primary methods for dissociating peptides collision-based methods: ion trap collisional activation...
TRANSCRIPT
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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)
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Ion Trap CAD
ContinuousResonant
(M/Z Selective)Kinetic
Excitation
Many Weak Collisions
With Helium Molecules
“Slowly Heat”Precursor
Ions
PreferentialCleavage
ofLabileBonds
Simultaneous Processes
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NoResonant
(M/Z Selective)Kinetic
ExcitationOf
Product Ions
Ion Trap CAD
Many Weak Collisions
With Helium Molecules
“Cool”Product Ions
“Cool”Product Ions
Remain Intact
Product Ions NOT Subject to Further Activation/Dissociation
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RF ION TRAP ELECTRODE STRUCTURES
LCQ-Type 3D Quadrupole Trap
LTQ-Type (2D) Linear Quadrupole Trap
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RADIO FREQUENCY THREE DIMENSIONAL QUADRUPOLE ION TRAP
Figure FromQuadrupole Mass Spectrometry and Its ApplicationsP.H. Dawson Ed., AIP Press
M/Z Selection/AnalysisTypicallyPerformed in Axial Dimension
x
y
z
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Activation Time• Extent of Conversion to Products
Normalized Collision Energy
• Strength of Excitation
Activation Q• Max Kinetic Energy
• Low M/Z Cutoff
Resonance Excitation For ion trap CAD
Default Low Mass Cutoff = .25/.908 = 28%
1/3.6th rule
)/( em
Vkq
rf
.908
.908 q axis
30-5 ms
itCAD Control Parameters
Precursoractivation
LMCO zmq
zm )/(908.
)/(
q axis
qactivationqactivation → fion → KEmax
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Phosphorylation is Phosphorylation is CAD labileCAD labile
labile PTMs•phosphorylation•glycosylation•sulfonation•nitrosylation
itCAD MS/MS
(M + 3H – H3PO4)++
+
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Also known as Multi-Stage Activation (MSA)
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Multi-Stage Activation (MSA)
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MSA example
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z
Figure FromQuadrupole Mass Spectrometry and Its ApplicationsP.H. Dawson Ed., Reprinted AIP Press 1995
x
y
Confinement in Axial Dimension Provided By OTHERDC or RF FieldsAt Ends of Device
RADIO FREQUENCY TWO DIMENSIONAL QUADUPOLELINEAR ION TRAP
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Detector
Detector
Detector
Radial Ejection Linear Ion Trap MS
Axial Ejection Linear Ion Trap MS
Resonant Radial Excitation
Radial Ion EjectionFor Detection
Axial Ion EjectionFor Detection
Common Linear Ion Trap Mass Spectrometers
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AXIAL INJECTION RF 3D Quadrupole Ion Trap
+
qlow ; M/Zhigh
qhigh ; M/Zlow
+
2 z0
RF Pseudo-Potential Well
0 V
HeliumBuffer/DampingGas ~2 mtorr
• Trapping Efficiency Strongly M/Z (q) Dependent
• Short Path Length For Stabilizing Collisions: 2 z0 < 16 mm typ.
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AXIAL INJECTION RF 2D Quadrupole Linear Ion Trap
+HeliumBuffer/DampingGas ~3 mtorr
0 V++
• Trapping Efficiency Not Strongly M/Z (q) Dependent.
• Long Path Length For Stabilizing Collisions: 2 L > 100 mm typ.
L
True DC AxialTrapping Potential Well
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~ Spherical Ion Cloud ~ Cylindrical Ion Cloud
x
y
z
R3D
x
yz
R2D
L
3D RF QuadrupoleIon Trap
2D RF QuadrupoleLinear Ion Trap
Estimating Relative Ion Storage Capacity3D Ion vs Linear (2D) Quadrupole Ion Traps
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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) : ~ 20-40 K ~1-2 K ~ 20
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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
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Neil Kelleher
Roman Zubarev
Fred McLafferty
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Roman Zubarev
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Ion/ion reactions in ion Ion/ion reactions in ion trapstraps
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, 1996McLuckey and Stephenson, Mass Spec Reviews, 1998
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Electron Transfer Electron Transfer Dissociation Dissociation
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++ +
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Phosphosite identification summary
Swaney, Wenger, Thomson, Coon. PNAS, 2009
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Probability of bond cleavage for CAD and ETD
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ETD allows freedom from trypsin
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Internal basic residues sequester charge
Dongre, Jones, Somogyi, Wysocki. JACS 1996
Kapp, Simpson et al. Analytical Chemistry 2003
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Sequence coverage - trypsinSequence coverage - trypsin
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Sequence coverage – 5 Sequence coverage – 5 enzymesenzymes
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Collision Activated Dissociationaka HCD
Kinetic Excitation
Collisions Convert Kinetic
Energy to Vibrational
Energy
Elevated Vibrational
Energy Causes Bond
Cleavage
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Q-TOFs andOrbitrap systemsOffer beam-type
CAD (HCD)
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HCD
Trap CAD
Mann et al., JPR 2010
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HCD
Trap CAD
Mann et al., JPR 2010
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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
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Mann et al., JPR 2010
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CAD-FTCAD-IT
HCD vs. ion trap CAD for phosphorylated tryptic peptides – Coon Lab data
HCD-FT
Fragment mass tolerance (Th)
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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
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• Decision trees can integrate all these methods
Heck et al., JPR 2011