages:
1) formation of aerosol droplets
2) solvent vaporization
3) ion analysis
Mass spectrometry of biomolecules 2010 114
Thermospray, TS
• Solution boils in the capillary tip, droplets are generated, then dry aerosol
and finally MH+ ions. Spectra similar as in the case of CI, molecular ions
prevail. Electrodes may be inserted into the source to further increase
ionization efficiency.
• Electronegative compounds may form negative ions. (An electron source
can be inserted into the chamber to promote formation of negative ions M-.)
• Use of volatile compounds, such as NH4Ac, to prevent capillary tip clogging.
heated metal
capillary
(~ 200 ºC)
spray of
droplets
(-)
eluent from
LC or CE
column
to MS
to mechanical
pump (100 Pa)
20
Mass spectrometry of biomolecules 2010 115
Particle Beam, PB
Typical PB setup for GC and LC. (www.micromass.co.uk )
Mass spectrometry of biomolecules 2010 116
Particle Beam
Principle
• Derived from the generator of monodisperse aerosol (R. C. Willoughby, R.
F. Browner, Anal. Chem., 56, 1984, 2626-2631).
• Similar to TSI and heated nebulizer. Additional beam of particles, usually
He. Separator of He atoms from ions.
• Additional EI source may be used to increase ion production. Results are
EI spectra (with higher noise due to presence of ions and molecules of
solvent).
Characteristics
• Spectra similar as in the case of TS. More fragments.
• Less sensitive than TS and ESI.
• Useful for thermally stable, nonionic compounds with medium mass.
Mass spectrometry of biomolecules 2010 117
Heated Nebulizer
• Spectra similar to CI spectra, usually capture of 1 proton ([M+H]+).
• Low fragmentation (soft ionization).
• Suitable for molecules with medium masses (~2000 Da).
• Structure? ... Additional collision cell needed (MS/MS).
heated
element
ions extracted
through apertures
into mass
analyzer
gas
gas
LC, CE discharge
N2
(drying gas)
atmospheric
pressure
Mass spectrometry of biomolecules 2010 118
Ion Spray, IS
• Only positive ions penetrate through electrodes, negative are removed.
• Formation of multiply charged ions [M+H]+, [M+2H]2+, [M+3H]3+, [M+4H]4+...
• Packed LC columns with a splitter, micro columns, capillary columns.
ions extracted
through
apertures into
mass analyzer
gas
gas
LC, CE
heated
element
N2
(drying gas)
atmospheric pressure
(-)(+)
Mass spectrometry of biomolecules 2010 119
Electrospray (ESI)*, Nanospray+
* Yamashita, M; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451.
* Yamashita, M; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671.
+ Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167.
Schematics
1–5 kV
infusion, mLC, CE
tip (quartz, metallic,
quartz, metal)
sheath liquid
(optional)
atmospheric
pressure
vacuum
counter electrode with an
aperture, "nozzle" (0 V)
heated
capillary
additional
gas –
optional
k MS
"skimmer"
100 Pa
Mass spectrometry of biomolecules 2010 120
ESI Principle
+
-
+
+
+
-
-
-
+
+
++
+
+
+++
+++
+
+
+ ++
+++
+
+
+
+ +
-
--
-
+
+
++
++
++
++
++
++
++
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ -
21
Mass spectrometry of biomolecules 2010 121
Mass spectrometry of biomolecules 2010 122
ESI Principle
• Formation of Taylor cone in electric field. Concentration of positive
charge in the cone, destabilization of the meniscus and emission of
droplets with excess of positive charge.
• Volume reduction and increase of surface charge density of the droplets
due to solvent evaporation.
• Unsymmetrical fission of charged droplets (Rayleigh stability limit);
original droplet loses ~15% of charge, but only 2% of volume.
• Droplet size: mm  nm. Number of charges in a droplet: 105  10.
(Note: Size of a macromolecule ~ nanometers.)
• Formation of secondary ions in gaseous phase, secondary reactions in
gaseous phase.
• Ion transfer into the mass spectrometer.
• No discharge; discharge is not desirable.
Mass spectrometry of biomolecules 2010 123
ESI Arrangement
• Additional (curtain) gas – N2 stream, heated capillary behind the entrance
aperture: better desolvation, reduction of cluster formation.
• Optional coaxial stream of liquid through an additional capillary.
• Needle: i.d. < 100 mm, o.d. 100 mm – 1 mm, tip < 100 mm.
• Distance tip – counter electrode: 1 – 3 cm. Flow rate < 10 mL/min.
• Nanospray: smaller dimensions,without additional coax. liquid and forced
flow, flow rate < 100 nL/min.
Mass spectrometry of biomolecules 2010 124
ESI Arrangement
• Arrangement of needle and aperture (nozzle)
... separation of ions from ballast
- on-axis
- off-axis
- diagonal (tilted) and orthogonal
- Z-spray
• Voltage connection
- through additional liquid (sheath flow)
- liquid junction
- metallic or metallized needle tip (sheathless interface)
Mass spectrometry of biomolecules 2010 125
ESI Characteristics
• Very soft ionization suitable for biomolecules.
• Very high mass limit, M.W. ~ 106 (m/z much lower). Ionization of heavy
polymers (also virus particle).
• Generation of multiply charged ions
Bell-shaped envelope. Typical for ion spray and electrospray.
The gaps between adjacent peaks are not equidistant (in contrast to
isotope pattern).
Example:
M.W. = 10 000 Da
z 4 5 6 7 8 9 10
m/z 2501 2001 1668 1429 1251 1112 1001
m/z
S
Mass spectrometry of biomolecules 2010 126
Generation of Multiply Charged Ions
+ Higher z reduces m/z  mass analyzer with lower m/z limit can be used
for ions with very high M.W.
+ Two adjacent [M+nH]n+ peaks sufficient for determination of m and z:
(m/z)n = (m+n)/n
(m/z)n+1 = (m+n+1)/(n+1)
+ Alteration of number of charges, z ?
• pH (solution): pH  z
pH  z or even negative charge promoted
• b- emitter or other e- source (electron capture  z )
- MS of mixture more complex (but can be evaluated). Single compounds
are present in several forms and give several peaks in the spectrum ...
complex spectra, reduced sensitivity.
- Satellite peaks, e.g adducts [M+Na]+, [M+K]+ etc.
22
Mass spectrometry of biomolecules 2010 127
ESI
• Application of electric field (nozzle-skimmer)  fragmentation in source.
• For structure analysis – additional chamber for collision dissociation after
the first mass analyzer.
• ESI signal dependent on analyte concentration, c(analyte); at very low
concentrations dependent on the amount of analyte (nanospray).
• Signal α c(analyte) (10-7 – 10-3 M), it reaches plateau at higher c(analyte).
• ESI closely related to other API:
e.g. APCI (Atmospheric Pressure Chemical Ionization)
(solvent acts as a reagent gas ... ionization similar to CI)
- heated needle
- zero voltage on needle
- additional elements:
- electrode for discharge in front of the entrance nozzle
- piezoelectric element for better nebulization
- nebulizer (ion spray sometimes called pneumatic ESI)
Mass spectrometry of biomolecules 2010 128
Factors Influencing ESI
• Type of analyte
• Needle, spray tip (dimensions, arrangement)
• Voltage between needle and counter electrode
• Solution composition (solvents, additives, salts, ion-pair reagents)
• Flow rates of sample, sheath liquid and drying (curtain) gas
• Temperature of the entrance capillary
Mass spectrometry of biomolecules 2010 129
ESI – Some Rules
• Use volatile buffers:
- CH3COOH
- HCOOH
- TFA (trifluoroacetic acid)
- NH4
+ salts of volatile acids
• Keep salt concentration < 20 mM
• Avoid use of sulfates, phosphates etc.
• Orthogonal or Z spray may partially help in the cases the rules mentioned
above cannot be applied.
• For positive ionization, pKa (electrolyte) < pKa (analyte) – 2
• For negative ionization, pKb (electrolyte) < pKb (analyte) – 2
• Proper sample preparation = easier analysis
desalting, removal of surfactant and other contaminants
Mass spectrometry of biomolecules 2010 130
Mass Analyzers. Mass Spectrometers: Ion Optics. Wein Filter.
Simulation of Ion Movement (Simion). Energy Analyzers (E).
Magnetic Sector (B). Quadrupole Filter (Q).
5
Mass spectrometry of biomolecules 2010 131
III. Mass Analyzers
• Ion optics basics. Simulation of ion movement
• Energy analyzer
• Mass analyzers
• Detection of ions and data acquisition
• Vacuum techniques
• Coupling of separation to MS. Microfabricated devices
• New techniques/instrumentation
Mass spectrometry of biomolecules 2010 132
Ion Optics
Analogy with light optics
slit slit
lens lens
prism, grating Wkin: energy analyzer, deflector
m/z: mass analyzer
mirror ion mirror
optical fiber ion guide
Differences from light optics
wavelength, l kinetic energy, Wkin
mass/charge, m/z
refraction index (const.) electric or magnetic fields (tunable, can be
altered even during experiment)
Intensity-independent space charge effects – mutual ion repulsion
23
Mass spectrometry of biomolecules 2010 133
Lorentz and Coulomb Forces
Lorentz force at magnetic induction B:
Fmag. = ze ( v x B )
Coulomb force at el. field intensity E:
Fel. = zeE
Total:
F = ze (E + v x B )
For simplicity only:
Fel. = zeE
Fmagn. = zevB
v
B
F
Mass spectrometry of biomolecules 2010 134
Elements of ion optics
Slit
 restriction of ion beam
 restriction vs. transmission
Deflector
 deflection of ions
 2 deflectors for x, y scans
+
+
+V
-V
Mass spectrometry of biomolecules 2010 135
Ion Lens (Electrostatic, Einzel Lens)
• analogy of classical lens
• focal length may be changed by variation of the voltage V
• higher transmission compared to slit
• focal length is not a function of m/z (for ions accelerated in the same ion
source). The lens is ideal only for ions with the same kinetic energy and
at reasonable numbers (no space-charge effect)
• the schematic above shows only one of the possible arrangements
+V
+
Mass spectrometry of biomolecules 2010 136
Ion Mirror (reflector, reflectron)
Kinetic energy vs. potential energy in electric field:
1) ion mirror: ion returns with the same velocity as the
velocity, at which the ion entered the mirror
2) energy filter: ions with sufficient velocity (energy)
penetrate through the second grid … mirror with a partial
transmission
Ion mirror: with grids or gridless, with one or two stages, nonlinear fields.
Use: e.g. for correction of initial energy dispersion in TOF MS.
+
+U0
vx
two grids
zeU
2
mvx

2
zeU
2
mvx

2
Mass spectrometry of biomolecules 2010 137
grids: definition of equipotential planes
rings: shielding of the ground potential (vacuum apparatus walls)
potential at the rings defined using a series of resistors and U3
U1 ... acceleration voltage
U3>U1(U2)
R1 R1 R1 R1 R1 R1 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2
Construction of Dual Stage Ion Mirror
Mass spectrometry of biomolecules 2010 138
Ion Characteristics
Characteristics of a single ion
m/z mass/charge
v velocity (kinetic energy, W = mv2/2)
t time
x, y, z coordinates
a angle (... direction)
t0 time of ion formation (index0 … ion formation)
Characteristics of ion groups
(usually for the ions of the same type, i.e. the same m/z)
… described by dispersions of v (W), x, y, z, t0, a
24
Mass spectrometry of biomolecules 2010 139
Ion Properties
Example: Initial velocity dispersion at MALDI
(Pv … fraction of ions moving at velocity interval <v, v+v> )
Pv
0 1000 2000 v0 (m/s)
protein
matrix
Mass spectrometry of biomolecules 2010 140
Simulation of Ion Movement
• Exact calculation of ion movement complex even for relatively simple
electric and magnetic fields.
• Simulation of ion movement: program Simion
Development of ion optics using the program Simion:
1.Input of geometry (schematics of electrodes).
2.Input electrode potential, define magnetic field.
3.Definition of ions (number n, v0 , x0, y0, z0, a0, f0 ).
4.Movement simulation  result (graphical representation, text).
Mass spectrometry of biomolecules 2010 141
Simion
Example: Simulation of electrostatic lens using program Simion.
Lens: 3 segments
diameter, d = 36 mm
length of segments, y1 = 28 mm, y2 = 26 mm, y3 = 32 mm
gaps between segments, l = 2 mm
ion origin in xyz [0, 0, 0] mm
potentials, U1 = 0; U2 = tunable; U3 = 0
Ions: number, n = 5
mass, m = 100
kinetic energy, W0 = 100 eV
coordinates xyz [0, -30, 0] mm
a0(i) = (-4 + 2i )o , where i = 0 … n – 1
Aim: Verify function of the lens for voltage of the middle ring, U2 = 0, 85,
100, 120 a 133 V.
Solution: Presented in the lecture
Mass spectrometry of biomolecules 2010 142
Wein Velocity Filter
• Transverse electric field between two flat electrodes
• Transverse magnetic field
• Electric intensity field vector E is perpendicular to the vector of magnetic
induction B
• Ions are drawn by electric and magnetic forces with opposite direction
+
v
+ B
+V/2
-V/2
L
detector
Mass spectrometry of biomolecules 2010 143
Wein Velocity Filter
For ion flying on axis of the filter:
zeE = zevB ( )
... only ions with specific speed pass through the filter.
In case all ions were accelerated by voltage U:
… Wein filter can be used for mass analysis (m/z).
B
E
v 
zeU
mv

2
2
U
E
eB
z
m
22
2

L
V
E 
Mass spectrometry of biomolecules 2010 144
Energy Analyzer; Electrostatic Analyzer (ESA, E)
-V
+V
r
+U
+
ion
source
ESA
entrance slit
exit slit
L
25
Mass spectrometry of biomolecules 2010 145
Energy Analyzer
Acceleration: , where .
Energy:
Radius of curvature:
• Radius of curvature is directly proportional to ion kinetic energy.
• Not a function of m/z.
• Even thick ion beam (parallel ion trajectories) are focused.
• Using electrodes curved also axially, ions can be focused in two
dimensions.
• Sector – shape of a slice, a portion of a circle
zeE
r
mv2

L
V
E
2

zeU
2
mv2

E
U2
r 
Mass spectrometry of biomolecules 2010 146
Mass Analyzers
Magnetic sector (MAG, B)
Quadrupole analyzer (Q, q)
Ion cyclotron (ICR-FT-MS)
Ion trap (IT), Linear trap (LT)
Time-of-flight (TOF)
Mass spectrometry of biomolecules 2010 147
Magnetic Sector (MAG, B)
+ + + +
+ +
r
(+)
+ ion
source
B entrance
slit
detector
magnetic sector
m2
m1
m1/z1 > m2/z2
Vector of magnetic induction B is perpendicular to the velocity vector of ions
streaming from the ion source into the sector.
Mass spectrometry of biomolecules 2010 148
Principle of Magnetic Sector
1.) Lorentz force = centrifugal force:
2.) Kinetic energy = acceleration energy:
r
mv
Bzev
2

v
eBr
z
m

zeU
mv

2
2
U
reB
z
m
2
22

Mass spectrometry of biomolecules 2010 149
Principle of Magnetic Sector
Scan types:
• Scan U, const. B. Problems with low extraction efficiency at low values
of U.
• Scan B, const. U. Difficult originally, prevailing nowadays.
• Move the exit slit, r. Const. U, B. Usually not used.
Peak switching
Stepwise change of one of the variables (B, U). Suitable for monitoring of
limited number of ion types.
U
reB
z
m
2
22

Mass spectrometry of biomolecules 2010 150
Tandem Electrostatic Analyzer – Magnetic Sector
Double focusing mass spectrometer
Forward-geometry, ESA-MAG, EB
1. energy analyzer: selections of ions with a certain kinetic energy
2. magnetic sector: mass analysis
B
MAG
-V
+V
ESA
+U
. . . .
. .
ion
source
detector
+
26
Mass spectrometry of biomolecules 2010 151
Tandem EB (ESA-MAG)
• Dispersion of ion characteristics (v, x, a) and instability of fields (B, U)
reduce quality of spectra.
• Placing ESA prior to MAG solves problem with velocity (kinetic energy,
Wkin) dispersion of ions entering into mass sector.
Practical EB geometries:
1. Nier-Johnson
90o ESA + 60o MAG
exit focused for given radius, r
suitable for scanning spectrometers
2. Mattauch-Herzog
31.8o ESA + 90o MAG
single focal plane for ions with different m/z
suitable for planar detectors (photographic plate, array)
3. Matsuda
suitable for compact instruments
Mass spectrometry of biomolecules 2010 152
Mattauch-Herzog Geometry
Mass spectrometry of biomolecules 2010 153
Nier-Johnson Geometry
Mass spectrometry of biomolecules 2010 154
Other Combinations of E and B
Reverse-geometry, BE
1. magnetic sector: mass filter
2. energy analyzer: sorting of ions according to kinetic energy
MIKES (Mass-Analyzed Ion Kinetic Energy Spectrometry)
the first MS/MS technique, tandem mass spectrometry (1973)
1. Magnetic sector allows passing of only ions with certain m/z.
2. Metastable ions may undergo decay in the region between magnetic and
energy analyzer.
3. Daughter ions from the decay are sorted according to kinetic energy in
the energy analyzer.
A variety of hybrid instruments based on E and B: EBE, BEB, EBEB,
BEBE ...
Mass spectrometry of biomolecules 2010 155
Quadrupole Filter (Q, q)
4 rods with hyperbolic (also round, square or other) cross-section
U ... DC voltage component
V ... AC (RF) voltage component
w … angular frequency, w = 2pf, f = 1 - 3 MHz, phase shift 180o
U+Vcos(wt)
-U-Vcos(wt)
+
r
y
z
x
Mass spectrometry of biomolecules 2010 156
Quadrupole Filter
xz plane: heavy ions pass
yz plane: light ions pass
... only ions with a specific m/z will pass through the quadrupole filter, all
other ions are deflected.
Note: important exception is m/z-unselective RF quadrupole
27
Mass spectrometry of biomolecules 2010 157
Mass spectrometry of biomolecules 2010 158
Ion Trajectories in Quadrupole Filter
xz xz
yz yz
Mass spectrometry of biomolecules 2010 159
Ion Trajectories in Quadrupole Filter
xz
xz
yz
yz
Mass spectrometry of biomolecules 2010 160
Mass spectrometry of biomolecules 2010 161
Mass spectrometry of biomolecules 2010 162
28
Mass spectrometry of biomolecules 2010 163
Stability Diagram
... graphic representation of the solution of the Mathieu equations, which
describe trajectories of ions in the quadrupole filters.
( ) 22
8
r
z
m
eU
a
w

( ) 22
4
r
z
m
eV
q
w

const. a/q
(const. U/V)
a
q
m
m-1m+1
transmission
region
Mass spectrometry of biomolecules 2010 164
Resolution
… is determined by the value (a/q) of the slope of the scan line
Mass spectrometry of biomolecules 2010 165
Properties of Quadrupole Filter
MS scan
r a f usually const.; U a V scanned simultaneously at const. U/V.
Upper m/z limit: 2 000 - 4 000.
[V, cm, MHz]
To increase the upper m/z limit: V, r, f.
( ) 22
0.316
max
fr
V
~
z
m
Mass spectrometry of biomolecules 2010 166
Properties of Quadrupole Filter
To increase resolution: (Resolution, R < 10 000, usually R < 2 000.)
• For acceleration voltage, Uacc (towards the quadrupole):
For given quadrupole length, l, the voltage Uacc must be low enough to
enable sufficient number of ion oscillations, tens to hundreds at the given
frequency, w, during the time of flight, t, through the filter.
• Better manufacturing, very low mechanical tolerances.
2
2
z v
eUm acc

t
l
v 
acceU
z
m
l
t
2
2

Mass spectrometry of biomolecules 2010 167
Quadrupole Filter - Notes
External source
• fringe field, especially at the entrance
- responsible for unstable trajectories; significant fraction of ions does not
get inside
- mass discrimination of heavy ions, which spend longer period in the
fringe field, are more influenced
• solution
- entrance electrostatic lens
- entrance RF quadrupole (q, RF only), hexapole or octopole
... only RF component: a = 0, U = 0  unselective filter, through
which ions of all m/z can pass
Triple quadrupole (QqQ)
• popular tandem mass spectrometer for collisional dissociation
• middle quadrupole (q, RF only) serves as a collision chamber
Mass spectrometry of biomolecules 2010 168
Ion Trap (IT). Linear Trap (LT). Fourier Transform Ion
Cyclotron (FT-ICR-MS). Orbitrap. Electrostatic Trap.
Simion: Examples
6
29
Mass spectrometry of biomolecules 2010 169
Quadrupole Ion Trap (Ion trap, IT)
1953 Wolfgang Paul (Paul trap) – almost unnoticed
1983 Finnigan – commercial instrument (one of best selling MS’s)
Schematics of ion trap
DC: constant voltage or ground Bruker, Model ESQUIRE-LC
AC: constant + alternate (RF) component, U + Vcos(wt)
DC DCAC
incoming
ions
detector
ring
lids
z
r
Mass spectrometry of biomolecules 2010 170
Mass spectrometry of biomolecules 2010 171
Mass spectrometry of biomolecules 2010 172
Stability Diagram (Ion Trap)
( ) 22
16
r
z
m
Ue
az
w

( ) 22
8
r
z
m
Ve
qz
w

m3 > m2 > m1 (z = 1)
qz  V/(m/z) for movement along z axis (a = 0, U = 0)
Scan: by increasing V. First lighter, then heavier ions
are expelled from the trap. (Also resonant ejection
helps to improve resolution.)
stability region:
ions oscillates inside trap, they
are trapped (storage mode)
instability region:
ions leave trap through apertures
in lids
az
qz
m3 m2 m1
Mass spectrometry of biomolecules 2010 173
Ion Trap
• Experiment stages: ionization, accumulation, scan a detection.
• Accumulation: ions are being trapped even during ionization pulse – the
result is very low detection limits. The total time of experiment depends on
following requirements:
Sensitivity ... Longer accumulation means higher sensitivity.
Scan time ... Wider m/z range, higher m means longer scan (see next page).
Resolution ... Longer scan means slower (and longer) scan.
• Resonance ejection
of ions of specific m/z by application of RF voltage on trap lids. A hole in
the stability diagram is created. It is used to increase resolution even in
regular scan mode.
(1983, G. Stafford: ―mass selective instability mode―)
• Buffer gas (low mass gas: He, 1 mTorr)
Much better results than with analyte itself.
Collisions of analyte with buffer gas molecules lead to better distribution of
energy among analyte ions and keeps ions in the center of the trap.
Advantageous in GC-MS.
Mass spectrometry of biomolecules 2010 174
30
Mass spectrometry of biomolecules 2010 175
Ion Trap
• MS/MS – trap can replace tandem of two mass spectrometers
CID, CAD: collision-induced (activated) dissociation
• High R … < 10 000, usually ~ 5 000
• Upper mass limit, m/zmax ~ 70 000 (resonance ejection)
• Miniaturization: ion micro trap, trap on a chip (also quadrupole filter)
+ total size only 1 cm
+ useful in space exploration
- much lower mechanical tolerances, demanding manufacturing
- low parameters (R, m/zmax )
Mass spectrometry of biomolecules 2010 176
Ion Trap
• Ion transmission
only half ions may be detected
• Physical restrictions
dimensions, voltage and frequency limited.
• Maximum dynamic range (106 for single ion type) limited:
- minimum by sensitivity (usually 10; even 1 ion may be detected)
- maximum by space charge effect: for number of trapped ions higher
than 106 the trap does not function properly – due to mutual ion
repulsion. Note that the charge limit involves sum of charges of all ion
types!
Mass spectrometry of biomolecules 2010 177
Linear Trap
Linear trap or ―2D trap‖ is based on quadrupole ion filter/ion guide. The
previously described ion trap is sometimes called ―3D trap‖.
Ions are injected into the quadrupole and trapped by elevating potential on
lids. (Ions oscillate in a potential well of RF-only quadrupole.) Later the
ions can be gradually ejected and detected through the rods or lids.
Two approaches of ion ejection from LT:
A. Schwartz, J.C., Senko, M.W., Syka, J.E.P., J. Am. Soc. Mass Spectrom.
13 , 2002, 659.
B. Hager, J.W., Rapid Comm. Mass Spec. 16, 2000, 512.
Mass spectrometry of biomolecules 2010 178
Linear Trap with Radial Ejection
detector I
+
detector II
+
Ions
are ejected from LT in radial direction.
(From: Schwartz, J.C., Senko, M.W., Syka, J.E.P.,
J. Am. Soc. Mass Spectrom. 13 , 2002, 659.)
Mass spectrometry of biomolecules 2010 179
Linear Trap with Axial Ejection
Ions are ejected from the LT (Q3) along quadrupole axis.
(From: J. C. Y. Le Blanc et al. Proteomics 3, 2004, 859-869.)
Mass spectrometry of biomolecules 2010 180
Advantages of 2D Ion Traps
Capture Efficiency: 50-75% (3-D: 5 % - 20%, and depends on mass)
Extraction Efficiency: 25-50% (3-D : 20%)
Sensitivity increased by a factor of 5-10
Ion Capacity: 20 - 30 times larger than 3-D
Linear range increased more than 2 orders, up to 106
Resolution: ~10 000 at scan rate 300 a.m.u./sec
Wider variety of ion handling (accumulation, scans...)
31
Mass spectrometry of biomolecules 2010 181
Differences b/w Quadrupole Filter and Traps
Quadrupole filter
- no ion storage
- only ion with certain m/z can pass through a filter at a time
... scanning instrument, loss of ions and hence lower sensitivity
(except in single ion monitoring)
... no space charge effects, hence higher dynamic range
Mass spectrometry of biomolecules 2010 182
Comparison of Quadrupole Analyzers
QIT QQQ LQIT
Sensitivity ++ - ++
Dynamic range - ++ +
Mass range - + +
MS3 ++ - ++
Neutral loss scan/Precursor scan - + +
m/z accuracy + - +
Resolution + - +
Mass spectrometry of biomolecules 2010 183
Ion Cyclotron (FT-ICR-MS)
(Fourier Transform - Ion Cyclotron Resonance - Mass Spectrometer)
…another type of ion trap
Cubic trap consisting of two pairs of electrodes and two lids
excitation
detection
x
z
y
B
Mass spectrometry of biomolecules 2010 184
FT-ICR-MS
Ion movement in magnetic field:
Angular (cyclotron) frequency:
r
mv
Bzev
2

( )z
m
Be
r
v
w
+
+ + +
+ + +
+ + +
+ + + B
r
v
+
Mass spectrometry of biomolecules 2010 185
FT-ICR-MS Characteristics
- (superconducting) magnet with very high magnetic induction B ~ 10 Tesla
- very low pressure, p < 10-7 Pa (UHV, ultra high vacuum)
... high cost ~ $700 000
- limited dynamic range ~ 103, 100 – 105 ions
+ very high precision and accuracy, m/z, ~ ppm
+ very high resolution, R ~ 106
+ multiplex (Fellgett) advantage of FT … ion signal is acquired for all m/z
during entire acquisition period  improvement of S/N 103x, reduction of
acquisition period ~106x (FT-ICR vs. ICR)
+ suitable for MSn (CID), commonly n = 2 – 3, even n = 10 demonstrated
Other geometries of cell: cylindric, hyperbolic (Penning)
Mass spectrometry of biomolecules 2010 186
Fourier Transform
... transformation of signal from time domain to frequency (m/z) domain
Ideal signal of ions with single m/z
Real FT-ICR-MS signal
• Collisions with background molecules and inhomogeneity of magnetic
field result in signal decrease. Higher p  more collisions  lower R.
• Lorentz peak shape.
t
St
m/z
S
FT
t
m/z
S
FT
St
32
Mass spectrometry of biomolecules 2010 187
FT-ICR-MS Principle
Ionization for FT MS
1. internal: analyzer = source (e.g. EI, LDI)
2. external: ionization in an external source and introduction to the cell
- ion guides, quadrupoles, electrostatic lens
- differentially pumped chambers (ions are formed at the first cell at higher
pressure and then introduced into analyzer cell through an aperture along
z-axis)
Excitation
1. Pulse ... high amplitude, extremely short duration
2. Chirp ... fast scan through frequencies in required interval
3. SWIFT ... Stored Waveform Inverse Fourier Transform
profile of the excitation waveform generated by iFT of required m/z profile
(iFT = inverse FT, transformation from frequency (m/z) to time domain
Mass spectrometry of biomolecules 2010 188
FT-ICR-MS Principle
Detection
1.nondestructive (inductive) – ions attract electrons in the detection plates
during passage nearby: non-destructive detection (FT-ICR)
2.destructive – ions strike the detection plates (ICR)
Data acquisition
• Higher sampling frequency  higher upper m/z limit.
Nyqist criterion: the highest achievable frequency = sampling frequency/2
• Longer acquisition period, t  higher resolution and lower m/z limit.
• Result ... necessity of storage of many data points
- heterodyne frequency mixer (shift of signal frequency down allows to
use lower sampling frequency)
Mass spectrometry of biomolecules 2010 189
FT-ICR-MS Principle
Z-trapping
• DC voltage ~1-5 V on the lids to keep ions inside (and not to leave in z-axis
direction.
• Presence of an additional electric field causes magnetron oscillations (~10
Hz) of ions in addition to cyclotron oscillation (~ MHz). The result of
combined movement are more complex calibration, peak shift and higher
loss of heavy ions.
Mass spectrometry of biomolecules 2010 190
FT-ICR-MS
Resolution
Upper m/z limit. From the mean quadratic speed of thermal movement,
,
which is responsible for pre-excitation of ions, it can be expressed:
.
In a trap characterized by B and r, m of ions that can be stored is lower
than
.
z
m
Bt
m
m
a

zeB
mkT
zeB
mv
r
2

m
kT
v
2

kT
)zeBr(
m
2
2

Mass spectrometry of biomolecules 2010 191
New Mass Spectrometers
Mass spectrometer of future?
... not a single solution
Trends:
• Withdrawal of magnetic sector instruments.
• Hybrid spectrometers
Modern spectrometers and their hybrids gain popularity (ion traps,
orthogonal TOF analyzers).
• New spectrometers?
Linear quadrupole traps (discussed earlier)
Electrostatic trap ‖Orbitrap―
Linear electrostatic trap
Mass spectrometry of biomolecules 2010 192
A. Makarov, HD Technologies Ltd., USA, ASMS Conference 1999
rings (+)
electrode (-)
Principle
• Ions are injected into axially symmetric electric field and circulate around
the middle electrode.
• Ion oscillations along the electrode axis are inductively detected, mass
spectra are obtained after Fourier transform, f = f(m/z).
Electrostatic Trap ”Orbitrap“
+
-
+
ion injection
ring (+)
electrode (-)
detection
+
++
33
Mass spectrometry of biomolecules 2010 193
Orbitrap Properties
+ Very high resolution – close to FT-ICR-MS (R = 50 000).
+ Very high precision and accuracy of m/z… close to FT-ICR-MS.
+ No magnet necessary.
+ No RF generators necessary.
- Ultrahigh vacuum needed as in the case of FT-ICR-MS.
Mass spectrometry of biomolecules 2010 194
Linear Electrostatic Trap
Detected frequency = f(m/z)
(Benner, Anal. Chem. 1997, 69, 4162-4168)
Trap electrodes
(ion mirrors)
Detection tube in potential well
electrons are attracted and repelled,
AC current is produced
Mass spectrometry of biomolecules 2010 195
Time-of-Flight Mass Spectrometer (TOFMS). Techniques for
Enhanced Resolution in TOFMS (Reflector, Delayed Extraction and
Orthogonal Extraction).
7
Mass spectrometry of biomolecules 2010 196
TOFMS History
TOF = Time-of-flight
m/z calculated from the time of ion travel
1946 TOF Principle
(W. E. Stephens, Phys. Rev., 1946, 69, 691)
1948 First instrument, „Ion Velocitron“
(A. E. Cameron, D. F. Eggers, Rev. Sci. Instrum. 1948, 19, 605)
1955 Ion source with 2 stages, time-lag focusing
(Wiley, W. C.; McLaren, I. H.; Rev. Sci. Instrum., 1955, 26, 1150)
Mass spectrometry of biomolecules 2010 197
TOFMS History
1973 Ion mirror
(B. A. Mamyrin, V. I. Karataev, D. V. Schmikk, and V. A. Zagulin, Sov.
Phys. JETP 1973, 37, 45)
1995 Delayed (pulse) extraction
(Whittal, R. M.; Li, L., Anal. Chem. 1995, 67, 1950-1954;
Brown, R. S.; Lennon, J. J., Anal Chem. 1995, 67, 1998-2003;
Vestal, M. L.; Juhasz, P.; Martin, S. A, Rapid Commun. Mass Spectrom.
1995, 9,1044-1050)
Mass spectrometry of biomolecules 2010 198
TOFMS Principle
1. Pulse formation of ions
2. Acceleration of ions
The same acceleration energy, W(el.) for ions with the same z.
Transformation W(el.) = W(kin.)
3. Drift (flight) of ions:
Separation of ions according to m/z; W(kin.) = mv2/2
4. Impact of ions on detector
Acquisition of signal in time, I(t)
5. Determination of m/z from the time of flight
Transformation I(t)  I(m/z)
34
Mass spectrometry of biomolecules 2010 199
(+)
ionization beam:
e.g. pulse e- beam
repeller,
~20 kV
drift zone, E = 0 detector
entrance and exit grids
TOFMS Geometry
Linear geometry
... the simplest arrangement
... ions are not focused optimally on the detector
Mass spectrometry of biomolecules 2010 200
detector
exit grid,
0 V
(+)
ionization beam:
e.g. laser pulse
repeller,
~20 kV
drift zone, E = 0
acceleration
grid, 0 V
extraction grid, ~17 kV
(+)
TOFMS Geometry
Wiley-McLaren linear geometry (1955)
... adjustment of the extraction electric field does not depend on the value of
the total acceleration voltage
... allows focusing of ions on the detector at the end of the flight tube
Mass spectrometry of biomolecules 2010 201
(+)
ionization beam:
e.g. pulse e- beam or
laser pulse
repeller,
~20 kV drift zone
(E = 0)
source
grids
grids and
electrodes
of reflector
ion
deflector
and gate
ion mirror,
>20 kV (+)
(-)
(+)
detector
TOFMS Geometry
TOF MS with ion mirror (reflector)
... for higher resolution
... for structural analysis (PSD)
Mass spectrometry of biomolecules 2010 202
flight tube
detector
ion source
laser
diffusion
pump
mechanical
pump
digital oscilloscope
delay generator
control unit
vacuum controller
high voltage source
MALDI TOFMS
Mass spectrometry of biomolecules 2010 203
Ion Source (detail)
Mass spectrometry of biomolecules 2010 204
1. Pulse ionization (pulse width ~ ns): fast generation of ion plume (LDI,
MALDI, PD, EI …)
2. Extraction and acceleration of ions in electric field
3. Separation of ions in drift zone at E = 0 (field-free region, flight tube)
4. Detection of ions, signal acquisition and transformation to m/z domain
acceleration energy: kinetic energy
length of drift zone
flight time
2
mv
Uez
2

t
L
v 
2
2
L
t
U
z
m
e2
TOF
S
(m/z)1<(m/z)2
TOFMS Principle
35
Mass spectrometry of biomolecules 2010 205
Exact relation includes also the time spent in in the ion source and the
detector regions:
voltage: Us 0 0 Ud
distances:
tTOF = ts + tL + td
Note: 1) ts, tL, td and hence tTOF are proportional to (m/z)1/2
2) for rough estimate tTOF: tTOF ~ tL (L >> s, d)
s L d
s
s
zeU
ms
t
2
2
 )UUU(
ze
m
U
d
t dss
d
d 4
2

s
L
zeU
mL
t
2
2

TOFMS Principle
Mass spectrometry of biomolecules 2010 206
m/z=(2eU/L2)t2

m/z=k1(t-t0)2

t=c0+c1(m/z)1/2
t=c0+c1(m/z)1/2+c2(m/z)
t=c0+ c-1(m/z)-1/2 +c1(m/z)1/2+c2(m/z)
correction of data acquisition trigger
correction of the original ion velocity before extraction pulse
correction of non-ideal extraction pulse shape
TOFMS Calibration
Mass spectrometry of biomolecules 2010 207
Principal Advantages of TOFMS
No theoretical upper m/z limit
Ideal for pulse ionization
Fellgett advantage: for each of pulses, entire spectrum recorded, no need
for scanning
Very short duration of acquisition of a spectrum (~10-4 s)
High ion transmission ... a prerequisite of high sensitivity
Simplicity
Mass spectrometry of biomolecules 2010 208
1. Ion source for gaseous samples
2. Ion source for condensed samples (desorption)
(+) (-)
+
e-, ions, laser …
(+) (-)
laser, ions, atoms …
Ion Sources for TOFMS
Mass spectrometry of biomolecules 2010 209
y
z
x+
source detector
Ideal Ion Source for TOFMS
All ions are created:
• in time t0 (with period of formation, t = 0)
• at distance x0 (x … flight axis of TOFMS)
(preferably in a single point [x0, y0, z0])
• with the same velocity vx0 (, which does not have to be zero,) along x-
axis.
(preferably vy0 = 0 a vz0 = 0)
Mass spectrometry of biomolecules 2010 210
Real Ion Source for TOFMS
Ions are characterized by initial dispersions of
time (time of creation t0),
space (position of creation x0, y0, z0),
velocity v0 (energy Wkin0) and
angle a (deviation of the flight x-axis).
Result: drop of resolution, R
Wiley-McLaren ion source for gaseous samples:
Adjustment of voltage on the first grid allows to find a compromise between
contribution of v0 a x0 dispersions to reach optimal resolution (ion
focusing into detector plane)
36
Mass spectrometry of biomolecules 2010 211
Pv
0 1000 2000 v0 (m/s)
analyte
matrix
laser
Ion Properties
Example: Dispersion of initial ion velocity in MALDI
(Pv … probability of occurrence of molecules with velocity in <v0, v0+v> )
Mass spectrometry of biomolecules 2010 212
Pulse Generation of Ions
1. Extraction using constant electric field (DC)
pulse ionization beam + constant extraction field
2a. Pulsed extraction, PE (delayed extraction, DE)
pulse ionization beam + pulse extraction field
2b. Orthogonal extraction
continuous generation (introduction) of ions + pulse extraction field
Mass spectrometry of biomolecules 2010 213
Enhancement of Resolution in TOFMS
1. High acceleration voltage
2. Ion mirror.
3. Pulse extraction
... important decision when buying TOFMS
Mass spectrometry of biomolecules 2010 214
Increase of acceleration voltage will minimize contribution of initial
velocity dispersion of analyte.
m
zeU
vv
22
0 
contribution
of electric field
contribution
of desorption
zeU
2
vm
2
mv2

2
0

1. High Acceleration Voltage
Mass spectrometry of biomolecules 2010 215
Př.: MALDI TOFMS of a peptide, m = 2000 Da, z = 1, v0 = 750 m/s,
v0(FWHM) = 500 m/s, L = 1 m. (2 ions: v01 = 500 m/s, v02 = 1000 m/s.)
U = 1 kV: t1 = 101.673 ms, t2 = 101.284 ms R ~ 130
U = 10 kV: t1 = 32.190 ms, t2 = 32.177 ms R ~ 1300
Pv
0 500 1000 v0 (m/s)
50%
100%
500
m/s
Influence of Acceleration Voltage
Mass spectrometry of biomolecules 2010 216
• Ions 1 and 2 with same m/z and velocities v1 a v2: faster ion penetrates
deeper into the ion mirror and its trajectory is longer.
• If adjusted properly, two ions will hit the detector II simultaneously.
(B. A. Mamyrin, V. I. Karataev, D. V. Schmikk, and V. A. Zagulin, Sov.
Phys. JETP 1973, 37, 45;
Mamyrin, B. A.; Shmikk, D. V.; Sov. Phys. 1979, 49, 762)
+
U3>U1
(+)
deflector
(-)
detector II
U1
source
ion mirror
detector I2
1
U2
2. Ion Mirror (reflector, reflectron)
37
Mass spectrometry of biomolecules 2010 217
Construction of Ion Mirror
Linear ion mirror ... E is constant along the ion mirror axis
Single stage ... intensity of electric field, E is constant in the entire ion
mirror (Alikhnov)
Dual stage ... ion mirror is divided in two regions with different E
(B. A. Mamyrin, V. I. Karataev, D. V. Schmikk, V. A. Zagulin, Sov. Phys.
JETP 1973, 37, 45)
Nonlinear ion mirror ... E varies along the ion mirror axis
Quadratic field (D. R. Jardine, J. Morgan, D. S. Alderdice, P. J. Derrick,
Org. Mass Spectrom. 1992, 27, 1077)
Curved field (T. J. Cornish, R.J.Cotter, Rapid Commun. Mass Spectrom.
1993, 7, 1037-1040)
+ additional patents (Japan, Soviet Union)
Mass spectrometry of biomolecules 2010 218
grids: definition of equipotential plane
rings: electric shielding of vacuum apparatus walls
voltage on the rings is defined by series of resistors
U3>U1(U2)
R1 R1 R1 R1 R1 R1 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2
Construction of Ion Mirror
Mass spectrometry of biomolecules 2010 219
Construction of Ion Mirror
Mass spectrometry of biomolecules 2010 220
...
2
2
2
3
1
2
8
1
2
2
2
1
2
2
1
2
2
0
2
00
0 


















as
v
a
a
s
L
a
s
as
v
a
a
s
L
a
s
a
v
tt
rr
TOF
Ur
(-)
detector
Us
s L1 (+)
L2 Lm
ion mirror
L = L1 + L2 ... drift zone, E=0
a ... acceleration in source, a=qEs /m
ar ... acceleration in mirror, a=qEr /m
(Moskovets, E. Rapid Commun. Mass Spectrom. 2000, 14, 150-155)







ra
a
s
L
a
s
t
2
2
1
2
0
tTOF = ts + tL + tr ... sum of periods ion spends in source, tube and mirror
Single Stage Ion Mirror
Mass spectrometry of biomolecules 2010 221
...
2
2
2
3
1
2
8
1
2
2
2
1
2
2
1
2
2
0
2
00
0 


















as
v
a
a
s
L
a
s
as
v
a
a
s
L
a
s
a
v
tt
rr
TOF
Increase of R by minimization of the 3rd term: 0
2
2
1 
ra
a
s
L







ra
a
s
L
a
s
t
2
2
1
2
0
source
drift zone
mirror
t0 ... tTOF ion with zero initial velocity, v0= 0
contribution of initial velocity, v0
Single Stage Ion Mirror
Mass spectrometry of biomolecules 2010 222
Dual Stage Ion Mirror
Two-stage ion mirror ... ion mirror is divided in 2 stages with different E
For positive ions:
• positive voltage on the middle grid, 0 > U2 > U3
- a variety of arrangements, various ratio of stage lengths,
e.g. ~1:2, ~2:3
- short 1st stage with higher E allows shortening of the entire
mirror length
• negative voltage on the middle grid:
- space focusing in the 1st stage
- energy focusing in the 2nd stage
38
Mass spectrometry of biomolecules 2010 223
tTOF = f(Wkin, U2, U3)
energy dispersion ... the main reason of peak broadening and low R
Mamyrin:
Solution and
U3
+
U2
Wkin
0),,( 32 


UUWf
W
kin
kin
0),,( 322
2



UUWf
W
kin
kin
Dual Stage Ion Mirror
Mass spectrometry of biomolecules 2010 224
Dual Stage Ion Mirror
+ Applicable to energy dispersion below ~20%.
E.g. for energy dispersion < 5% theoretical resolution ~100 000
- Only ions that penetrate deep into the ion mirror (85-100%) are focused.
- Mass spectra of ions with wider energy dispersion have to be recorded at
several values of the potential on the mirror. The resulting spectrum is
stitched from these several segments.
Mass spectrometry of biomolecules 2010 225
Aim: tTOF independent on their Wkin for ions of all m/z
In quadratic field
ion reflection  f(Wkin0)
z ... axis of potential change
a ... parabola minimum
k a C ... constants
f ... frequency of oscillations
(A. A. Makarov, E. N. Raptakis, and P. J. Derrick, Int. J. Mass Spectrom.
Ion Processes 1995, 146/147, 165.)
Caz
k
zU  2
)(
2
)(
)(2
2
azk
m
q
dt
zd

Nonlinear Ion Mirror
Mass spectrometry of biomolecules 2010 226
Nonlinear Ion Mirror
Other practical fields:
axially symmetric hyperbolic potential
planar hyperbolic potential
axially symmetric hyperlogarithmic potential
Mass spectrometry of biomolecules 2010 227
linear field
nonlinear (curved,
quadratic) field
(T. J. Cornish, R. J. Cotter ―Non-linear field reflector―, US Patent 5 464 985)
Nonlinear Ion Mirror
Mass spectrometry of biomolecules 2010 228
Usually quadratic field or its approximation
Creation of nonlinear field
using unequal resistors in series
using unequal spacers between rings and/or grids
+ simultaneous focusing of ions with wide dispersion of Wkin , e.g. products
of post-source fragmentation, for entire m/z range
- more complex calibration
- ideal only for ions moving on the axis of the reflector
... only limited R can be achieved in practice
Nonlinear Ion Mirror
39
Mass spectrometry of biomolecules 2010 229
Extraction field is turned on after a delay:
1. Laser pulse at t = 0
2. Expansion of ions at zero extraction field for a period t (delay)
3. Extraction field is turned on at t = t, with fast rise time
(Brown, R. S.; Lennon, J. J.; Anal. Chem. 1995, 67, 1998)
t
V(repeller)
V(grid)
V
15 kV
0
0
t
12 kV
3. Pulsed Extraction
(Delayed Extraction Time-Lag Focusing)
Mass spectrometry of biomolecules 2010 230
repeller grids detectordrift zone
1.
t = 0:
2.
t = t :
3.
t = tof:
+
+
E = 0
E > 0
+
+
+
+
e.g. 12 kV 12 kV 0 kV
Pulsed Extraction
e.g. 15 kV 12 kV 0 kV
Mass spectrometry of biomolecules 2010 231
Pulse extraction for continual ion sources
Analyte stream is extracted by pulse extraction field orthogonal to
the entering ion beam.
Usually with ion mirror for higher resolution.
repeller,
pulse
voltage, U1
(+)
analyte ion beam
from a continual ion source
drift zone, E = 0 detector
exit grid,
0 V
acceleration
grid, 0 V
extraction grid, U2
Orthogonal Extraction
Mass spectrometry of biomolecules 2010 232
Other Factors Influencing Resolution in TOFMS
• non-ideal ion source
• ion optics
• tube length dilatation
• properties of detector
• sampling frequency of A/D converter
• sample preparation
• acceleration voltage fluctuations
E.g. acceleration voltage fluctuations:
m = const. Ut2 
dt given by max. frequency of AD converter, detector speed and time
dispersion of ion generation
dU given by high voltage power supply (drifts and noise)







t
td
2
U
Ud
.
m
md
R 1
const
Mass spectrometry of biomolecules 2010 233
Strict requirement on parallel arrangement
MCP with narrow channels
L
drift zone+
+
MCP
detectorion source
+
+
3 mm
L
drift zone
MCP (detail)
10o
ion source
Importance of Alignment in TOFMS
Mass spectrometry of biomolecules 2010 234
Resolution:
E.g. for R = 15 000 and L = 3 m:
L 100 mm
Note: MCP with channel angle 10o and diameter 10 mm:
L = 10 mm/tan(10o) = 57 mm
m
m
R


m
z
eU
t
L
2
2
2
R
L
L

2
t
t
R


2
t = L/v

Importance of Alignment in TOFMS
40
Mass spectrometry of biomolecules 2010 235
Influence of Grids
Grids ... electrodes transparent for ions
... in ion source, ion mirror and in front of detectors
MS with grids
+ precise definition of potential
- ion losses (impact, deflection, reaction)
- secondary ion formation on grids, sputtering of grid material
- field penetration
Gridless MS
- higher resolution without losses on grids
- more complex design (extra curvature of ion trajectories, lens effect)
For details, see: T. Bergmann, T. P. Martin, H. Schaber Rev. Sci. Instrum.
1989, 60, 347.
Mass spectrometry of biomolecules 2010 236
TOF with grids Gridless TOF
Note: grids do not have to always mean drop of resolution
1441
m/z
0
100
200
300
400
IonSignal(a.u.)
1445 1449
Influence of Grids
Mass spectrometry of biomolecules 2010 237
system geometry extraction R
TOFMS linear DC 500
DE-TOFMS linear pulse 5 000
rTOFMS reflector DC 10 000
DE-rTOFMS reflector pulse 20 000
oTOFMS reflector pulse 10 000
Comparison of TOFMS Systems
Mass spectrometry of biomolecules 2010 238
ion
mirror
ion optics
detector (+)
Q1: precursor selection
Q2: CID
Q3: ion guide & focusing
API, AP MALDI...
laser (MALDI)
Ion beam is wide after
exit from Q3 (space
dispersion - x0), but
ions have negligible
energy dispersion - v0
in the direction of flight
Tandem Mass Spectrometer QTOF
Mass spectrometry of biomolecules 2010 239
Hybrid Ion Source for QTOF
Exchangeable ion source
... continual and pulse ion techniques
Example: QTOFMS: API
AP MALDI
Mass spectrometry of biomolecules 2010 240
Source: Micromass/Waters
Hybrid QTOF MS for API and MALDI
41
Mass spectrometry of biomolecules 2010 241
Source: Micromass/Waters
Hybrid QTOF MS for API and MALDI
Mass spectrometry of biomolecules 2010 242
TOF: Other Geometries
(+)
+ detector- detector
+
(-)
TOF
MS for simultaneous detection of positive and negative ions
Mass spectrometry of biomolecules 2010 243
360o magnetic sector - no separation in space
- separation in time
m1 m2 (m2>m1)
+ B
TOF: Other Geometries
Mass spectrometry of biomolecules 2010 244
Technical Notes
Target (MALDI): 384 spots on target sized as microtitration well plate,
sufficient area for collection of eluent from a separation column
Ion optics: with or without grids (gridless)
Collision chamber in ion source: increase of fragmentation degree
length of the drift zone (flight tube): typically 1-2 m, but even 10 cm or 5 m
Ion guide – it may be placed in the tube to increase ion transmission (wire
in the flight axis with ~ -50 V for positive ions)
Mass spectrometry of biomolecules 2010 245
MALDI Target
Mass spectrometry of biomolecules 2010 246
Other Technical Notes
Detectors: microchannel plate, MCP
usually double MCP
electron multiplier
hybrid detector (scintilating layer + photomultiplier)
Detection electronics: AD converter (8 bits, 0.5 - 4 GS/s)
TD converter + segmented detector
42
Mass spectrometry of biomolecules 2010 247
Applera Mariner API oTOF
Voyager MALDI TOF
4700 MALDI TOF/TOF
QSTAR API, MALDI QoTOF
Bruker Biflex, Proflex, Reflex MALDI TOF
Autoflex, Omniflex, Ultraflex MALDI TOF (LIFT)
JEOL AccuTOF API oTOF
LECO Renaissance API (ICP) TOF
Jaguar API oTOF
Micromass/Waters M@LDI MALDI TOF
QTOF API oTOF
QTOF Ultima API, MALDI oTOF
Thermo/Finnigan Tempus EI, CI oTOF
Kratos/Shimadzu Kompact, Axima MALDI TOF
etc. etc.
Commercial TOFMS
Mass spectrometry of biomolecules 2010 248
Comparison of Common Mass Spectrometers
MS max. m/z R
quadrupole filter 4 000 2 000
magnetic sector 20 000 50 000
ion traps 10 000 5 000
FT-ICR-MS 100 000 100 000
TOFMS 1 000 000 10 000
Note:
The values in the table are approximate, as they describe average
instruments, the parameters of research-grade systems might be
significantly higher (differences in order of magnitudes).
Mass spectrometry of biomolecules 2010 249
TOFMS Summary
• Unlimited theoretical upper m/z limit. Practical limit: ionization and
detection efficiency, metastable ion decay.
• Entire spectrum recorded at once.
• High speed of data acquisition (1 spectrum in ~10-4 s).
E.g. insulin (m/z = 5735) accelerated by 15 kV overcomes 1-m drift zone
in ~ 50 ms.
• High ion transmission, especially in linear mode (> 50 %).
• High resolution (R > 10 000).
• Simplicity and relatively low cost.
• Fragmentation techniques available (ISD, PSD, TOF/TOF ... next lecture)
Mass spectrometry of biomolecules 2010 250
TOFMS Perspective
Advantages of TOFMS
upper m/z limit, speed and high sample throughput, high sensitivity and
resolution, relatively lost cost
Huge spread of TOFMS in the last decade
MALDI + PE TOF, rTOF MS
API, AP MALDI + oTOF MS
New techniques for MS/MS (TOF-TOF, LIFT, QTOF)
Competition
LT, FT-ICR and hybrid MS
Mass spectrometry of biomolecules 2010 251
Ion Dissociation (CID, SID, ECD, ETD, IRMPD). Tandem Mass
Spectrometry (MS/MS). In-Source Decay (ISD), Post-Source
Decay (PSD). New Techniques (TOF-TOF, LIFT).
8
Mass spectrometry of biomolecules 2010 252
1. ionization  mixture of ions
2. first MS  selection of parent ion, precursor ion
3. fragmentation of ions (e.g. in collision cell)
4. second MS  analysis of fragmentation products, daughter ions
Arrangement
1. in space – two or more discrete m/z analyzers in a series
2. in time – consecutive ionization, precursor selection, fragmentation and
scan in a single m/z analyzer (ion trap)
Tandem Mass Spectrometry
(Tandem MS, MS/MS, MSn)
ionization MS1 precursor ion MS2 daughter ionsfragmentation
43
Mass spectrometry of biomolecules 2010 253
Fragmentation Classification
Dissociation ... monomolecular reaction, t(induction) << t(dissociation)
Fragmentation can be induced by:
1. Collision w/ atom or molecule
collision-induced dissociation, CID
2. Collision with surface
surface-induced dissociation, SID
3. Photon (photodissociation, PD)
e.g. infrared multiphoton dissociation, IRMPD using CO2 laser
4. Electron (electron capture dissociation, ECD)
Note: It is difficult sometimes to determine the exact cause of ionization,
e.g. the decay in MALDI (in- and post-source decay, ISD and PSD) may
be induced by both collisions and photons.
Mass spectrometry of biomolecules 2010 254
Fragmentation Induced by Collisions
1. collisions with molecules of background (collision) gas in collision cell
at elevated pressure, p ~100 Pa
CID, collision-induced dissociation
CAD, collisionally activated dissociation
2. excessive excitation during ionization, e.g. high laser power at MALDI
ISD, in-source decay ... in TOFMS
PSD, post-source decay … in TOFMS
3. collisions with surface: SID, surface-induced dissociation
- surface: layer of an organic compound (polymer or monolayer of small
organic molecules, e.g. alkanthiols) on a suitable substrate (Au)
- collisions as a result of acceleration by electric field, e.g. voltage
nozzle-skimmer
Mass spectrometry of biomolecules 2010 255
Ion Stability
1. Stable: lifetime, t > 10-6 s
Ion flights through entire MS without decomposition.
2. Metastable: lifetime, t ~ 10-7 - 10-6 s
Ion is decays during flight in the m/z analyzer.
3. Unstable: lifetime, t < 10-7 s
Ion fragments in the ion source.
Note: This is historical classification according to the time ions spend in
magnetic sector.
Reactions: unimolecular, bimolecular
Mass spectrometry of biomolecules 2010 256
Ion Collisions
Fragmentation
• intended fragmentation aimed at elucidation of analyte structure
• usually with atoms of rare gases
Elastic collisions
Kinetic energy is conserved.
Inelastic collisions
Part of kinetic energy is converted into inner ion energy:
Ein <= E(Mt/(Mt + Mi))
t = target, i = ion
Use heavy target to transfer more energy on ion.
1 eV/ion ~ 100 kJ/mol (100 kJ/5 g tatranky)
Mass spectrometry of biomolecules 2010 257
Low-Energy Collisions
Collision Energy: 1 – 100 eV
vibrational excitation, t (ion-target interaction) ~ 10-14 s
Collision efficiency
usually sufficient due to many collisions in collision cell
Instrumentation
triple quadrupole filter, ion traps, hybrid MS
The most widespread technique nowadays.
Mass spectrometry of biomolecules 2010 258
High-Energy Collisions
Collision energy: keV
electron nature of excitation, t (ion- target interaction) ~ 10-15 s
converted energy ~ 1 – 3 eV
Collision efficiency
He – reduces angular scatter of products
(scatter has negative impact on m/z analysis)
Ar, Xe – enables more efficient energy conversion
Instrumentation
hybrid sector instruments, TOF/TOF
44
Mass spectrometry of biomolecules 2010 259
Example: McLafferty Rearrangement
Electron impact-induced breakage of carbonyl compounds with hydrogen in
g position and formation of enolic fragment and olefin:
F. W. McLafferty, Anal. Chem. 31, 82 (1959).
D. G. I. Kingston et al., Chem. Rev. 74, 215 (1974); K. Biemann, Mass Spectrometry (New
York, 1962) p 119;
Djerassi et al., J. Am. Chem. Soc. 87, 817 (1965); 91, 2069 (1969); 94, 473 (1972)
M. J. Lacey et al., Org. Mass Spectrom. 5, 1391 (1971); G. Eadon, J. Am. Chem. Soc. 94,
8938 (1972); F. Turecek, V. Hanus, Org. Mass Spectrom. 15, 8 (1980).
Mass spectrometry of biomolecules 2010 260
Mechanism of McLafferty Rearrangement
Mass spectrometry of biomolecules 2010 261
Why MS/MS?
1.Structure elucidation of organic compounds
2.Analysis of mixture of analytes as a substitute of separation – MS
combination.
• All ions are ionized simultaneously at MS/MS. CID and daughter-ion
scan are done for selected precursors.
• Attention! Mutual ion suppression in complex mixtures during
ionization.
3.Improvement of S/N
• Higher selectivity  reduced noise
• Single ion monitoring
Mass spectrometry of biomolecules 2010 262
Mass spectrometers for MS/MS
1. Triple quadrupole filter (Triple quad, QQQ, TQ, Q3)
2. Ion traps (IT, LIT, FT-ICR)
3. BE (magnetic sector – electrostatic analyzer) with reverse geometry
4. TOF/TOF MS
5. Hybrid spectrometers, such as QTOF, IT-TOF, EBQ etc.
Classification of tandem mass spectrometry
1. in space – 1, 3, 4, 5
2. in time – 2, 5 (IT-TOF)
Mass spectrometry of biomolecules 2010 263
Triple quadrupole, QQQ, QqQ, Q3
Classical instrument for low-energy CID (MS/MS, MS2)
Q1: MS1
q2: RF-only, collision chamber
Q3: MS2
Q1 q2 Q3
Mass spectrometry of biomolecules 2010 264
Scan Types in MS/MS
1.Single Ion Monitoring
Set Q1: only ABC+ pass
Set Q3: only one of ABC fragments pass – e.g. AB+, A+, BC+…
… very selective and sensitive proof of ABC
2.Daughter Ion Scan
Set Q1: only ABC+ pass
Scan Q3: spectrum of ABC+ fragments
… structure elucidation
3.Parent Ion Scan, Precursor Ion Scan
Scan Q1: spectrum of original ions ABC+, ABF+, DEF+ etc.
Set Q3: detect only specific fragment, e.g. AB+
… for identification of a group of similar compounds (with the same
functional group or structure motif)
4.Neutral Loss Scan
Scan Q1 and Q3 simultaneously while keeping constant difference
between the m/z of transmitted ions
… loss of the same neutral (compounds with the same functional group.)
45
Mass spectrometry of biomolecules 2010 265
Other Mass Spectrometers for MS/MS
Tandem magnetic and electrostatic sector with reverse geometry (BE)
1.Isolation of precursor ABC+ in B.
2.Fragmentation in collision chamber.
3.Kinetic energy analysis in E.
Daughter ions are characterized with W; W = f(m/z).
More sophisticated combinations: EBE, EBEB.
EBqQ
EB: precise precursor selection
q1: RF-only (collision cell)
Q2: selection/analysis of products
Tandem quadrupole filter - oTOFMS (QTOFMS)
TOFTOF
Mass spectrometry of biomolecules 2010 266
Other Mass Spectrometers for MS/MS
Tandem IT - TOFMS (IT-TOFMS)
IT: accumulation and MSn option in the 1st stage.
TOF: sensitive detector with high mass resolution&accuracy as the 2nd stage.
Ion traps: IT, FT-ICR-MS
• MSn option
• the same spectrometer as for MS, only software upgrade needed
• the same price
• Procedure:
1. isolation of precursor (after accumulation of all ions)
2. excitation of precursor (amplitude boost) for a longer period
3. product scan (or back to the 1st step for MSn, n >2)
Tandem LT – FT-ICR-MS
plenty of scan types and operational modes
Mass spectrometry of biomolecules 2010 267
... for structural analysis and/or identification
In-source decay (ISD)
Post-source decay (PSD)
Collision-induced dissociation (CID, TOF/TOF)
TOF in Tandem MS
Mass spectrometry of biomolecules 2010 268
In-Source Decay (ISD)
• Also called In-Source Fragmentation (ISF)
• Technique for study of molecular structure
• Elevated laser power at MALDI leads to excessive ―heating" (vibrations)
of molecules/ions of analyte and fragmentation of analyte in the source
• Intensity of fragments << intensity of parent ion ([M+H]+)
• Pulse extraction necessary to:
• reach sufficient resolution and sensitivity
• prolong ion stay in the ion source (more collisions)
Mass spectrometry of biomolecules 2010 269
ISD Characteristics
+ Ion mirror not necessary.
- Pulse extraction needed.
- Preparation of clean analyte required (no option of precursor selection).
- May require special sample preparation (e.g. higher salt concentration)
Use: MALDI TOFMS of peptides, saccharides
Mass spectrometry of biomolecules 2010 270
H2N - CH- C-
OR1
NH - CH- C-
OR2
NH - CH- C-
OR3
NH - CH- COOH
R4
x3
a1
y3
b1
z3
c1
x2
a2
y2
b2
z2
c2
x1
a3
y1
b3
z1
c3
Examples of ISD
Fragmentation rules … according to relative strength of bonds.
Typical fragmentation products of peptides:
c, y, z
46
Mass spectrometry of biomolecules 2010 271
ISD. Analyte: 20 pmol KGF analogue. Matrix: sinapinic acid. Laser: N2.
(V. Katta, D. T. Chow, M. F. Rohde Anal. Chem., 70, 4410-4416, 1998.)
Mass spectrometry of biomolecules 2010 272
ISD
Analyte: oxidized bovine insulin, chain B
Matrix: thiourine
Laser: Er:YAG (l = 2936 nm)
(http://medweb.uni-muenster.de/institute/impb/research/hillenkamp/publicat/abs-cm99.html)
Mass spectrometry of biomolecules 2010 273
Post-Source Decay (PSD)
• Ion selector (ion gate): selection of precursor (m/z interval 1 - 20 Da).
• Analysis of product ions in ion mirror:
- Fragment ions of analyte are formed during flight through the drift
zone (as a result of excessive excitation), e.g.:
- ABC+  AB+ + C
- ABC+  A+ + BC etc.
- Kinetic energy conservation for the first equation:
- mABCv2/2 = mABv2/2 + mCv2/2.
• The heavier the fragment ion is, the higher kinetic energy it has and the
deeper it penetrates into the ion mirror  longer flight time.
Mass spectrometry of biomolecules 2010 274
Precursor ABC+
Fragmentation 1
Fragmentation 2
ABC+
AB+
C
A+
BC
v
v
v
v
v
222
222
vmvmvm AABABC 

Mass spectrometry of biomolecules 2010 275
Clasical ion mirror allows focusing of only product ions with < 20 %
energy dispersion and hence in < 20 % m/z interval. Total PSD
spectrum has to be put together from few PSD spectrum increments
obtained at different reflector potential.
Quadratic ion mirror allows focusing of products ions in wide m/z interval.
+U2 , (U2 > U1)0
+
(+)
deflector/selector
(-)
detector 2
+U1
source
ion mirror
detector 1
for neutrals
(BC, C etc.)
ABCH+
ABH+CH+
Ion Mirror as an Energy Analyzer in PSD TOFMS
Mass spectrometry of biomolecules 2010 276
MALDI PSD of a Derivatized Peptide YLYELAR
m/z
100 200 300 400 500 600 700 800 900 1000 1100
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
[Abs. Int. * 1000]
a Y
y R A
Spectrum composed from spectral increments recorded at different ion
mirror potential. Only ions that penetrate deep into the mirror were
focused. Dual stage ion mirror used. (Source: Z. Zdráhal)
47
Mass spectrometry of biomolecules 2010 277
MALDI PSD of a Peptide
1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 m /z
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
a .i.
MALDI PSD: Dual stage ion mirror. (Source: Z. Zdráhal)
Mass spectrometry of biomolecules 2010 278
MALDI PSD of a Peptide
200 400 600 800 1000 1200 1400 1600
0
20
40
60
80
B
a1
b1
a2y3
b2
y4
a3
y5
b3
a4
y6
b4
b5
y7
a6
b6
y8
a7
b7
[M+H]
+
Intensity[%]
Mass/Charge
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
MALDI PSD, a quadratic-field reflector used.
(O. Šedo et al. Anal. Chim. Acta 2004, 515, 261-269)
Mass spectrometry of biomolecules 2010 279
Collision-Induced Dissociation (CID) in TOFMS
Fragmentation results of collision of precursor ion with gas molecule
Collision chamber
- additional collision chamber in TOF ion source (negligible effect)
- inserted between two TOF analyzers (TOF/TOF)
- in hybrid instrument (e.g. QqTOF)
Mass spectrometry of biomolecules 2010 280
TOF-based MS/MS
(Medzihradszky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falick, A. M.;
Juhasz, P.; Vestal, M. L.; Burlingame, A. L. Anal. Chem. 2000, 72, 552).
TOF/TOF
Mass spectrometry of biomolecules 2010 281
6 kV LIFT cell
15 kV (pulse)
ion mirror
22 kV
source
15 kV (pulse)
LIFT cell:
entrance
grid
exit grid
tube
with grids
LIFT - TOFMS
Mass spectrometry of biomolecules 2010 282
Vacuum Instrumentation. Detectors. Data Acquisition. Signal
Converters. Coupling Separations to Mass Spectrometry (online,
off-line, microdevices)
9
48
Mass spectrometry of biomolecules 2010 283
Ion Detection
Faraday cup
Electron multiplier
Channeltron
Microchannel plate, MCP
―Daly‖ detector
Array detectors
Photographic plate
Hybrid detectors, e.g. MCP-diode array
Mass spectrometry of biomolecules 2010 284
Faraday Cup
- Very small currents
E.g.: 100 ions/s  current = 1.6 x 10-17 A
-100 V
-10 V
+
Mass spectrometry of biomolecules 2010 285
Electron Multiplier
• Analogy of photomultiplier; conversion dynode (instead of photocathode)
followed by a series of dynodes with decreasing potential and a collector.
Electron multiplier is not encapsulated in a glass bulb.
• Conversion of ions to electrons on the first dynode (e.g. Cu-Be alloy).
Multiplications of electrons on dynodes. Electron capture on collector,
generation of current.
• Gain ~ 106
+
etc.
dynodes
conversion
dynode, -2 kV
collector, 0 V
Mass spectrometry of biomolecules 2010 286
Channeltron
• Glass tube with a layer of semiconducting PbO inside and applied voltage
on the ends. Current flowing through the PbO layer forms potential
gradient along the tube (rather than dynodes kept at discrete potential
values). Electron multiplication similar to electron multiplier.
• Conversion dynode necessary for detection of positive ions.
• Gain ~ 106
-3
kV
-100 V
0 V
Mass spectrometry of biomolecules 2010 287
Microchannel Plate (MCP)
Dimensions of MCP
• thickness ~1 mm
• diameter 1 - 10 cm.
Microchannels
• slightly tilted,
• diameter ~ 3 - 20 mm.
• covered with a PbO layer,
... formation of gradient along
microchannels (electron
multiplication on continuous dynodes)
• Chevron structure in sets of more MCP’s.
Array detector with large sensitive area useful in TOFMS
Gain of single MCP ~103, dual MCP ~106 or higher.
+
+
+
+
+
-2.2 kV
-1.2 kV
-200 V
collector
0 V
2 MCP’s w/microchannels
Mass spectrometry of biomolecules 2010 288
“Daly” Detector
Ion  electron(s)  4 photons (phosphorus)
+ Very low noise.
+ Very high gain (photomultiplier usually works in photon counting mode).
- Light interferes.
- Very low pressure (p < 10-5 Pa) necessary.
photomultiplier
+
thin metal film
phosphorus layer
polished metal
electrode, -30 kV
49
Mass spectrometry of biomolecules 2010 289
Array Multichannel Detectors
… e.g. for magnetic sector
Photographic plate
- historical detector, time-consuming processing
Combination of MCP and diode array
Incorporation of optical fiber bunch for higher positional resolution.
Other detectors ... combination of the detectors discussed before
E.g. MCP and phosphorus scintillator … isolation of high voltage
optical fiber
bunch
2 MCP’s
phosphorus
diode
array
+
+
Mass spectrometry of biomolecules 2010 290
Data Acquisition in MS
ions
electrons
current
voltage
recording device
charge, q
conversion (conversion efficiency,amplification)
I = q/t (t ... time)
U = IR (R ... impedance)
Different schematic
for photographic plate
and scintillating detectors
Mass spectrometry of biomolecules 2010 291
Mass Spectrum
y-axis: current, voltage, charge, ions, counts???
a.u. (arbitrary units) or relative intensity
0
10
20
30
40
50
60
70
80
90
100
%Int.
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500
Mass/Charge
normalization of ion signal
Mass spectrometry of biomolecules 2010 292
Mass Spectrum Acquisition
I(m/z) = f(X) = f(t)
Scanning instruments Scanned X Scan period
MAG magnetic sector B, U ~ 1 s
Q quadrupole filter U, V, f ~ 0.1 s
IT, LT ion traps U, V, f ~ 0.1 s
Direct signal acquisition in time:
TOF ~ 100 ms
FT-ICR ion cyclotron ~ 1 s
Other non-scanning instruments may use array detectors.
Mass spectrometry of biomolecules 2010 293
Recording Devices
digital converters
• A/D converter (analog to digital)
• T/D converter (time to digital)
photographic plate
chart recorder
analog oscilloscope with a camera
Mass spectrometry of biomolecules 2010 294
A/D Converter (ADC)
Measurement (digitization) of voltage
Basic parameters
number of bits (resolution)
sampling frequency, number of samples pre second [Sample/s]
max. frequency (cut-off frequency)
polarity: unipolar (negative), bipolar
input voltage range
max. input voltage
number of data point (memory length, buffer size)
stability
etc.
50
Mass spectrometry of biomolecules 2010 295
A/D Converter
Example:
2-bit converter with input voltage range 0-3V and sampling frequency 10 S/s
number of levels = 22:
period, T = 1/10 = 0.1 s
SA (V)
3
0
0 0.2 0.4 0.6 t (s) 0 0.2 0.4 0.6 t (s)
SD (#)
2
1
level high-bit low-bit
0 0 0
1 0 1
2 1 0
3 1 1
Mass spectrometry of biomolecules 2010 296
Signal Precision and Accuracy
Precision and (accuracy) of measurement given by number of bits of ADC
Example: for 8-bit ADC, 28 = 256 levels:
deviation might be up to 1/2 of the difference between two adjacent levels
min. rel. deviation > (2x(number of levels - 1))-1 = (1/2x255)-1 ~ 0.2 %
number of bits 1 8 12 16 24
number of levels 2 256 4 096 65 536 16 777 216
min. rel. deviation (%) 50 0.2 0.01 8x10-4 3x10-6
dynamic range
(rel. deviation < 10%)
- 50 800 13 000 3 000 000
Mass spectrometry of biomolecules 2010 297
Selection of ADC
Possible reasons:
low signal level
converter with low number of bits
100 102 104
0
2
4
6
8
10
Iontovýsignál
m/z
100 101 102 103 104 105
0
50
100
150
200
250
Iontovýsignál
m/z
Solution:
• higher detection range
• converter with higher number of bits
• signal accumulation (averaging)
Mass spectrometry of biomolecules 2010 298
Acquisition Speed
... given by sampling frequency of ADC
Factors: scan period
scan range
required resolution
required quality of peaks (number of data points/peak)
Example 1:
Requirements on a Q: scan period = 0.1 s, unit resolution in m/z range =
200 – 2 000, >10 pts/peak. What is min. sampling frequency?
min. sampling frequency = (2 000-200)x10/0.1 = 180 ksample/s
memory length = (2 000-200)x10 = 18 000 samples
… for 16-bit converter: 36 kByte (1 data point = 19 bits or 2 bytes)
Mass spectrometry of biomolecules 2010 299
Example 2: How fast ADC is needed for TOFMS in order to record ion
flying 20 ms with resolution 2000, provided the peak should be
characterized by 10 points?
t = 20 ms, R = 2 000
R = m/m = t/(2t)
t = t/2R = 20 ms/4000 = 5 ns (FWHM)
entire (~10 points) … 10 ns  tsample = 1 ns (1 ns/sample)
sampling frequency, f = 1/ tsample = 1 Gsample/s
ADC Speed
Signal
t
50%
100%
5 ns
20 ms
t
Mass spectrometry of biomolecules 2010 300
Influence of Sampling Frequency of ADC
(in TOFMS)
5000
10000
15000
20000
25000
5000
10000
15000
20000
25000
Ionsignal(a.u.)
Time of flight (ms)
2 Gsample/s 1 Gsample/s 500 Msample/s
4000
6000
8000
10000
12000
14000
16000
18000
20000
10 ns10 ns10 ns
51
Mass spectrometry of biomolecules 2010 301
ADC: Current State of Art
High number of bits: 24 bits
• but only at low sampling frequency (1 kHz)
High sampling frequency: 20 Gsamples/s
• but only with low-bit ADCs (8 bits)
High performance
• data accumulation (averaging) directly in PC card
• on-board compression (algorithms without and with data loss)
• fast data transfer from card to PC
... PCI card in computer, direct PC memory access
Mass spectrometry of biomolecules 2010 302
Single-shot spectrum Averaged spectrum Compressed spectrum
Data flow
12 Mbyte/s 7.8 Mbytes/s 0.17 Mbyte/s
5 pulses/sample 1 avg. MS/sample 1 comp. MS/sample
25 000 pts/MS 25 000 pts/MS ~700 pts/MS
Application: CE–MS, simultaneous 8 CE eluents, 12 comp. MS/s/eluent
 31.3 Mbyte/3-minute CE
Hardware averaging and compression using multiple digital signal
processors (FastFlight, EG&G).
Use of Accumulation and Compression
in TOFMS
Mass spectrometry of biomolecules 2010 303
Available ADCs
Digital oscilloscopes
up to 20 Gsamples/s
max. deviation of t: 1 ps
bandwidth up to 8 GHz
Tektronix, LeCroy, Agilent etc.
PC cards (PCI)
up to 5 Gsample/s, 8bit, with accumulation
Aqiris, Signatec, GaGe etc.
www.acqiris.com
www.lecroy.com
Mass spectrometry of biomolecules 2010 304
T/D converter (TDC)
Pulse counters
Impact of ion on detector  pulse  amplifier  discriminator  counter
TDC (time to digital converter)
1-bit A/D converter
2 levels: 0 a 1
Parameters:
time resolution
dead time
number of channels
etc.
Mass spectrometry of biomolecules 2010 305
0 20 40 60 t (ms)
0 20 40 60 t (ms)
Accumulation of n spectra
Analog spectrum
(1 pulse)
Digitized spectrum
(1 pulse)
SA (V)
100 %
0
0 20 40 60 t (ms)
SD1 (#)
0
1
SDn (#)
0
n
Accumulated digitized spectrum
(n pulses)
threshold signal level
Mass spectrometry of biomolecules 2010 306
TDC
Only one of a group of ions is used during one acquisition period (single
channel TDC).
Suitable for acquisition of relatively low signals with high repetition rate.
Relatively low cost (compared to ADC).
Applications:
TOFMS with high frequency of extraction pulses (>kHz)
ESI - oTOFMS
AP-MALDI – oTOFMS
TOFMS with low numbers of ions
PD – TOFMS
52
Mass spectrometry of biomolecules 2010 307
TDC
TDC with multiple channels
... to increase dynamic range
... using a multiple channel TDC in combination with a segmented detector
Classical TOF detector
T/D 1 bit
MCP
Mass spectrometry of biomolecules 2010 308
TDC
Segmented TOF detector
Ions hit randomly four segments with similar probabilities
...total ion fluence might be higher
Seg. MCP
T/D 1 bit
T/D 1 bit
T/D 1 bit
T/D 1 bit
4 bits
Mass spectrometry of biomolecules 2010 309
TDC
Current state of the art
Time resolution ~10 ps at millisecond range
Montáž do stojanu nebo do PC (PCI sběrnice)
Manufacturers: LeCroy, Kore Technologies etc.
www.lecroy.com
www.kore.co.uk
Mass spectrometry of biomolecules 2010 310
Informatics & Bioinformatics
Hardware: IBM PC platform most popular
Software: Windows XP
Exponential growth of performance: Moore law
Example:
Evaluation of MS/MS spectra using SEQUEST program
SCX - LC - MS/MS of Y2H yeast digest (the next slide)
Mass spectrometry of biomolecules 2010 311
SCX-LC
MS/MS
Mass spectrometry of biomolecules 2010 312
PC Cluster for evaluation of SCX-LC-MS/MS
1x master PC (2 processors P4, 3 GHz)
8 x slave PCs (2 processors P4, 3 GHz)
interconnected with 1-Gbit network
53
Mass spectrometry of biomolecules 2010 313
PC Cluster for evaluation of SCX-LC-MS/MS
5 minute search = + 5 oC temperature
Mass spectrometry of biomolecules 2010 314
Vacuum Instrumentation Background
Evangelista Torricelli (1608-1647)
Pressure unit: 1 Torr = 1 mm Hg
Pressure, p
p = force/area [Pa, N/m2]
1 atm = 760 Torr = 101 325 Pa = 101.325 bar = 14.70 psi
1 Torr = 133 Pa
Vacuum
gaseous state with p < 101325 Pa
Mean free path of a molecule
Mean path of a molecule (atom, ion, particle) travels between 2 collisions
l = (2ps2n )-1
l(cm) = 0.66/p(Pa) ... only rough estimation for air at 25°C
Mass spectrometry of biomolecules 2010 315
Gas Flow Classification
1. Turbulent flow (p > 10 kPa)
very short l
many collisions between molecules
2. Viscous, continuous flow (p = 1000 - 0.1 Pa, „rough" vacuum)
l = mm - cm:
still much shorter compared to dimension with vacuum apparatus
more collisions molecule – molecule than molecule – wall
3. Molecular flow (p < 0.01 Pa, ―high" or ―ultra high‖ vacuum, UHV)
l > m:
collisions of molecules with walls of vacuum apparatus prevail
usual situation in mass spectrometer
(exceptions: collision cells, CI, ion mobility spectrometry)
Mass spectrometry of biomolecules 2010 316
Gas Flow Regimes
Gas Flow Rate, Q
[Q] = Pa m3/s, Pa L/s
C … conductance of a tube with diameter D and length L
C = f(D, L, p, gas, T), [C] = L/s
At molecular flow regime, conductance is not a function of pressure p:
C  D3/L (approximation)
At viscous flow, conductance C depends on pressure p:
C  pD4/L (approximation)
Design of a vacuum apparatus
 Short thick connectors  C  faster pumping.
 In serial connections, the thinnest tube (bottleneck) limits pumping
of the entire system: 1/Ctot = S1/Ci
Mass spectrometry of biomolecules 2010 317
Pumping Speed
Pumping speed, S [S] = L/s
1/Schamber = 1/Spump + 1/Ctot
Appropriate design: Ctot > Spumpa
i.e., using expensive efficient pump together with long narrow tubes or
hoses does not make sense
Notes:
• In molecular flow regime pump does not suck gas. Pump acts as a trap;
molecules that come to the pump will not return to chamber.
• Pumping speed varies with gas type (drops with gas M.W.).
• Pressure is not constant throughout the system, positioning of pressure
sensor matters.
• Outgassing. Time needed for reaching sufficient vacuum is prolonged by
presence of volatile compounds adsorbed (water, sample, finger prints)
and absorbed in the system (gases and water in gaskets, plastics).
p
Q
S 
Mass spectrometry of biomolecules 2010 318
Vacuum Pumps
Mechanical pumps (rotary oil, membrane, roots)
Diffusion pump
Turbomolecular pump
Cryotrap, sorption traps, chemisorptions traps
54
Mass spectrometry of biomolecules 2010 319
Rotary Oil Pump
• One, two or even more stages.
• Rotor immersed in oil
- min. pressure limited by oil vaporization
- trap for oil vapors needed (to keep apparatus clean - oil contamination!)
- trap for oil mist on outlet needed (to keep operator’s lungs clean)
- periodic oil changes required
• Pressure > 0.1 Pa … ―rough" vacuum, pumping speed, S: 1 – 500 L/s
(typical)
• The most common rough pump in commercial systems.
• Other mechanical pumps: membrane pump (no contamination, but higher
limit p and lower pumping speed)
Mass spectrometry of biomolecules 2010 320
Turbomolecular Pump
• Series of turbines rotating at extremely high speed (up to 50 000 rpm).
Gas molecules are reflected by turbine blades. Perimeter speed of
turbine blades > speed of gas molecules.
• Dependence of inlet and outlet pressure on gas molecular weight:
ln (pout/pin)  m
• UHV pump, limit pressure ~10-8 Pa
• Pumping speeds up to ~ 3000 L/s
• Commonly used UHV pump in commercial systems.
Mass spectrometry of biomolecules 2010 321
Diffusion Pump
• Special non-volatile oil heated with a heater at the base of the pump,
heated oil vapors flow upwards through a central tube, stream as circular
jets downwards on cooled walls, condense and flow down to the base.
Molecules of pumped gas are drawn by oil jets.
• Often used in combination with a trap cooled with water or liquid nitrogen;
this may prevent oil backstreaming or increase pumping speed (solvent
vapors).
• UHV (ultra-high vacuum) pump, limit pressure ~10-6 Pa, pumping speed
up to ~ 10 000 L/s.
• Reliable, low maintenance.
• Slow start, backstreaming.
Cryotrap
• Sorbent (e.g. charcoal) cooled down to 4 K (liquid He).
• Regeneration necessary.
Mass spectrometry of biomolecules 2010 322
UHV System Heating
Molecules of background gas (especially water) adsorb on inner walls of
mass spectrometer. Slow desorption of gas molecules continues to elevate
pressure and prolongs pumping time. Pumping can be fastened by heating
of the entire system (e.g. by wrapping the system using a resistively-heated
tape), which shift equilibrium from adsorption to desorption.
Mass spectrometry of biomolecules 2010 323
Pressure Sensors (Pressure Gauges)
Hydrostatic pressure gauge
• Pressure determined from difference of liquid levels.
• Range down to 1 kPa, or 0.1 Pa for special constructions.
Mechanical gauge
• Determination of pressure from deviation of elastic membrane.
• Reading of the deviation - mechanical
- change of capacitance (range 105 – 10-2 Pa)
Thermocouple gauge (Pirani)
• Bimetallic thermocouple and resistively heated filament.
• Heat transfer from filament to thermocouple dependent on pressure and
type of gas.
• Range p: 100 Pa - 0.1 Pa or 100 kPa - 0.1 Pa (convectron).
Mass spectrometry of biomolecules 2010 324
Pressure Gauges
Ion gauge
Bayard & Alpert: ion tube with heated cathode.
• Three electrodes in a glass bulb: spiral (+), collecting wire (-) in the center
of spiral and heated filament (-) outside the spiral (heated cathode).
• The same setup as in the case of open ion source: electron from heated
filament are extracted towards the spiral, ionize gas, ions and electrons
are captured on the wire and current is measured. p = f(current). Note:
current depends on gas composition!
• Pressure range: 10-2 - 10-10 Pa (high vacuum, UHV)
Penning: ion tube with cold cathode.
• ions are formed in electric discharge in magnetic field.
• Pressure range: 1 - 10-4 Pa, some constructions down to 10-10 Pa.
Note: commercial spectrometers employ thermocouple and ion gauges.
55
Mass spectrometry of biomolecules 2010 325
Coupling Separation to Mass Spectrometry
Why separation?
... analysis of very complex samples, mixtures of many analytes, e.g.
common sample in proteomics contains >102 peptides
Simple MS or even MS/MS is not powerful enough for several reasons:
• high probability of occurrence of 2 or more analytes with the same m/z
• overlap of isotopic envelopes of analytes with close m/z
• mutual ion suppression
• limited dynamic range of mass spectrometer
• resolution of the 1st stage at MS/MS often unsatisfactory (R1 ~ 500)
• removal of contaminants
• source of additional information about analytes
Mass spectrometry of biomolecules 2010 326
Ion Suppression in Peptide Mixtures
600 800 1000 1200 1400 1600 1800 2000
0.00
0.05
0.10
0.15
0.20
0.25
600 800 1000 1200 1400 1600 1800 2000
0.00
0.05
0.10
0.15
0.20
0.25
AI
AIII
AG 1-14
AF 1-7
AF 3-8
AII
AII (off scale)
m/z
IonIntensity(V)
c(peptides) = 1 mM
c(peptides) = 1 mM
besides c(AII) = 50 mM
Mass spectrometry of biomolecules 2010 327
Classification of Interfaces
Gas  vacuum
• jet separator for analyte enrichment in carrier gas (particle beam)
• membrane interface
• capillary column or classical column + splitter
Liquid  vacuum
• API ionization techniques (ionization directly from liquid at atmospheric
pressure)
• Flow probes (FAB)
• Sample deposition on a target
- moving belt (deposition at atmospheric pressure – transport,
differential pumping - ionization)
- Fraction collection
Mass spectrometry of biomolecules 2010 328
Separation - ESI MS
Most common techniques:
2D GE - MS
• 2-dimensional gel electrophoresis on polyacrylamid gel
• band excision and protein processing
• common planar technique for protein separation
RPHPLC – ESI MS
• liquid chromatography on reverse phase – ESI MS
• common column technique for peptide analysis
Data dependent scan ... one MS scan followed by few MS/MS scans
(discussed again later)
Mass spectrometry of biomolecules 2010 329
Separation of biomolecules: MALDI or ESI ?
ESI MS
+ Separation compatible
+ Commercially available, routinely used
+ ESI-MS/MS well established
+ High sensitivity
+ Easy automation
- No sample archiving only on-line
- Minor components not analyzed in MS-MS mode
- Quantification vs. MS-MS
- LC gradient often slow
MALDI MS
+ Simpler spectra
+ Separation decoupled from mass spectrometry
+ Capability of sample archiving
Mass spectrometry of biomolecules 2010 330
MALDI Interface
Common strategy:
1. Deposition of liquid sample on target
2. Sample drying
3. Target insertion into mass spectrometer and analysis
Regime
• On-line
• Off-line
• In-line
Addition of MALDI matrix
• Mixing with analyte solution (sheath flow, T, liquid junction)
• Deposition of analyte solution on target precoated with matrix layer
Collection of eluent
• Discrete fractions
• Continuous streak
56
Mass spectrometry of biomolecules 2010 331
MALDI Interface
Off-line
Targets with hydrophilic spots (anchorchip).
Micromethods using piezoelectric pipetors and microtargets.
Deposition using electrospray
On-line
Flow probe with or without frit
Nebulizer for aerosol generation
In-line
ROBIN Interface
Deposition on target at subatmospheric pressure, moving belt interface
Off-line vs. on-line
+ Separation decoupled from mass analysis
+ Sample archiving
Mass spectrometry of biomolecules 2010 332
Sample Archiving (MALDI)
Strategy for separation - MALDI MS and MS/MS
1. Deposition of eluent on target
2. MALDI MS analysis (<10% sample consumed)
3. Analysis of MALDI MS data
4. MALDI MS-MS selected peaks (detail analysis)
(The overall procedure may be interrupted anytime after the first step.)
Mass spectrometry of biomolecules 2010 333
Flow Probe (on-line)
(Whittal, R. M., Russon, L. M., Li, L. J. Chromatogr. A 1998, 794, 367-375. )
Mass spectrometry of biomolecules 2010 334
(Fei, X., Wei, G., Murray, K. K. Anal. Chem. 1996, 68, 1143-1147. )
Nebulizer for Aerosol Generation (on-line)
Mass spectrometry of biomolecules 2010 335
Moving Belt Interface (in-line)
Interface for continuous introduction of nonvolatile sample in volatile solvent
into mass spectrometer
(McFadden W. H., Schwartz H. L. and Evans S. J. Chromatogr. 122, 389,
1976.)
to vacuum pumps
IR heating
warm N2 LC eluent
heated
element
slits
moving polyimide belt
ion
source
Mass spectrometry of biomolecules 2010 336
Orsnes, H., Zenobi, R. Chem. Soc. Rev. 2001, 30, 104-112.
ROBIN (Rotating Ball Inlet), in-line
57
Mass spectrometry of biomolecules 2010 337
source chamber (p ~ 10-6 Torr)
probe with
capillary
interface flange
source
coil
repeller
repeller center
(tape guide)
deposition
support
propelled
shaft
target
coil
rubber
wheel
2ND GENERATION VACUUM DEPOSITION INTERFACE
atmospheric
pressure
Mylar tape
Vacuum or Subatmospheric Deposition
(in-line, off-line)
Mass spectrometry of biomolecules 2010 338
Automated Fraction Collection (off-line)
E.g. commercially available Probot (www.lcpackings.com)
Mass spectrometry of biomolecules 2010 339
Microfluidic Devices and Mass Spectrometry
Why connect micro and macro? ... possible advantages of mdevices:
• integration of more steps, ―lab on a chip― (sample preparation,
purification, injection, separation...)
• paralell analysis (repeated motif on a single device)
• low cost – single use mdevice (serial production)
• all chemistry on the mdevice, MS as a detector
Basic types:
1.2D array
• affinity array: first selective preconcentration and/or capture of specific
compounds, then MALDI MS directly from the mdevice
• array of mvials with tips for ESI: higher reproducibility
2. Fluid mdevices (mdevices with channels)
• systems for paralell analysis – for ESI, MALDI ...
• systems integrating several steps
Mass spectrometry of biomolecules 2010 340
Micromethods
(Laurell, T.; Nilsson, J.; Marko-Varga, G. Trends Anal. Chem. 2001, 20, 225-231. )
Mass spectrometry of biomolecules 2010 341
Example of mdevice for Paralell Analysis
common liquid junction
separation channels
capillaries for infusion
into mass spectrometer
capillaries for sample injection
from a microtitration well plate
(Courtesy of T. Rejtar)
Mass spectrometry of biomolecules 2010 342
mdevice vs. Conventional Device
58
Mass spectrometry of biomolecules 2010 343
Applications: Proteins and Peptides. Protein Identification:
Peptide Mapping, Sequence Tag, Accurate Mass Tag.10
Mass spectrometry of biomolecules 2010 344
Biological Applications of MS
Genome, proteome, metabolome.
Small organic molecules, biomolecules, drugs, petrochemical products.
Biopolymers (DNA, proteins, carbohydrates).
Synthetic polymers.
Proteomics
Characterization of peptides and proteins – protein complement of genome.
Nowadays the main and the most perspective application of mass
spectrometry.
Genome  proteome. Level of protein expression (gen  protein) varies.
Genome is static, proteome dynamic: expression depends on type and
function of protein, location in cell, state and health of cell. Function of
organism is directly related to proteome.
Mass spectrometry of biomolecules 2010 345
Proteomics
Proteome analysis much more complex than genome analysis:
- more building blocks (amino acids)
- higher variability – modification of amino acids
- no existing amplification method for proteins analogical to PCR
- low levels of many proteins (e.g. regulatory proteins)
Differential proteomics
Determination of relative protein expression (presence or absence) in
v influenced and healthy organism (organ, tissue, cell).
Functional proteomics
Determination of all interactions (protein-protein, protein-DNA, etc.) in given
organism (organ, tissue, cell).
Mass spectrometry of biomolecules 2010 346
IUPAC: Amino Acids
Alanine Ala A CH3-CH(NH2)-COOH
Arginine Arg R H2N-C(=NH)-NH-[CH2]3-CH(NH2)-COOH
Asparagine Asn
N
H2N-CO-CH2-CH(NH2)-COOH
Aspartic acid Asp
D
HOOC-CH2-CH(NH2)-COOH
Cysteine Cys C HS-CH2-CH(NH2)-COOH
Glutamine Gln Q H2N-CO-[CH2]2-CH(NH2)-COOH
Trivial name Symbols Formula
Glutamic acid Glu
E
HOOC-[CH2]2-CH(NH2)-COOH
Glycine Gly G CH2(NH2)-COOH
Histidine His H
Mass spectrometry of biomolecules 2010 347
Isoleucine Ile I C2H5-CH(CH3)-CH(NH2)-COOH
Leucine Leu L (CH3)2CH-CH2-CH(NH2)-COOH
Lysine Lys K H2N-[CH2]4-CH(NH2)-COOH
Methionine Met M CH3-S-[CH2]2-CH(NH2)-COOH
Phenylalanine Phe F C6H5-CH2-CH(NH2)-COOH
Proline Pro P
Serine Ser S HO-CH2-CH(NH2)-COOH
Trivial name Symbols Formula
IUPAC: Amino Acids
Mass spectrometry of biomolecules 2010 348
Threonine Thr T CH3-CH(OH)-CH(NH2)-COOH
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V (CH3)2CH-CH(NH2)-COOH
Trivial name Symbols Formula
IUPAC: Amino Acids
59
Mass spectrometry of biomolecules 2010 349
Development of Techniques for Ionization of
Peptides and Proteins
1963 LDI Laser Desorption/Ionization (R. Honig)
1969 FD Field desorption (H. D. Beckey)
1974 PD Plasma desorption (R. D. McFarlane)
1976 SIMS Secondary ion mass spectrometry (A. Benninghoven)
1981 FAB Fast atom bombardment (M. Barber)
1984 ESI Electrospray (J. B. Fenn)
1988 MALDI Matrix-Assisted Laser Desorption/Ionization
(M. Karas & F. Hillenkamp, K. Tanaka)
1994 nano-ESI Nanoelectrospray (M. S. Wilm, M. Mann)
Mass spectrometry of biomolecules 2010 350
Current Ionization Techniques in Proteomics
FAB
max. m ~ 10 000 Da
LD ~ 20 pmol (classical FAB), < 1 pmol (CF-FAB)
ESI
max. m ~ 100 000 Da (z >> 1)
LD < 10 fmol (routine)
LD ~ amol or 10-9 M (selected applications)
MALDI
max. m ~ 106 (practically unlimited – TOF analyzer)
relatively least vulnerable to contaminants
LD < 10 fmol (routine)
LD ~ amol or 10-9 M (selected applications)
Mass spectrometry of biomolecules 2010 351
Mass Spectrometers in Proteomics
MALDI MS high sample throughput
TOF
ESI MS-MS structure elucidation
QqTOF, IT, FT ICR
New instrumentation MALDI TOF-TOF, MALDI LIFT TOF
MALDI QqTOF
• fast identification in MS mode
• option of later detail analysis in MS/MS mode
(result-dependent analysis)
Mass spectrometry of biomolecules 2010 352
Separation Methods in Proteomics
GE
gel electrophoresis on polyacrylamide gel
HPLC
high performance liquid chromatography on reverse phase
IEC
ion-exchange chromatography
AC
affinity chromatography
CE
capillary electrophoresis
Centrifugation
common centrifugation, gradient centrifugation
Mass spectrometry of biomolecules 2010 353
Gel Electrophoresis (GE)
2D SDS PAGE
2D: 2-dimensional electrophoresis
1. dimension: according to pI (isoelectric focusation)
2. dimension: according to size of SDS after denaturation
(charge/size = const.)
PA: polyacrylamide gel as separation medium
• Useful for separation of tens of thousands proteins, for very simple
mixtures 1D GE sufficient
• Fast identification and protein characterization on 2D gel is the most
common analysis of current proteomics
• Approximately 5% proteins and 30% peptides exhibit abnormal migration
• PTM’s influence apparent protein m (errors up to 50%)
• Problematic protein transport from gel matrix (electroblot on a membrane)
Mass spectrometry of biomolecules 2010 354
Example: 2D GE of Human Plasma
Zdroj: Expasy
60
Mass spectrometry of biomolecules 2010 355
RP HPLC
• Column: classical, capillary (f ~300 mm) or nano LC (f ~75 mm)
• Packing: C18, particle size: 3 – 10 mm (RP ... reverse phase)
• Gradient elution, e.g.:
- organic phase: ACN (acetonitrile) + 0.1% TFA (trifluoroacetic acid)
- aqueous phase: 0.1% TFA
- start: 10% organic phase + 90% aqueous phase
- end: 80% organic phase + 20% aqueous phase
• Organic solvent promotes peptide solubility, enables detection in UV
(230 – 240 nm) and evaporates fast, which is convenient for fraction
collection, e.g. in MALDI MS.
• Standard technique of column separation for peptides and proteins
Mass spectrometry of biomolecules 2010 356
Example: RP HPLC of BSA Digest
(Source: ThermoFinnigan, 2002)
Mass spectrometry of biomolecules 2010 357
Enzymatic Protein Digestion
protein peptide mixture
Reasons for moving from high m/z region to low m/z region:
1. Better parameters of mass spectrometers (higher resolution, precision
and accuracy of m/z determination, sensitivity)
2. Narrower isotopic envelope (simpler spectra, higher sensitivity)
Note.:
digestion = enzymatic cleavage
digest = mixture of protein fragments, products of digestion
modifications before digestion:
• reduction of -S-S- bridges using, e.g., dithiothreitol (DTT)
• alkylation –SH using, e.g., iodoacetamide (IAA)  m: + 57 Da
• purpose: „unfolding of protein― ... better approach for enzyme
enzyme
Mass spectrometry of biomolecules 2010 358
Enzymatic Protein Digestion
Most commonly used enzyme is trypsin (modified, e.g. TPCK to suppress
chymotrypsin activity, methylation etc.).
Other enzymes and reagents: lysine, chymotrypsin, CNBr etc.
Products of enzymatic cleavage
• specific fragments of analyzed protein
E.g. trypsin cleaves on C-terminus of amino acids K or R, if P is not a
neighbor. (Actual rules more complex):
N terminus-X-X-X-X-X-K-Y-Y-Y-Y-Y-Y-C terminus (YP)
• non-specific fragments of analyzed protein
• artifacts (modification of amino acids, e.g. oxidative, due to PA gel etc.)
• fragments of enzyme (autolysis)
• keratin fragments ( )
Mass spectrometry of biomolecules 2010 359
Enzymatic Protein Digestion in 2D Gel
1. Wash the gel slices for at least 1 hr in 500 microliters of 100 mM ammonium bicarbonate. Discard the
wash.
2. Add 150 microliters of 100 mM ammonium bicarbonate and 10 microliters of 45 mM DTT. Incubate at 60
degrees centigrade for 30 min.
3. Cool to room temp and add 10 microliters of 100 mM iodoacetamide and incubate for 30 min in the dark
at room temperature.
4. Discard the solvent and wash the gel slice in 500 microliters of 50% acetonitrile/100 mM ammonium
bicarbonate with shaking for 1 hr. Discardthe wash. Cut the gel into 2-3 pieces and transfer to a 200
microliter eppendorf style PCR tube.
5. Add 50 microliters of acetonitrile to shrink the gel pieces. After 10-15 min remove the solvent and dry the
gel slices in a rotatory evaporator.
6. Re-swell the gel pieces with 10 microliters of 25 mM ammonium bicarbonate containing Promega
modified trypsin (sequencing grade) at a concentration such that a substrate to enzyme ratio of 10:1 has
been achieved. (If the amount of protein is not known, add 0.1-0.2 microgramsof modified trypsin in 10
microliters of 25 mM ammonium bicarbonate).After 10-15 minutes add 10-20 microliters of additional
buffer to cover the gel pieces. Gel pieces need to stay wet during the digest. Incubate 4 hrs to overnight
at 37 degrees Centigrade.Proceed to step 8 if further extraction of the gel is desired (recommended)otherwise
continue with step 7.
7. Approximately 0.5 microliters of the supernatant may be removed for MALDI analysis and/or the
supernatant acidified by adding 10% TFA to a final concentration of 1% TFA for injection onto a narrowor
microbore reverse phase column. (If necessary the sample's volume may be reduced~1/3 on a
rotatory evaporator.)
8. Extraction (Optional)- Save supernatant from step 7 in tube X, and extract peptides from gel twice with
50 microliters of 60%acetonitrile/0.1% TFA for 20 min. Combine all extracts in tube X (using the same
pipet tip to minimize losses), and speed vac to near dryness.Reconstitute in 20 microliters of appropriate
solvent. Proceed with chromatography or MALDI analysis.
(http://www.abrf.org/ResearchGroups/ProteinIdentification/EPosters/pirgprotocol.html)
Mass spectrometry of biomolecules 2010 360
Strategy of Analysis in Proteomics
Basic Analysis Types
1. Identification (confirmation of presence of a known protein in sample)
2. Relative quantification in differential proteomics
3. Sequencing of unknown protein/peptide (de novo sequencing)
Sample is usually mixture of many proteins/peptides
     
Analysis is based on combination of separation with MS and MS/MS
Identification
Amino acid sequence of many proteins has already been described and it is
not necessary to analyze it again.
61
Mass spectrometry of biomolecules 2010 361
Strategy of Analysis in Proteomics
Top-down
• protein isolation
• protein processing, enzymatic cleavage
• analysis of peptides (MS, MS/MS)
Bottom-up
• enzymatic cleavage
• separation of peptides
• MS and MS/MS analysis of peptides
Mass spectrometry of biomolecules 2010 362
Some Useful Terms
genotype genetic ―equipment‖ of an organism, disposition
phenotype actual state of organism, result of interaction
with environment
in vivo in living organism
in vitro outside living organism, in artificial environment
http://www.meta-library.net/gengloss
Mass spectrometry of biomolecules 2010 363
Protein Identification
Information known prior analysis:
• Sample source (organism)
• Isoelectric point (pI) from 2D PAGE
• Molecular weight of protein from 2D PAGE, or possibly from MALDI MS
• N- and/or C- terminal portion of sequence (Edman degradation)
... The information is not sufficient for protein identification
Methods for protein identification
A. 2D GE + X + MS (peptide mass fingerprinting, peptide mapping)
B. 2D GE + X + RPHPLC + MS and MS/MS (sequence tag, AMT)
C. X + 2D LC (e.g. IEC and RHPLC) + MS and MS/MS
D. X + AC (afinity chromatography) + MS and MS/MS (IMAC, ICAT)
E. Strategy using X and isotopic reagents/standards
X ... selective enzymatic cleavage
Mass spectrometry of biomolecules 2010 364
Protein Identification
• MS is used to obtain information specific for protein
• Comparison of specific characteristics of analyzed protein with a protein
library (containing specific characteristics) of known proteins.
Specificity is based on:
1.Analysis of more peptides related to the protein
peptide mass fingerprinting (peptide mapping), MS analysis of
products of enzymatic cleavage of the protein
2.Detailed analysis of a single peptide of the protein
a) sequence tag, MS-tag – MS/MS analysis of a portion of the protein
(fragmentation of a peptide in gaseous phase)
b) accurate mass tag, AMT – determination of amino acid composition of
a portion of the protein (accurate m of product of enzymatic cleavage)
• Requires accurate MS analysis and a quality database
• MS-based identification is much faster and more sensitive than chemical
methods, such as Edman degradation)
• Negative result of database search may mean new protein discovery!!!
Mass spectrometry of biomolecules 2010 365
Peptide Mass Fingerprinting, PMF
(Peptide Mapping)
Confirmation of protein identity using MS of products of enzymatic cleavage
1. Separation (and possibly quantification) of proteins: 2D GE
2. Band excision, modifications, enzymatic cleavage
cells,
tissue
band excision
2D gel
electrophoresis
protein
extract isolated protein
(in gel)
peptide fragments
(protein digest)
chemical modification and
selective cleavage
(e.g. trypsin, CNBr)
Mass spectrometry of biomolecules 2010 366
Peptide Mass Fingerprinting, PMF
(Peptide Mapping)
3. MS analysis and evaluation of results
peptide fragments
(protein digest)
MALDI MS
ESI MS
m/z
search in protein database
(identification of known proteins)
separation - ESI MS
m/z
ESI MS-MS
of selected peptides
structure
(mutations, new proteins)
62
Mass spectrometry of biomolecules 2010 367
Example of Database Search: MS Fit
Input:
Mass spectrometry of biomolecules 2010 368
Example of Database Search: MS Fit
Output:
MS Fit – Protein Identification from m/z values of peptide fragments using
MS Fit program on http://prospector.ucsf.edu
Mass spectrometry of biomolecules 2010 369
Drawbacks of PMF
• Sample with a single protein needed, if possible, max. 2-3 proteins before
cleavage
• m/z accuracy < 100 ppm or even better (<10 ppm) needed
• Better m/z accuracy  more confident answer from database search
• Absence of peptides in digest usually lower problem than presence of
unexpected peptides
• Usually 4-5 peptides (in conjunction with high mass accuracy) sufficient
for confident protein identification
Mass spectrometry of biomolecules 2010 370
PMF: Theory and Practice
Extra (unexpected) peaks
• non-selective cleavage (e.g. due to chymotryptic activity of trypsin)
• impurities (e.g. keratins)
• unsatisfactory protein isolation (additional proteins in band)
• enzyme autolysis
• post-translational modifications (PTM’s), artifacts
Missing peaks
• low-soluble peptides
• adsorption of peptides
• mutual suppression of peptides
• non-selective cleavage
• insufficient digestion (sterical restrictions)
• PTM’s, artifacts
Mass spectrometry of biomolecules 2010 371
Sequence-Tag Method (MS-Tag)
• Protein identification based on knowledge of a portion of short portion of
protein sequence (sequence-tag, MS-tag), usually sequence of a single
peptide
• Precise determination of amino acid sequence of a protein fragment from
digestion – usually using ESI-MS/MS or MALDI-PSD-MS mass spectrum
or peak list
• Required length of sequence 3 or more amino acids
• The longer sequence is known, the more unambiguous identification
• The known portion of sequence is being searched for in protein databases
• Other information of protein: organism, M.W., pI (both from 2D GE), etc.
Mass spectrometry of biomolecules 2010 372
Basic Strategy of MALDI MS and LC - ESI MS
Source: ThermoFinnigan 2002
peptidové mapování
MS/MS: MS-tag
63
Mass spectrometry of biomolecules 2010 373
2D LC/MS/MS Schematics
Source: ThermoFinnigan 2002
Mass spectrometry of biomolecules 2010 374
Experimental Setup for 2D LC/MS/MS (1)
1D: Chromatography on ionex
2D: RHPLC (2 HPLC columns ...
higher productivity)
Source: ThermoFinnigan 2002
Mass spectrometry of biomolecules 2010 375
Experimental Setup for 2D LC/MS/MS (2)
Source: ThermoFinnigan 2002
Mass spectrometry of biomolecules 2010 376
Control of 2D LC/MS/MS Experiment
Source: ThermoFinnigan 2002
Mass spectrometry of biomolecules 2010 377
Results of 2D LC/MS/MS Experiment
Source: ThermoFinnigan 2002
Mass spectrometry of biomolecules 2010 378
LC-ESI MS and MS/MS:
Dynamic analysis LC-ESI MS/MS (data-dependent analysis):
• One MS sken followed by few MS/MS scans of main precursors
• In the case of more complex mixtures even MS3 scans possible (e.g. for
detection of phosphorylation)
64
Mass spectrometry of biomolecules 2010 379
Accurate Mass Tag (AMT)
• Identification of protein on basis of knowledge of accurate mass of a
single peptide fragment (after enzymatic digestion of the protein)
• Accurate determination of protein mass of peptide using FT ICR MS
• Typical number of amino acid residues in peptide chain ~ 10
• With precision of determination m/z < 1 ppm, there is high probability that
the measured mass correspond only to a single amino acid composition
as shown on the next slide
atom H C N O S
m [Da] 1.0078246 12.000000 14.00307 15.994915 31.972072
Mass spectrometry of biomolecules 2010 380
AMT
(T. P. Conrads, G. A. Anderson, T. D. Veenstra, L. Paša-Tolić and R. D. Smith
Anal Chem. 2000, 72, 3349-3354.)
Mass spectrometry of biomolecules 2010 381
Proteins and peptides. Isotope Labeling. ICAT. Sequence
Determination. Post-translational Modification.
11
Mass spectrometry of biomolecules 2010 382
Other Strategies of Proteome Analysis
• Not aimed at complex proteome analysis
• Based on column separation techniques (rather than on 2D GE)
• Simplification of the original mixture – selection of specific
proteins/peptides, e.g. containing cystein (ICAT) or phosphorylated
amino acid (IMAC) etc.
• Possible losses due to incomplete analysis are not dramatic and are
compensated for by simpler and shorter analysis
• Additional tricks, such as use of isotope labels provide quantitative
information about analyte
Mass spectrometry of biomolecules 2010 383
Isotope Labels MS of biomolecules
Unstable isotope labeling
• in radiochemistry
Stable isotope labeling
• common in MS
• basic approaches
- use of inner standards labeled with isotope
- incorporation of compound labeled with isotope into organism
(useful for differential proteomics of lower organisms)
- derivatization of analyte using reagent labeled with isotope
Mass spectrometry of biomolecules 2010 384
Some Methods and Abbreviations
GIST Global Internal Standard Technology (2H, 13C, 15N)
ICAT Isotope-Coded Affinity Tags
(cystein-containing peptides capture on affinity columns)
PhIAT Phosphoprotein Istope-coded Afinity Tag (fosforylované peptidy)
IMAC Ion Metal Afinity Chromatography
(phosphopeptide capture on affinity columns)
AQUA Absolute QUAntification
(synthesized isotopically labeled peptides as internal standards)
SILAC Stable Isotope Labeling with Amino acids in Cell culture
(culture growth in normal and enriched media)
MUDPIT Multidimensional Protein Identification Technology
(SCX – RHPLC – MS/MS)
etc. etc.
65
Mass spectrometry of biomolecules 2010 385
ICAT (Isotope-Coded Affinity Tags)
ICAT ... isotope-coded affinity tags (R. Aerbersold)
Determination of differences in protein expression from relative signal of
peptides
1. Protein fractionation
2. Enzymatic digestion of entire sample
3. Isotope labeling (ICAT)
4. Mixing and MS analysis
Mass spectrometry of biomolecules 2010 386
ICAT
Mass spectrometry of biomolecules 2010 387
ICAT
Note
• Proteins can be labeled before digestion (step # 2 and 3 exchanged)
Drawbacks of 1st generation ICAT
• Mass difference of the isotope labels was equal to 8, which might lead to
interferences in MS/MS spectra
• Slightly different retention of analytes labeled with light and heavy
reagent. Result: the ratio b/w light and heavy form cannot be found from
the ratio of ion intensities during HPLC MS/MS; integration across entire
LC peak was necessary
2nd generation ICAT
• Use 9 13C atoms in the link (instead of 8 2H atoms)
• The same elution profile of light and heavy forms and less interferences
Mass spectrometry of biomolecules 2010 388
ICAT Characteristics
Advantages
+ Suitable for a range of protein samples (body fluids, cells, tissues,
cultures)
+ Significant simplification of mixture
+ Compatibility with other methods suitable for analysis of minor proteins
Disadvantages
- Relatively large label (~500 Da)
- Not suitable for proteins without cystein (e.g. 8% in the case of yeast)
Mass spectrometry of biomolecules 2010 389
Determinations of Protein/Peptide Sequence
Classical analysis
- Edman degradation
- Determination of terminals (N, C)
Disadvantages: analysis time, relatively high cost and sample consumption
Current strategy: combination of more methods
- preparative separation
- enzymatic digestion
- isotope labeling
- MS
- MS/MS, ISF, PSD
- MSn
Mass spectrometry of biomolecules 2010 390
Determination of Peptide Sequence using MS
Combination of chemical cleavage and MS
1. Generation of mixture of peptide fragments differing by one amino
acid:
a) phenyl isothiocyanate + A1A2A3..  phenylisothihydantoinA2A3.. + A1
phenyl isothiocyanate + A2A3..  phenylisothihydantoinA3.. + A2
etc.
phenyl isothiocyanate in low amounts as terminating reagent forms
small fraction of phenylcarbamate of each peptide
b) alternative strategy uses application of carboxypeptidase for different
time periods or in different amounts  formation of different digests
2. MALDI MS of peptide fragment mixtures.
amino acid is determined from distance of adjacent peaks of the same
type, sequence from the peak order
66
Mass spectrometry of biomolecules 2010 391
Patterson, D. H., Tarr, G. E., Regnier, F. E. and Martin, S. A., Anal. Chem.1995, 67, 3971.
Mass spectrometry of biomolecules 2010 392
Determination of Peptide Sequence using MS/MS
CID
• low-energy CID currently most popular fragmentation method of peptides
• IT, TQ, QTOF
ISD
• requires isolation of pure analyte
• MALDI TOF
PSD
• lower quality of spectra compared to CID, sometimes spectra stitching
needed
• MALDI TOF
Note
• high-energy CID, SID, photodissociation, dissociation after electron capture
are not used routinely yet
• Leu/Ile … isomers, Gln/Lys ... isobars
Mass spectrometry of biomolecules 2010 393
Peptide Fragmentation
Fragmentation of 1 or 2 bonds in peptide chain
- fragments containing N terminus (a, b, c)
- fragments containing C terminus (x, y, z)
- these ions may be formed even after breakage of 2 bonds of the chain
- loss of NH3, H2O, CO2
Fragmentation of side chain (amino acid residue)
- usually loss of a portion of side chain of amino acid
- fragments type d, w, v
Inner fragments
- do not contain either N or C terminus ... lower analytical significance
Immonium ions
- useful information about amino acid presence; m(IM)= m(AKres) - 27
Mass spectrometry of biomolecules 2010 394
Schematics of Peptide Fragmentation
immonium ions of amino acids:
H
2
N - CH - C -
OR
1
NH - CH - C -
OR
2
NH - CH - C -
OR
3
NH - CH - COOH
R
4
x
3
a
1
y
3
b
1
z
3
c
1
x
2
a
2
y
2
b
2
z
2
c
2
x
1
a
3
y
1
b
3
z
1
c
3
H+
Roepstorff & Fohlman, Biomed. MS 1984, 11, 601.
Mass spectrometry of biomolecules 2010 395
Fragments of Peptides
Mass spectrometry of biomolecules 2010 396
Schematics of Peptide Fragmentation
67
Mass spectrometry of biomolecules 2010 397
Other Types of Peptide Fragments
Mass spectrometry of biomolecules 2010 398
amino acid m(mono) m(immonium) m(accompanying ions)
A 71.03712 44
R 156.10112 129 59,70,73,87,100,112
N 114.04293 87 70
D 115.02695 88 70
C 103.00919 76
E 129.04260 102
Q 128.05858 101 56,84,129
G 57.02147 30
H 137.05891 110 82,121,123,138,166
I 113.08407 86 44,72
L 113.08407 86 44,72
K 128.09497 101 70,84,112,129
M 131.04049 104 61
F 147.06842 120 91
P 97.05277 70
S 87.03203 60
T 101.04768 74
W 186.07932 159 77,117,130,132,170,171
Y 163.06333 136 91,107
V 99.06842 72 41,55,69
m ... amino
acid residuum
Mass spectrometry of biomolecules 2010 399
Calculation of Molecular Weight
of Peptide/Protein
Sum of amino acid residua masses
+
Mass of terminals:
usually 1 Da (H) for N terminus (-NH2)
and 17 Da (OH) for C terminus (-COOH)
+
Mass of proton, 1 Da – ion is usually in [M+H]+ form
(but e.g. 23 Da for adduct [M+Na]+ etc.)
Mass spectrometry of biomolecules 2010 400
CID Fragmentation
Types of ions in CID
1. b and y ion series
2. accompanying peaks
–17, NH3 loss from Gln, Lys, Arg
–18, H2O loss from Ser, Thr, Asp, Glu
a (tj. b – 28), satellite fragment series of b type (CO loss)
3. immonium ions of amino acids.
4. internal fragments – especially from Pro in the direction of C terminus
Precursor selection in CID MS/MS:
z = 2: [M+2H]2+
Ion with two charges provides higher quality CID spectra compared to ions
with z = 1 a 3
Mass spectrometry of biomolecules 2010 401
ISD fragments
Fragmentation rules … according to relative bond strength.
Typical products of peptide fragmentation:
c, y, z
H2N - CH - C -
OR
1
NH - CH - C -
OR
2
NH - CH - C -
OR
3
NH - CH - COOH
R
4
x3
a
1
y3
b
1
z3
c
1
x2
a
2
y2
b
2
z2
c
2
x1
a
3
y1
b
3
z1
c
3
H+
Mass spectrometry of biomolecules 2010 402
ISD. Analyte: 20 pmol KGF analog. Matrix: sinapinic acid. Laser: N2.
(V. Katta, D. T. Chow, M. F. Rohde Anal. Chem., 70, 4410 -4416, 1998.)
68
Mass spectrometry of biomolecules 2010 403
ISD
Analyte: oxidized B chain of bovine insulin
Matrix: thiourea
Laser: Er:YAG (l = 2936 nm)
(http://medweb.uni-
muenster.de/institute/impb/research/hillenkamp/publicat/abs-cm99.html)
Mass spectrometry of biomolecules 2010 404
PSD Fragments
Typical products of peptide fragmentation: a, b, y, z, d.
Accompanying peaks –17 (loss of NH3) a –18 (loss of H2O).
Immonium ions of amino acids.
Internal ions – especially from Pro to the C terminus
(B. Spengler J. Mass Spectrom. 32, 1019-1036, 1997)
Mass spectrometry of biomolecules 2010 405
immonium ions of amino acids:
Mass spectrometry of biomolecules 2010 406
PSD, source: B. Spengler J. Mass Spectrom. 32, 1019-1036, 1997
Mass spectrometry of biomolecules 2010 407
PSD of bombesin, source: Bruker Daltonics, GmbH 2000
Mass spectrometry of biomolecules 2010 408
Evaluation of MS/MS spectra of peptides
Manual and a semiautomatic deduction of sequence
• localization of ion series of b and y type (accompanying series a)
• identification of immonium ions
• identification of inner fragments
• interpretation complicated by occurrence of other fragments
• not all peptides are product of specific digestion, e.g. not all tryptic
fragments are terminated with with R or K on C-terminus
... unambiguous determination of sequence of unknown peptide from CID is
often impossible
Automated determination of sequence
• MS/MS spectrum of unknown peptide is compared with a database of
computer-generated spectra of all peptides, which have m/z of precursor
• e.g. algorithm Sequest (J. R. Yates III et. al., 1994)
... much more successful than the deductive approach
69
Mass spectrometry of biomolecules 2010 409
Determination of Protein Sequence
Procedure
1. Enzymatic digestion
production of more types of digests to generate overlapping fragments
- various length of digestion
- various enzymes (trypsin, V8 ...)
- random cleavage, ―shotgun sequencing‖
2. Determination of at least partial sequence of peptides
RPHPLC ESI MS/MS
3. Deduction of sequence using PC
total sequence determined from combined portions of peptide sequences
Mass spectrometry of biomolecules 2010 410
Helpful Techniques for Sequence Determination
Protein hydrolysis in H2
18O
identification of terminus: C-terminus will not be labeled
Hydrolysis of protein in mixture of H2
18O a H2
16O (1:1)
1. MS: dublets of tryptic peptides (except fragment of protein C-terminal)
2. MS/MS: both dublet peaks of a peptide selected as precursor for MS/MS;
only fragments containing C terminus of a peptide will generate doublet
fragments ... easier to survey spectra
Esterification (methylation) of carboxyl groups of a peptide
m = +14 / carboxyl group (-COOH  -COOCH3)
comparison of original and resulting spectra
similar technique based on derivatization of amino group of a peptide
Note 1: exchange reaction may run even on C terminus
Note 2: more complex isotope patterns with masses M (2x16O), M+2
(1x16O,1x 2x18O) a M+4 (2x18O)
Mass spectrometry of biomolecules 2010 411
Determination of Protein Sequence
Example:
Various extent of fragmentation
due to various length of
digestion and use of isotope
labels
Determination of terminus:
hydrolysis in H2
18O
(C-terminus will not be labeled)
Note: exchange reaction may
run even on C terminus
HK
time:
Mass spectrometry of biomolecules 2010 412
Mutation Detection
Detection of exchange/absence of an amino acid(s) in protein chain
Procedure:
1. Enzymatic digestion
2. Analysis of mass spectra
- missing peptides
- superfluous peptides
3. MS/MS analysis of unknown (superfluous) peptides and their comparison
with normal proteins/peptides, analysis of peak shifts
Mass spectrometry of biomolecules 2010 413
Post-Translational Modifications (PTM)
• modifications after translation RNA  protein on ribosome
• cannot be explained based on genome
• related significantly with protein function
• more than 200 PTM types described (database Delta Mass)
• often only small fraction of protein modified  sensitivity
• requires selective isolation of peptides with modified amino acid
• bond between peptide and modifying group often weak
Mass spectrometry of biomolecules 2010 414
Post-Translational Modifications (PTM)
Common PTMs:
• phosphorylation
• glycosylation
• acylation (fat acid esters, acetyl)
• attachment of glycosylphosphatidyl inositol
• proteolytic products
• carboxylation (of glutamic acid)
• deamidation of asparagine and glutamine
Some modifications might be quite complex, e.g. oligosaccharide
modifications of glycoproteins (many types of sugars and many sites on
protein that can accommodate sugars – O, N). For structural elucidation,
combined methods using MSn and enzymatic cleavage might be used (Nglycosidase,
O-glycanase etc.).
70
Mass spectrometry of biomolecules 2010 415
Phosphorylation
• reversible PTM related to cellular regulatory mechanisms
• monoesteric bond of phosphoric acid group to side chain hydroxyl of
1. serine (167 Da)
2. threonine (181 Da)
3. tyrosine (243 Da)
MS/MS identification of phosphorylation
1. Loss of H3PO4 (neutral loss scan: 98 Da)
usually provides also ion [M+H-98]+
2. Detection of ion PO3
- (79 Da) in negative mode
Parent ion scan for m/z (product) = 79
Other negative ions: H2PO4
- (97 Da), PO2
- (63 Da)
3.Fragment peak shifts in MS/MS spectra
 NH  CH  C 
O
R
HO  P  OH
O
O
Mass spectrometry of biomolecules 2010 416
Phosphorylation
Identification procedure:
1. Preparation of mixture of proteolytic peptides (containing phosphopeptides)
2. Separation of phosphopetides using e.g. HPLC, CE, affinity
chromatography, such as Immobilized Metal Affinity Chromatography
(IMAC), see Porath, J. Protein Expression Purif. 1992, 3, 263.
3. MS/MS analysis of phosphopeptides
Relatively stable monoester bond
 peak shifts in MS/MS spectra (m = + 80 Da)
amino acid M (residue) M (monoester H3PO4)
serine 87 167
threonine 107 187
tyrosine 163 243
Mass spectrometry of biomolecules 2010 417
Example: ESI MS/MS of Oligopeptide with Monophosphorylated Serine
Phe Gln Pse Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys
y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1
b2 b3 b4 b5 b6
Phe Gln Pse Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys
y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1
b2 b3 b4 b5 b6
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z0
100
%
x2.5
y15
y14
y13
y12
y11
y10
y9
y8
y7
y6
y5
y4y3
y1
MH2
2+ H3PO4
MH2
2+
pSer
b2
y2
b3 b5
b6b4
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z0
100
%
x2.5
y15
y14
y13
y12
y11
y10
y9
y8
y7
y6
y5
y4y3
y1
MH2
2+ H3PO4
MH2
2+
pSer
b2
y2
b3 b5
b6b4
Mass spectrometry of biomolecules 2010 418
Example: ESI MS/MS of Oligopeptide with Monophosphorylated Serine
Information sources:
1. Molecular peaks MH2
2+ a (M - H3PO4)H2
2+, mass difference m = 98
(m/z = 49)
2. b-ions: m/z (b3-b2)= 167
3. y-ions: m/z (y14-y13)= 167
(credit: K. R. Jennings)
Phe Gln Pse Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys
y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1
b2 b3 b4 b5 b6
Phe Gln Pse Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys
y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1
b2 b3 b4 b5 b6
Mass spectrometry of biomolecules 2010 419
Disulfide Bridge Analysis. Proteins. MS databases.
DNA, Saccharides, Synthetic Polymers.12
Mass spectrometry of biomolecules 2010 420
Disulfide Bridge Analysis
cysteine (-SH)  cystin (-S-S-), intra- and inter- molecular bridges
Number of cysteines – using alkylation of cysteine
 e.g. alkylation of a single cysteine with vinylpyridine increase mass of
protein/peptide by 105 Da  number of cysteines = m/105
Localization of cysteines in protein chain
Enzymatic cleavage and splitting of the digest into 2 aliquots:
1. aliquot ... MS analysis
2. aliquot ... reduction -S-S- (e.g. with dithioerythritol), then MS analysis
Comparison of the 2nd spectrum with the 1st spectrum:
1. m = 2 Da  peptide with an intramolecular -S-S- bond
2. the first peak disappear, two other peaks with lower m/z show up 
intermolecular bridge between 2 peptides
Knowledge of sequence of amino acid (MS/MS) enables exact localization
of -S-S- bridge in protein chain.
71
Mass spectrometry of biomolecules 2010 421
Influence of S-S Bridges
in ESI-MS
Structures of lysozyme (bsheet and a
helix) stabilized by 4 S-S bridges
Reduction with 1,4-dithiothreitol (DTT)
removes S-S bridges

„Unfolded― protein with more exposed
basic amino acid residues

Higher number of charges
HK
Mass spectrometry of biomolecules 2010 422
pH increase:
„Unfolded― protein with
more exposed basic
amino acid residues
HK
Mass spectrometry of biomolecules 2010 423
CA=carbonicanhydrase,Cy=cytochrome
Valaskovic,G.A.;Kelleher,N.L.;McLafferty,
F.W.Science1996,273,1199.
Examples: Nanospray MS
Mass spectrometry of biomolecules 2010 424
Noncovalent Interactions
• Interactions with low mass ligands (+metal ions), other proteins,
oligomers etc.
• The relation between occurrence of the complex in (g) and (l) phase is
not always clear
• Complexes dissociate during ionization and MS analysis
Example:
1.Complexes of bovine hemoglobine are stabilized in form of tetramer by
cross-linking with glutaraldehyde (bridges between lysine residues). Then
MALDI MS (see next slide)
2.ESI for complexes of proteins with ligands (e.g. metaloproteins with
drugs) ... soft ionization, which barely removes water molecules. Study of
interactions between 2 proteins is more difficult – use of soft extraction
conditions, favorable for formation of complexes in solution.
Mass spectrometry of biomolecules 2010 425
Tetramer
known from solution
HK
Mass spectrometry of biomolecules 2010 426
Scientific Databases on Internet
The databases below are just a few examples of many:
NCBI (National Center for Biotechnology Information)
Medline: www.ncbi.nlm.nih.gov
Scirus: www.scirus.com
ScienceDirect: www.sciencedirect.com
Databases for organic chemistry
NIST Chemistry WebBook (NIST)
Spectra Online (ThermoGalactic)
Spectral Data Base System, SDBS (NI AIST)
72
Mass spectrometry of biomolecules 2010 427
Scientific Databases on Internet
Internet sources for protein identification using MS
• Eidgenossische Technische Hochschule (MassSearch)
www.cbrg.inf.ethz.ch
• European Molecular Biology Laboratory (PeptideSearch)
http://www.mann.embl-
heidelberg.de/GroupPages/PageLink/peptidesearchpage.html
• Swiss Institute of Bioinformatics (ExPASy) www.expasy.ch/tools
• Matrix Science (Mascot) www.matrixscience.com
• Rockefeller University (PepFrag, ProFound) prowl.rockefeller.edu
• Human Genome Research Center (MOWSE) www.seqnet.dl.ac.uk
• University of California (MS-Tag, MS-Fit, MS-Seq) prospector.ucsf.edu
• Institute for Systems Biology (COMET) www.systemsbiology.org
• University of Washington (SEQUEST)
http://fields.scripps.edu/sequest/index.html
Mass spectrometry of biomolecules 2010 428
Scientific Databases on Internet
Other links:
UMIST (pepMAPPER) http://wolf.bms.umist.ac.uk/mapper/
European Molecular Biology Laboratory, Heidelberg (PeptideSearch)
www.narrador.embl-heidelberg.de
Global Proteome Machine
http://h112.thegpm.org/tandem/thegpm_tandem.html
Protein (peptide) databases
Genpept – NCBI GenBank
NBRF - National Biomedical Research Foundation
Swissprot - Swiss Institute of Bioinformatics
Owl - Leeds Molecular Biology Database Group
Delta Mass – protein posttranslational modification database
Mass spectrometry of biomolecules 2010 429
Useful Tools on Internet
Tools
MS-Comp – suggestion of possible combinations of amino acids, mass
table of dipeptides
MS BLAST 2 – short sequences (6 amino acids)
BLAST a FASTA – protein homology
GlycoSuite DB, GlycoSciences – saccharides analysis
Mass calculators (GPMaw, SHERPA, PAWS, MW Calculator)
etc.
Mass spectrometry of biomolecules 2010 430
MS of Nucleic Acids and Oligonucleotides
• Ionization: usually more efficient for negative ions. Analysis typically in
negative mode.
• Ionizaton technique: MALDI (usually IR MALDI)
• Less successful than in the case of proteomics
• Higher extent of fragmentation and more salt adducts
• Proper desalting essential … prevention of formation of a series of
adducts with Na and K
• MALDI of heavy DNA (>100 kDa): linear TOF MS, IR laser, pulse
extraction
Mass spectrometry of biomolecules 2010 431
MALDI MS of Nucleic Acids and Oligonucleotides
Typical matrices
3-hydroxypicolinic acid
2’,4’,6’-trihydroxyacetophenon
picolinic acid
Typical applications
• characterization of synthetic and biologic oligonucleotides (determination
of M.W.)
• analysis of PCR products, mutation analysis (absence or exchange of
nucleotides)
• DNA sequencing
- Sanger sequencing (classical method)
- Sequencing using exonuclease
- Fragmentation (in gaseous phase)
Mass spectrometry of biomolecules 2010 432
MALDI MS of Nucleic Acids and Oligonucleotides
• MALDI MS spectra of very heavy NA (>2 thousands nucleotides)
• m/z accuracy: < 1% ... the best of current methods
• excellent sensitivity: < 1 fmol
Berkenkamp, S; Kirpekar, F; Hillenkamp, F Science 1998, 281, 260-262.
73
Mass spectrometry of biomolecules 2010 433
IR MALDI MS of RNA
HK
Mass spectrometry of biomolecules 2010 434
Sequencing NA using Exonuclease and MALDI MS
(Juhasz, P.; Roskey, M. T.; Smirnov, I. P.; Haff, L. A.; Vestal, M. L.; Martin, S.
A.; Anal. Chem. ; 1996; 68(6); 941-946)
Mass spectrometry of biomolecules 2010 435
Oligonucleotides Fragmentation in Gaseous Phase
Schematics of fragmentation of oligonucleotides in gaseous phase and
marking of fragments
O  P  O
O
OH
B2B1
HO O  P  O
O
OH
O  P  O
O
OH
B3 B4
OH
w3
a1
x3
b1
y3
c1
w2
a2
w2
b2
x2
c2
w1
a3
x1
b3
y1
c3
z1
d3
z3
d1
y2
d2
Mass spectrometry of biomolecules 2010 436
Saccharides Analysis
• Soft ionization methods: MALDI, ESI
• MSn for determination of sequence and structure
• Enzymes used for partial cleavage of saccharides prior MS analysis
• Common monosacharides: glucose, manose, galactose, fucose, Nacetylglucosamine,
N-acetylgalactosamine and N-acetylneuramine acid
(sialic acid).
• Determination of complete structure of oligosaccharides is more difficult
than in the case of proteins and nucleic acids
- result of isomeric nature of sugars and possible branching
- knowledge of building blocks and sequence is not sufficient for
structural elucidation. Type, location and optical configuration of each
glycosidic bond needed
Mass spectrometry of biomolecules 2010 437
Nomenclature for MS/MS Oligosaccharides
• Nomenclature of MS fragments with charge on non-reducing side: A, B
and C; fragments with charge on reducing side: X, Y and Z depending
whether a circle or a glycosidic bond is cleaved.
• Lower index of B, C, Y and Z fragment ions determines number of
cleaved glycosidic bonds, upper index of A and X fragment ions
determines bonds that were cleaved. Lower indexes a, b etc. give
cleaved branch of nonlinear saccharides.
• Fragments B, C, Y and Z together with mass differences can be used for
determination of sequence and branching.
• MS is suitable for determination of optical isomery of glycosidic bond.
Method is based on selective oxidation of b-anomer of derivatized
hexoses using CrO3; ketoester is formed.
Mass spectrometry of biomolecules 2010 438
MSn of Oligosaccharides
Ion fragments produced upon (a) MS 2 , (b) MS 3 and (c) MS 4 of
permethylated maltoheptaose.
Source: Weiskopf, A. S.; Vouros, P. and Harvey, D. J. Rapid.
Commun. Mass Spectrom. 1997, 11, 1493–1504.
74
Mass spectrometry of biomolecules 2010 439
MSn of Oligosaccharides
Mass spectrometry of biomolecules 2010 440
Mass spectrometry of biomolecules 2010 441
MSn of Oligosaccharides
Mass spectrometry of biomolecules 2010 442
MSn of Oligosaccharides
Mass spectrometry of biomolecules 2010 443
MSn of Oligosaccharides
MSncharacterizationoftheHexNAc5Hex5–Iisomer
fromchickenovalbumin.(a)MS3–m/z1169.5
937.7,(b)MS4—m/z1169.5937.71333.7
Mass spectrometry of biomolecules 2010 444
Lipids
Fat acids, acylglycerols, derivates of cholesterol, phospholipids and
glycolipids
FAB, DCI (desorption chemical ionization), MALDI
Negative mode ([M-H]- ions detected)
Fragmentation
phospholipid negative ion
fragments of sacharides
75
Mass spectrometry of biomolecules 2010 445
Analysis of Synthetic Polymers
MALDI TOFMS
high Mmax, simple spectra, number of charges z = 1
alternative of GPC
Analysis
Building unit (monomer)
Terminal groups
Absolute molar weight
Polydispersion (mean m, dispersion width)
Challenges
Adduct formation with Na+, K+
Mass discrimination … ionization and detection efficiencies = f(m)
Correct transformation from time to mass domain
Example: Carman Jr, H.; Kilgore, D.; Eastman Chemical Company, USA,
ASMS 1998
Mass spectrometry of biomolecules 2010 446
Source:Esser,E.etal.Polymer2000,41,4039–4046
Mass spectrometry of biomolecules 2010 447
Other Topics
... all topics were not covered, for example:
Fragmentation – chemistry of reactions in gaseous state
Ion mobitlity mass spectrometry
Isotope dilution/enrichment, quantification in MS
Preparative MS, history: preparation of 235U
etc. etc.
Mass spectrometry of biomolecules 2010 448
Questions and Consultation
Training for examination
A consultation meeting can be scheduled in beginning of January
Mass spectrometry of biomolecules 2010 449
Questions and Consultation
13
Mass spectrometry of biomolecules 2010 450
Jan Preisler
Dept. Analytical Chemistry 43, tel.: 541 129 271, preisler@chemi.muni.cz
Please download updated study material in pdf format:
http://147.251.29.118/MSBio/MSBio.htm
Please report any discrepancies/errors in the pdf document to me.
Thank you.
Other Consultations in my office.
Exam dates
December? January? February?
V. Questions and Consultation
76
Mass spectrometry of biomolecules 2010 451
1. Compare MALDI and ESI (advantages vs. disadvantages).
2. What can be said about 2 different spectra of the same (single) peptide?
(m/z-axis origin is not in zero, only a portion of the spectra is displayed.)
3. For 2 adjacent peaks of the same compound in ESI-MS, following values
were determined: (m/z)1~ 1001 and (m/z)2 ~ 1501. Determine m.
4. Time of flight of ion C2H8O2N+ in TOFMS is 14.8 ms. Calculate the mass
of a singly-charged ion, which hits the detector at 8.94 ms? What organic
ion can it be?
5. What variables do have impact on resolution in TOFMS? Positive or
negative influence?
6. Compare numbers of ions that can be detected during acquisition of a
single spectrum using FT-ICR-MS and quadrupole filter?
Exemplary Questions
m/z m/z
S S
Mass spectrometry of biomolecules 2010 452
Exemplary Questions
7. Why instruments that can monitor both isotopes at the same time are
preferred for isotope ratio determination?
8. Can a quadrupole filter be used for analysis of a protein with M.W. 30
kDa?
9. What ionization method and what mass spectrometer would you
suggest for:
a) explosive detection on airport
b) sequencing of short peptides
c) semiquantitative determination of ~70 samples in geologic sample
d) identification of elemenal impurities in thin surface layer of sample
10. What is the origin of Lorentzian peak profile in FT-ICR-MS?
11. What is mutual orientation of equipotential level of U and electric field
intensity vector E?
12. What will be the difference between velocities of two ions with m = 100
a.m.u., z = 2, initial velocity v01 = 100 m/s and v02 = 200 m/s after
acceleration by 1 kV and 10 kV?
Mass spectrometry of biomolecules 2010 453
Exemplary Questions
13. What determines the practical upper m/z limit in TOF MS?
14. Compare challenges in determination of peptides and DNA oligomers
using MS.
15. You are about about to analyze peptides/proteins in a soup. Adding of
what compound will you try to avoid prior to MS analysis? How would
you modify the soup and what ionization technique will you use?
16. What detector fits ion trap better: MCP, channeltron, electron multiplier,
photographic plate or Faraday cup?
17. What is the influence of time dispersion (of ion formation) on resolution
of ion trap?
18. What is m of amino acid A, its residuum (in peptide chain) and its
immonium ion?
19. Explain the plateau on the graph of number of peptides vs. m/z
accuracy (AMT method) between 200 and 700 ppm accuracy.
20. Compare advantages and disadvantages of 2DGE and column
separation techniques in proetomics.
Mass spectrometry of biomolecules 2010 454
Exemplary Questions
21. What parameters of mass spectrum may improve if signal is recorded
using a) FT ICR MS, b) TOF MS, c) IT for longer period?
22. Why does mass spectrometer need to be evacuated prior to any
measurement?
23. What is the most significant reason of limited resolution in MALDI TOF
MS? Explain principles of techniques leading to resolution
improvement in MALDI TOF MS.
24. What is the difference between units u, Da a Th?
25. What is the difference between energy and velocity dispersions of ions?
26. In what region of TOF mass spectrometer are analytically important
fragments generated during a) MALDI ISD, b) MALDI PSD?