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)

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

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

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

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

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 50-100% 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

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

Also known as Multi-Stage Activation (MSA)

Multi-Stage Activation (MSA)

MSA example

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

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

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

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.

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

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

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 More pathways on the way Swaney, Wenger, Thomson, Coon. PNAS, 2009

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

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 Elevated Vibrational Energy Causes Bond Cleavage Collisions Convert Kinetic Energy to Vibrational Energy Kinetic Excitation

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