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 Preferential Cleavage of Labile Bonds Simultaneous Processes

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

q

axis

q



k V rf

(

m

/

e

)

q

axis Resonance Excitation For ion trap CAD

q

activation 30-5 ms .908

.908

Default Low Mass Cutoff = .25/.908 = 28% 1/3.6

th rule itCAD Control Parameters Activation Time

• Extent of Conversion to Products •

Normalized Collision Energy

Strength of Excitation •

Activation Q

Max Kinetic Energy

q

activation →

f

ion → KE max •

Low M/Z Cutoff

(

m

/

z

)

LMCO



q activation

(

m

.

908 /

z

)

Precursor

Phosphorylation is CAD labile

labile PTMs

• phosphorylation • glycosylation • sulfonation • nitrosylation

itCAD MS/MS

(M + 3H – H 3 PO 4 ) +++

Also known as Multi-Stage Activation (MSA)

Multi-Stage Activation (MSA)

MSA example

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

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 • Trapping Efficiency Strongly M/Z (q) Dependent 2 z 0 q high ; M/Z low q low ; M/Z high • Short Path Length For Stabilizing Collisions: 2 z 0 < 16 mm typ.

0 V

RF Pseudo-Potential Well

AXIAL INJECTION RF 2D Quadrupole Linear Ion Trap

+ Helium Buffer/Damping Gas ~3 mtorr

L

+ • Trapping Efficiency Not Strongly M/Z (q) Dependent. + 0 V 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 z y 2D RF Quadrupole Linear Ion Trap z y L x x R 3D ~ Spherical Ion Cloud R 2D ~ Cylindrical Ion Cloud

Trapping Efficiency Summary Trapping Efficiency:

2D-LTQ 3D-LCQ Increase

~ 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

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