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

ContinuousResonant

(M/Z Selective)Kinetic

Excitation

Many Weak Collisions

With Helium Molecules

“Slowly Heat”Precursor

Ions

PreferentialCleavage

ofLabileBonds

Simultaneous Processes

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

RF ION TRAP ELECTRODE STRUCTURES

LCQ-Type 3D Quadrupole Trap

LTQ-Type (2D) Linear Quadrupole Trap

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

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

Phosphorylation is Phosphorylation is CAD labileCAD 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

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

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

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.

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

~ 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

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

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

Neil Kelleher

Roman Zubarev

Fred McLafferty

Roman Zubarev

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

Electron Transfer Electron Transfer Dissociation 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 - trypsinSequence coverage - trypsin

Sequence coverage – 5 Sequence coverage – 5 enzymesenzymes

Collision Activated Dissociationaka HCD

Kinetic Excitation

Collisions Convert Kinetic

Energy to Vibrational

Energy

Elevated Vibrational

Energy Causes Bond

Cleavage

Q-TOFs andOrbitrap systemsOffer 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

CAD-FTCAD-IT

HCD vs. ion trap CAD for phosphorylated tryptic peptides – Coon Lab data

HCD-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