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)
Ion Trap CAD
Continuous
Resonant
(M/Z Selective)
Kinetic
Excitation
Many Weak
Collisions
With Helium
Molecules
“Slowly Heat”
Precursor
Ions
Simultaneous Processes
Preferential
Cleavage
of
Labile
Bonds
Ion Trap CAD
No
Resonant
(M/Z Selective)
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
RF ION TRAP ELECTRODE
STRUCTURES
LCQ-Type 3D
Quadrupole Trap
LTQ-Type (2D)
Linear Quadrupole Trap
RADIO FREQUENCY
THREE DIMENSIONAL QUADRUPOLE ION TRAP
z
y
x
Figure From
Quadrupole Mass Spectrometry and Its Applications
P.H. Dawson Ed., AIP Press
M/Z Selection/Analysis
Typically
Performed in Axial
Dimension
Resonance Excitation For
ion trap CAD
itCAD Control Parameters
q axis
q k
.908
Vrf
( m / e)
.908
qactivation
Default Low Mass Cutoff = .25/.908 = 28%
1/3.6th rule
• Extent of Conversion to Products
Normalized Collision Energy
• Strength of Excitation
30-5 ms
q axis
Activation Time
Activation Q
• Max Kinetic Energy
qactivation → fion → KEmax
• Low M/Z Cutoff
qactivation
( m / z ) LMCO 
( m / z ) Precursor
.908
Phosphorylation is
CAD labile
labile PTMs
•
•
•
•
phosphorylation
glycosylation
sulfonation
nitrosylation
itCAD
MS/MS
(M + 3H –
H3PO4)+++
Also known as Multi-Stage Activation (MSA)
Multi-Stage Activation (MSA)
MSA example
RADIO FREQUENCY
TWO DIMENSIONAL QUADUPOLE
LINEAR ION TRAP
z
y
x
Figure From
Quadrupole Mass Spectrometry and Its Applications
P.H. Dawson Ed., Reprinted AIP Press 1995
Confinement in
Axial Dimension
Provided By OTHER
DC or RF Fields
At Ends of Device
Common Linear Ion Trap Mass Spectrometers
Radial Ejection
Linear Ion Trap MS
Detector
Axial Ejection
Linear Ion Trap MS
Resonant Radial Excitation
Detector
Detector
Radial Ion Ejection
For Detection
Axial Ion Ejection
For Detection
AXIAL INJECTION
RF 3D Quadrupole Ion Trap
+
Helium
Buffer/Damping
Gas ~2 mtorr
2 z0
qhigh ; M/Zlow
+
qlow ; M/Zhigh
0V
RF Pseudo-Potential Well
• Trapping Efficiency
Strongly M/Z (q)
Dependent
• Short Path Length
For Stabilizing
Collisions:
2 z0 < 16 mm typ.
AXIAL INJECTION
RF 2D Quadrupole Linear Ion Trap
Helium
Buffer/Damping
Gas ~3 mtorr
+
L
0V
+
+
• Trapping Efficiency
Not Strongly M/Z (q)
Dependent.
True DC Axial
Trapping Potential Well
• Long Path Length For
Stabilizing Collisions:
2 L > 100 mm typ.
Estimating Relative Ion Storage Capacity
3D Ion vs Linear (2D) Quadrupole Ion Traps
3D RF Quadrupole
Ion Trap
2D RF Quadrupole
Linear Ion Trap
z
z
y
L
y
x
R3D
~ Spherical Ion Cloud
x
R2D
~ Cylindrical Ion Cloud
Trapping Efficiency Summary
2D-LTQ
Trapping Efficiency:
~ 55-70%
3D-LCQ Increase
~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
# Charges (11000 Th/Sec) :
3D-LCQ Increase
~ 20-40 K ~1-2 K
~ 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
Roman Zubarev
Neil Kelleher
Fred McLafferty
Roman Zubarev
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
Electron Transfer Dissociation
+
+
+
+
+
-
+
+
-
-
-
+
-
+
-
-
Phosphosite identification summary
Swaney, Wenger, Thomson, Coon. PNAS, 2009
Probability of bond cleavage for CAD and ETD
ETD allows freedom from trypsin
Internal basic residues sequester charge
Dongre, Jones, Somogyi, Wysocki. JACS 1996
Kapp, Simpson et al. Analytical Chemistry 2003
Sequence coverage - trypsin
Sequence coverage – 5 enzymes
Collision Activated Dissociation
aka HCD
Kinetic
Excitation
Collisions
Convert
Kinetic
Energy to
Vibrational
Energy
Elevated
Vibrational
Energy
Causes
Bond
Cleavage
Q-TOFs and
Orbitrap systems
Offer beam-type
CAD (HCD)
HCD
Trap CAD
Mann et al., JPR 2010
HCD
Trap CAD
Mann et al., JPR 2010
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
Mann et al., JPR 2010
HCD vs. ion trap CAD for
phosphorylated tryptic
peptides – Coon Lab data
HCD-FT
CAD-IT CAD-FT
Fragment mass tolerance (Th)
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
• Decision trees can
integrate all these
methods
Heck et al., JPR 2011