4. Collision-based methods: Electron-based methods:
Primary methods for dissociating peptides
Collision-based methods:
Ion trap collisional activation – itCAD
Beam-type collisional activation – CAD aka (HCD)
Electron-based methods:
Electron capture dissociation (ECD)
Electron transfer dissociation (ETD)

5. Ion Trap CAD Many Weak Collisions With Helium Molecules “Slowly Heat”
Precursor Ions
Continuous
Resonant
(M/Z Selective)
Kinetic Excitation
Preferential
Cleavage of
Labile
Bonds
Simultaneous Processes

6. Ion Trap CAD No Many Weak Collisions Resonant (M/Z Selective) “Cool”
Kinetic Excitation Of
Product Ions
Many Weak Collisions
With Helium Molecules
“Cool”
Product Ions
“Cool”
Product Ions
Remain Intact
Product Ions NOT Subject to Further Activation/Dissociation

7. RF ION TRAP ELECTRODE STRUCTURES
LCQ-Type 3D
Quadrupole Trap
LTQ-Type (2D)
Linear Quadrupole Trap

8. THREE DIMENSIONAL QUADRUPOLE ION TRAP
RADIO FREQUENCY
THREE DIMENSIONAL QUADRUPOLE ION TRAP
M/Z Selection/Analysis
Typically
Performed in Axial Dimension x y z
Figure From
Quadrupole Mass Spectrometry and Its Applications
P.H. Dawson Ed., AIP Press

9. itCAD Control Parameters Default Low Mass Cutoff = .25/.908 = 28%
Resonance Excitation For
ion trap CAD
itCAD Control Parameters
Activation Time
Extent of Conversion to Products
Normalized Collision Energy
Strength of Excitation
Activation Q
Max Kinetic Energy
Low M/Z Cutoff
q axis
.908
30-5 ms
q axis
.908
qactivation
Again, there are many methods of activating ions in an ion trap. We use a method which results in very high efficiency. It again utilizes the resonance process by applying a voltage across the endcaps which oscillates at a frequency which matches the an ions frequency of motion. However, instead of using amplitudes of the resonance voltage which will drive the ions out of the trap, a lower amplitude is applied. This amplitude is only sufficient to excite the ions to larger trajectories and is often called a “tickle voltage”. This term indicates the nature of this applied field for increasing the energy of the ions such that subsequent collisions with the neutral He atoms causes fragmentation. The optimum frequency for fragmenting ions by this method is a compromise of several effects. To reach the highest kinetic energies for collisions, the highest operating Q value should be used for fragmentation. However, if lower mass ions are formed, they would then be fundamentally unstable and not be trapped. Consequently, a lower operating Q value must be used to allow sufficient kinetic energy for fragmentation but still allow a large mass range to be trapped. Also, trapping efficiency of ions is also dependant on the Q value of the ions. Our default activation Q value is .25, which is a good compromise point which typically results in % fragmentation efficiency and allows fragments down to 28% of the parent mass to be detected. The activation is applied for 30 ms. Activation of multiply charged parent ions may form fragments which have higher m/z values than the parent ion. For stable ions which need more energy, a higher Q value can be chosen at the expense of fragment mass range. Conversely, if the ions of interest are fragile and low mass fragments are of interest, then a lower Q value can be utilized.
qactivation → fion → KEmax
Default Low Mass Cutoff = .25/.908 = 28%
1/3.6th rule

10. Phosphorylation is CAD labile
itCAD MS/MS
labile PTMs
phosphorylation
glycosylation
sulfonation
nitrosylation
(M + 3H – H3PO4)+++

11. Also known as Multi-Stage Activation (MSA)

12. Multi-Stage Activation (MSA)

13. MSA example

14. TWO DIMENSIONAL QUADUPOLE LINEAR ION TRAP
RADIO FREQUENCY
TWO DIMENSIONAL QUADUPOLE
LINEAR ION TRAP z y x
Confinement in
Axial Dimension Provided By OTHER
DC or RF Fields
At Ends of Device
Figure From
Quadrupole Mass Spectrometry and Its Applications
P.H. Dawson Ed., Reprinted AIP Press 1995

15. Common Linear Ion Trap Mass Spectrometers
Radial Ejection
Linear Ion Trap MS
Axial Ejection
Linear Ion Trap MS
Detector
Resonant Radial Excitation
Detector
Detector
Radial Ion Ejection
For Detection
Axial Ion Ejection
For Detection

16. RF 3D Quadrupole Ion Trap
AXIAL INJECTION
RF 3D Quadrupole Ion Trap
Trapping Efficiency
Strongly M/Z (q)
Dependent
Short Path Length
For Stabilizing
Collisions:
2 z0 < 16 mm typ. +
Helium
Buffer/Damping
Gas ~2 mtorr
2 z0
qhigh ; M/Zlow
qlow ; M/Zhigh +
0 V
RF Pseudo-Potential Well

17. RF 2D Quadrupole Linear Ion Trap
AXIAL INJECTION
RF 2D Quadrupole Linear Ion Trap
Helium
Buffer/Damping
Gas ~3 mtorr + L
0 V +
True DC Axial
Trapping Potential Well
Trapping Efficiency
Not Strongly M/Z (q)
Dependent.
Long Path Length For
Stabilizing Collisions:
2 L > 100 mm typ.

18. Estimating Relative Ion Storage Capacity
3D Ion vs Linear (2D) Quadrupole Ion Traps
3D RF Quadrupole
Ion Trap
2D RF Quadrupole
Linear Ion Trap x y z
R2D L x y z
R3D
~ Spherical Ion Cloud
~ Cylindrical Ion Cloud

19. Trapping Efficiency Summary
2D-LTQ 3D-LCQ Increase
Trapping Efficiency: ~ 55-70% ~5% ~ 11-14x
Detection Efficiency: ~50-100% ~50% ~ 1-2x _________________________________________________
Overall Efficiency: ~35-55% ~2.5% ~14-22x
Scanning Ion Capacity
(Spectral Space Charge Limit)
2D-LTQ 3D-LCQ Increase
# Charges (11000 Th/Sec) : ~ K ~1-2 K ~ 20

20. Introduction of the linear ion trap improved itCAD performance for phosphopeptide identification. This is primarily because it offered ~ 20X boost in ion capacity so that the low level fragment ions are more often detectable, even if at low abundance

22. Roman Zubarev
Neil Kelleher
Fred McLafferty

24. Roman Zubarev

25. Ion/ion reactions in ion traps
Proton transfer
(M + 3H)3+ + A–  (M + 2H)2+ + HA
Anion attachment
(M + 3H)3+ + A–  (M + 3H + Y)2+
Electron transfer
(M + 3H)3+ + A–•  (M + 3H)2+• + A
Stephenson and McLuckey, JACS, 1996
McLuckey and Stephenson, Mass Spec Reviews, 1998

26. Electron Transfer Dissociation

27. +

28. + + +

29. +

30. + -

31. - - + -

32. + -

33. - - +

41. Phosphosite identification summary
More pathways on the way
Swaney, Wenger, Thomson, Coon. PNAS, 2009

42. Probability of bond cleavage for CAD and ETD
More pathways on the way

45. ETD allows freedom from trypsin

46. Internal basic residues sequester charge
Dongre, Jones, Somogyi, Wysocki. JACS 1996
Kapp, Simpson et al. Analytical Chemistry 2003

47. Sequence coverage - trypsin

48. Sequence coverage – 5 enzymes

49. Collision Activated Dissociation
aka HCD
Elevated
Vibrational Energy Causes Bond
Cleavage
Collisions Convert Kinetic Energy to Vibrational Energy
Kinetic Excitation

50. Q-TOFs and
Orbitrap systems
Offer beam-type
CAD (HCD)

51. HCD
Trap CAD
Mann et al., JPR 2010

52. HCD
Trap CAD
Mann et al., JPR 2010

53. Which dissociation method is best for phosphoproteomics?
Depends on who you ask.
Excellent results can be achieved with any of these methods
The deepest coverage is achieved by using all three

54. Mann et al., JPR 2010

56. HCD vs. ion trap CAD for phosphorylated tryptic peptides – Coon Lab data
HCD-FT
CAD-IT
CAD-FT
Fragment mass tolerance (Th)

57. Why the varied results?
I believe it’s a matter of comfort/compatibility with a specific method
Dissociation parameters can be highly optimized (e.g., AGC, inject time, etc.)
Database searching algorithm can make very large differences
Site localization methods

59. Decision trees can integrate all these methods
Heck et al., JPR 2011