1. CH 908: Mass Spectrometry Lecture 7 Tandem mass spectrometry
Prof. Peter B. O’Connor

2. Tandem Mass Spectrometry or MS/MS
Isolation
Fragmentation
Isolation
Fragmentation
Benefits:
Extremely high specificity
More structural information
Limitations:
Isolation window
Fragmentation efficiency
Ion Losses
MS/MS/MS, or MS3
MS/MS

3. Tandem Mass Spectrometry
“Tandem in Time” – FTMS, QITMS
“Tandem in Space” – Triple quad, TOF/TOF, sector

4. OUTLINE Tandem in space instruments Tandem in time 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

5. Magnetic sector instruments
Ions are deflected and accelerated down a curved path to the detector.

7. 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.

8. 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.

10. Figure 6. MALDI tandem time-of-flight mass spectrometer.
Laser
Source
deflector
Collision Cell (Vc)
Vr ≈ Vs + + + + + + +
first field free drift region + + Vs +
Delay Generator + + +
second field free drift region
Detector
Oscilloscope
Figure 6. MALDI tandem time-of-flight mass spectrometer.

12. Aerosol mass spectrometer

13. 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.

14. Triple quadrupole

15. MS3

16. MS/MS scan modes

17. Ions in an Oscillating Electric Field
A± = U ± Vsin(ωt)
“Matthieu eqn”
az = 8eU/mω2r2
qz = 4eV/mω2r2
z stability
r stability
0.5
1.0
1.5 qz
Operating
Line
b=1.0
qz=.908
Stable
z & r az
0.2
0.0
-0.2
-0.4
-0.6
0.4 + -
qz a V/m
qz a fion
az a U/m

18. z stable r stable az qz qz = 0.908 A B B A r and z stable D
0.5
1.0 qz az
0.2
0.0
-0.2
-0.4
-0.6
z stable B A
r and z
stable D
az = 0.02, qz = 0.7
az = 0.05, qz = 0.1 C C D
r stable
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.

19. Ejection Frequency

20. QITMS: Theory of MS/MS
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
qz isolation = 0.80
qz excitation = 0.25

21. Scan Function for MS/MS on QIT
Time
Ion
Injection
Isolation
Excitation
m/z analysis
RF Amplitude
Tailored
Waveform
Resonance
Excitation /
Ejection
Amplitude
Beir, M.E. and Schwatz, J.C. in Electrospray Ionization Mass Spectrometry

22. 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.

23. Excitation/Isolation methods in FTICR

24. 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:

25. Correlated Harmonic Excitation Frequency (CHEF)

26. 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):

27. 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

28. 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)

29. Collisionally Activated Dissociation also called Collision Induced Dissociation (CID)+ N2
Ion’s smack into neutral gas molecules and break up
Energy of the collision is controlled by changing the kinetic energy of the ion.
Fragments scatter radially
By far the most common MS/MS technique
slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation.
SORI-CAD, ITMSn, Triple quad, TOF/TOF, etcetera

30. Figure 14. Quadrupole Time-of-Flight Hybrid
Oscilloscope
Laser
Delay Generator
Source S
Pusher (Vp) + + + + + + + + + Q0 Q1 Q2 V
(RF-only)
(mass filter)
(RF-only) + + +
Focusing
Collision Cell
D (field free drift region) + + +
Vr ≈ Vp
Figure 14. Quadrupole Time-of-Flight Hybrid

31. Collisional Activation in a QIT
A± = U ± Vsin(ωt)
“Matthieu eqn”
az = 8eU/mω2r2
qz = 4eV/mω2r2
z stability
r stability
0.5
1.0
1.5 qz
Operating
Line
b=1.0
qz=.908
Stable
z & r az
0.2
0.0
-0.2
-0.4
-0.6
0.4 + -
qz a V/m
qz a fion
az a U/m

32. Collisional Activation inside an FTICR
Gauthier, J. W., T. R. Trautman, et al. (1991). "Sustained off-resonance irradiation for CAD involving FTMS. CAD technique that emulates infrared multiphoton dissociation." Anal. Chim. Acta 246:
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:

34. Photo-Dissociation + + +* hυ Ion absorbs photon(s) and breakhυ
Ion absorbs photon(s) and break
Energy of the fragmentation is controlled by changing the photon’s wavelength.
No scattering, except for multiply charged ions
slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation (depends on wavelength).
IRMPD, UVPD, BIRD

35. 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.

36. 157 nm photodissociation

38. 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.

39. Infrared Multiphoton Dissociation

40. Surface induced dissociation+ +
Ion smack into a surface, break, and rebound
Energy of the fragmentation is controlled by changing the ion kinetic energy.
Fragments scatter radially
slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation.
Ions are lost by neutralization at the surface (much better with perfluorinated surfaces)

41. SID in an FTICR

42. Surface induced dissociation

43. Electron Capture Dissociationm+ n+
(n-1)+* + e-
Multiply charged ions capture a slow electron
Energy of the fragmentation is determined by coulombic recombination.
no scattering, but if both fragments are charged, coulombic repulsion will occur
Fast fragmentation method involving a radical rearrangement in the region of the backbone carbonyl (for proteins)
Generates very predicable and very even sequence ladder
Nobody knows how it works on things other than proteins

44. Odd vs. Even Electron Fragmentation
Even electron = proton rearrangements
Odd electron = radical rearrangements
Non-ergodic fragmentation = FAST!!

45. ECD Spectrum

46. Substance P ECD RPKPQQFFGLM-NH2 c5 c10 c7 c6 c4 c8 c9 * a7 z9 w2 300
[M+2H]+• c6 c4 c8 c9 * a7 z9 w2
300
600
900
1200
1400
400
500
700
800
1000
1100
1300
m/z

47. RPKPQQFFGLM 674.8 = [M+2H]2+ 1348.7 = [M+2H]+• Z9•/Z9 C7•/C7 C9 C10

48. 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?

49. CH908: Mass spectrometry
Lecture 1
Fini…

51. Magnetic-Sector Mass Spectrometry

52. 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.

53. 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

54. 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).

55. 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

56. 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)

57. 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

58. 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
CHEF, multi-CHEF
In each case:
Isolation resolution
Limitations
Requirements
Selectivity