CH 908: Mass Spectrometry
Lecture 7
Tandem mass spectrometry
Prof. Peter B. O’Connor
Tandem Mass Spectrometry or MS/MS
Isolation
Fragmentation
Isolation
Fragmentation
Benefits:
1.Extremely high
MS/MS/MS,
MS/MS
or MS3
specificity
2.More structural
information
Limitations:
1.Isolation window
2.Fragmentation
efficiency
3.Ion Losses
Tandem Mass Spectrometry
“Tandem in Time” – FTMS, QITMS
“Tandem in Space” – Triple quad, TOF/TOF,
sector
OUTLINE
• Tandem in space instruments
–
–
–
–
Sectors
Triple quads
Q-tofs, tof/tofs, unique instruments (pentaquads)
Orbitrap (sort-of)
• Tandem in time instruments
– Ion traps
• Classic 3D
• Linear ion trap
– FTICR
• MS/MS methods
–
–
–
–
CAD
ECD (plus ETD, EID, EED, etc)
PD (UVPD, IRMPD)
SID
Magnetic
sector
instruments
Ions are deflected and accelerated down a curved path to
the detector.
Magnetic-Sector Mass
Spectrometry
In summary, by varying the voltage or magnetic field of the
magnetic-sector analyzer, the individual ion beams are
separated spatially and each has a unique radius of
curvature according to its mass/charge ratio.
High resolution isolation requires very stable high voltage
power supplies, magnetic field, and very narrow slits
(micron)
Isolation resolution of 103 – 104 is possible, but it comes
at the cost of sensitivity. Usually a mass window of ~5 Da
wide is selected.
Time of Flight Mass
Spectrometry (TOF-MS)
•Separates ions based on flight time
High resolution isolation requires very stable high voltage
power supplies for the source, high timing accuracy and
rapid response in the TIS (picoseconds)
Usually limited to an isolation resolving power of 102.
Usually a mass window of ~5 Da wide is selected.
Laser
Source
deflector
Collision Cell (Vc)
+
+
Vs
first field free drift region
Delay Generator
second field free drift region
Detector
Oscilloscope
Figure 6. MALDI tandem time-of-flight mass spectrometer.
Vr ≈ Vs
Aerosol mass spectrometer
Time of Flight Mass
Spectrometry (TOF-MS)
•Separates ions based on flight time
•Timed ion selector used for separation
•In MALDI, metastable ions have the same flight time as
precursor ions, so it is often impossible to completely select
the precursor ion.
Triple quadrupole
MS3
http://dx.doi.org/10.1016/0168-1176(90)80017-W
MS/MS scan modes
Ions in an Oscillating Electric Field
A± = U ± Vsin(ωt)
“Matthieu eqn”
az
-0.4
Operating
z stability Line
-0.2
b=1.0
qz=.908
0.0
Stable
z&r
-0.2
+
-0.4
+
-0.6
+
az = 8eU/mω2r2
r stability
0.5
1.0
1.5
qz = 4eV/mω2r2
qz
• qz a V/m
• qz a fion
• az a U/m
qz = 0.908
az
A
B
0.2
z stable
B
A
0.0
r and z
stable
D
-0.2
az = 0.02, qz = 0.7 az = 0.05, qz = 0.1
C
C
D
r stable
-0.4
-0.6
0.0
0.5
1.0
qz
az = -0.2, qz = 0.2 az = -0.04, qz = 0.2
Figure 12. Mathieu stability diagram with four stability points marked.
Typical corresponding ion trajectories are shown on the right.
Ejection Frequency
QITMS: Theory of MS/MS
qz isolation = 0.80
qz excitation = 0.25
• Isolation waveform is
applied to mass select
precursor ion
• A dipolar resonant
excitation amplitude is
applied to the
endcaps
• The selected ion gains
energy and undergoes
collisions with He
atoms and dissociates
via CID
• The fragment ions
with stable trajectories
are trapped and mass
analyzed
Scan Function for MS/MS on QIT
Ion
Injection
Isolation
m/z analysis
Excitation
RF Amplitude
Tailored
Waveform
Resonance
Excitation /
Ejection
Amplitude
Time
Beir, M.E. and Schwatz, J.C. in Electrospray Ionization Mass Spectrometry. 1997 259.
Ion trap isolation
• Resonant ejection of particular ions (or ranges) is the
standard method of isolation.
• The resonant pulse can be created in many ways.
• Resonant ejection of one ion usually involves
simultaneous ejection of other (lower m/z) ions.
• Isolation resolution can be as high as 103, but is rarely
used above 102 – or a 3-10 Da window.
• Ion recovery efficiency after resolution is the highest
possible with mass spectrometry.
Excitation/Isolation methods in
FTICR
Stored-Waveform Inverse Fourier Transform
Marshall, A. G., T.-C. L. Wang, et al. (1985). "Tailored Excitation for Fourier Transform Ion
Cyclotron Resonance Mass Spectrometry." J. Amer. Chem. Soc. 107: 7893-7897.
Correlated Harmonic Excitation Frequency
(CHEF)
High resolution ion isolation
Isolation of single isotopes of
ubiquitin (8.6 kDa) and
carbonic anhydrase (29 kDa)
was demonstrated.
O'Connor, P. B. and F. W. McLafferty (1995). "High Resolution Ion Isolation with the Capacitively
Coupled open cell." J. Am. Soc. Mass Spectrom. 6(6): 533-535.
FTICR ion isolation
• Resonant ejection of particular ions (or ranges) is the
standard method of isolation.
• The resonant pulse can be created in many ways –
sweep, SWIFT, FNF, CHEF, etc.
• Resonant ejection frequencies are largely independent
• Isolation resolution can be as high as 105, but is rarely
used above 103 –1 Da window.
• Most of these isolation methods result in off-resonant ion
excitation which can lead to fragmentation or poor
performance due to magnetron expansion
Fragmentation Methods
Breaking up a molecule requires putting energy into it's vibrational
modes or causing a reaction that breaks a bond.
• Collisional Activation (CAD or CID)
• Photodissociation (IRMPD and UVPD)
• Surface Induced Dissociation (SID)
•Electron ion reactions – ECD, ETD, EID, EDD, AI-ECD, …
•Metastable Atom dissociation (MAD)
Collisionally Activated Dissociation
also called Collision Induced Dissociation (CID)
N2
N2
+
N2
N2
N2
N2
+
N2
0
N2
• Ion’s smack into neutral gas • By far the most common
molecules and break up
MS/MS technique
• Energy of the collision is
controlled by changing the
kinetic energy of the ion.
• Fragments scatter radially
• slow fragmentation method,
deposits vibrational energy
throughout the molecule prior to
fragmentation.
•SORI-CAD, ITMSn, Triple
quad, TOF/TOF, etcetera
Oscilloscope
Delay Generator
Laser
Source
S
Pusher (Vp)
+
+
+
Q0
(RF-only)
Focusing
+
Q1
(mass filter)
+
Q2
(RF-only)
Collision Cell
D (field free drift region)
V
+
Vr ≈ Vp
Figure 14. Quadrupole Time-of-Flight Hybrid
Collisional Activation in a QIT
A± = U ± Vsin(ωt)
“Matthieu eqn”
az
-0.4
Operating
z stability Line
-0.2
b=1.0
qz=.908
0.0
Stable
z&r
-0.2
+
-0.4
+
-0.6
+
az = 8eU/mω2r2
r stability
0.5
1.0
1.5
qz = 4eV/mω2r2
qz
• qz a V/m
• qz a fion
• az a U/m
Collisional
Activation inside
an FTICR
Gauthier, J. W., T. R. Trautman, et al. (1991). "Sustained offresonance irradiation for CAD involving FTMS. CAD technique that
emulates infrared multiphoton dissociation." Anal. Chim. Acta 246:
211-225.
Mirgorodskaya, E., P. B. O'Connor, et al. (2002). "A General Method
for Precalculation of Parameters for Sustained Off Resonance
Irradiation/Collision-Induced Dissociation." Journal of the American
Society for Mass Spectrometry 13: 318-324.
Photo-Dissociation
+*
+
+
0
hυ
• Ion absorbs photon(s) and
break
•slow fragmentation method,
deposits vibrational energy
throughout the molecule prior to
• Energy of the fragmentation
fragmentation (depends on
is controlled by changing the
wavelength).
photon’s wavelength.
•IRMPD, UVPD, BIRD
• No scattering, except for
multiply charged ions
UV photodissociation
• High energy environment, cleaves the
backbone yielding a- and x- radical
cationic species which further dissociate
• If too much energy or wrong wavelength
(193 nm), only immonium ions are
observed.
157 nm photodissociation
IRMPD
• Ions are heated using a CO2 laser until
they dissociate.
• Fragments follow lowest energy pathways
which means preferential cleavages
• Fragments remain in the laser beam and
continue to absorb resulting in secondary
fragments.
Infrared Multiphoton Dissociation
Surface induced dissociation
+
0
+
• Ion smack into a surface,
break, and rebound
•slow fragmentation method,
deposits vibrational energy
throughout the molecule prior to
• Energy of the fragmentation
fragmentation.
is controlled by changing the
ion kinetic energy.
•Ions are lost by neutralization
at the surface (much better with
• Fragments scatter radially
perfluorinated surfaces)
SID in an FTICR
Surface induced dissociation
Electron Capture Dissociation
n+
+ e-
(n-1)+*
m+
0
• Multiply charged ions
capture a slow electron
•Fast fragmentation method
involving a radical
rearrangement in the region of
• Energy of the fragmentation
the backbone carbonyl (for
is determined by coulombic
proteins)
recombination.
•Generates very predicable and
• no scattering, but if both
very even sequence ladder
fragments are charged,
coulombic repulsion will
•Nobody knows how it works
occur
on things other than proteins
Odd vs. Even Electron
Fragmentation
• Even electron = proton rearrangements
• Odd electron = radical rearrangements
• Non-ergodic fragmentation = FAST!!
ECD Spectrum
Substance P ECD
RPKPQQFFGLM-NH2
M2+
c5
c10
c7
[M+2H]+•
c6
c4
c8
*
a7
w2
300
400
500
600
700
800
z9
900
m/z
c9
1000
1100
1200
1300
1400
674.8 = [M+2H]2+
18000
16000
1348.7 = [M+2H]+•
14000
RPKPQQFFGLM
12000
10000
Z9•/Z9
8000
C7•/C7
6000
C9 C
10
C5•/C5
4000
C6•/C6
C4•/C4
C8•/C8
2000
0
400
600
800
1000
1200
1400
Self Assessment
• How do you isolate ions in a TOF instrument?
An Ion Trap? An FTICR?
• Isolation and fragmentation of ions in an ion trap
(using CAD) results in losses of ions below
~30% of the precursor mass. Why?
• For proteins/peptides, CAD results in what two
main fragment types?
• For proteins/peptides, ECD results in what two
main fragment types?
CH908: Mass spectrometry
Lecture 1
Fini…
Magnetic-Sector Mass
Spectrometry
Magnetic-Sector Mass
Spectrometry
THEORY:
The ion source accelerates ions to a kinetic energy given by:
KE = ½ mv2 = qV
Where m is the mass of the ion, v is its velocity, q is the charge on
the ion, and V is the applied voltage of the ion optics.
Magnetic-Sector Mass
Spectrometry
•The ions enter the flight tube and are deflected by the magnetic
field, B.
•Only ions of mass-to-charge ratio that have equal centripetal and
centrifugal forces pass through the flight tube:
mv2 /r = BqV, where r is the radius of curvature
Magnetic-Sector Mass
Spectrometry
mv2 /r = BqV
•By rearranging the equation and eliminating the velocity term using
the previous equations, r = mv/qB = 1/B(2Vm/q)1/2
•Therefore, m/q = B2r2/(2V)
•This equation shows that the m/q ratio of the ions that reach the
detector can be varied by changing either the magnetic field (B) or
the applied voltage of the ion optics (V).
Time of Flight Mass
Spectrometry (TOF-MS)
THEORY:
•KE=qV when electrons are accelerated through an
electric field
•KE of ion is ½mv2, so qV= ½mv2 and velocity is
inversely proportional to mass
•Transit time (t) is L/v, where L is drift tube length
and v is velocity
•So t=L/(2V/m/q)½ can be solved for charge-mass ratio
Time of Flight Mass
Spectrometry (TOF-MS)
HOW IT’S DONE:
• Reflectron is series of rings or grids that serves to focus
ions to improve resolution
• Exact values of L and V do not need to be known if two or
more ions of known mass are used as mass calibration points
• Produces a mass spectrum as a function of time (can be
measured every 10 nsec)
Time of Flight Mass
Spectrometry (TOF-MS)
ADVANTAGES:
•Good for kinetic studies of fast reactions and for use
with gas chromatography to analyze peaks from
chromatograph
•Can register molecular ions that decompose in the flight
tube
Outline: Isolation Methods
•
•
•
•
•
•
Sectors – slits
TOF – timed ion selector
Orbitrap – not possible – why?
Quadrupoles – matthieu stability diagram
Ion traps
– Resonant ejection – frequencies?
– sweeps
– Swift
– Filtered noise field
FTICR
–
–
–
–
–
Resonant ejection
Sweeps
SWIFT
Filtered noise field
CHEF, multi-CHEF
In each case:
Isolation resolution
Limitations
Requirements
Selectivity