CH 908: Mass SpectrometryLecture 7

Tandem mass spectrometry

Prof. Peter B. O’Connor

Tandem Mass Spectrometry or MS/MS

MS/MSMS/MS/MS, or MS3

Benefits: 1.Extremely high specificity2.More structural information

Limitations:1.Isolation window2.Fragmentation efficiency3.Ion Losses

Isolation

FragmentationIsolation

Fragmentation

Tandem Mass Spectrometry“Tandem in Time” – FTMS, QITMS

“Tandem in Space” – Triple quad, TOF/TOF, sector

OUTLINE• Tandem in space instruments

– Sectors– Triple quads– Q-tofs, tof/tofs, unique instruments (pentaquads)– Orbitrap (sort-of)

• Tandem in time instruments– Ion traps

• Classic 3D• Linear ion trap

– FTICR• MS/MS methods

– CAD– ECD (plus ETD, EID, EED, etc)– PD (UVPD, IRMPD)– SID

Magnetic sector instruments

Ions are deflected and accelerated down a curved path to the detector.

Magnetic-Sector Mass Spectrometry

In summary, by varying the voltage or magnetic field of the magnetic-sector analyzer, the individual ion beams are separated spatially and each has a unique radius of curvature according to its mass/charge ratio.

High resolution isolation requires very stable high voltage power supplies, magnetic field, and very narrow slits (micron)

Isolation resolution of 103 – 104 is possible, but it comes at the cost of sensitivity. Usually a mass window of ~5 Da wide is selected.

Time of Flight Mass Spectrometry (TOF-MS)

•Separates ions based on flight time

High resolution isolation requires very stable high voltage power supplies for the source, high timing accuracy and rapid response in the TIS (picoseconds)

Usually limited to an isolation resolving power of 102.

Usually a mass window of ~5 Da wide is selected.

second field free drift region

first field free drift region

Figure 6. MALDI tandem time-of-flight mass spectrometer.

Laser

Vs

Source

Oscilloscope

++

Detector

Vr ≈ Vs

deflector

+ +

+

+

+

++

++++

Collision Cell (Vc)

Delay Generator

Aerosol mass spectrometer

Time of Flight Mass Spectrometry (TOF-MS)

•Separates ions based on flight time

•Timed ion selector used for separation

•In MALDI, metastable ions have the same flight time as precursor ions, so it is often impossible to completely select the precursor ion.

Triple quadrupole

MS3

http://dx.doi.org/10.1016/0168-1176(90)80017-W

MS/MS scan modes

Ions in an Oscillating Electric Field

• qz V/m• qz fion • az U/m

z stability

r stability0.5 1.0 1.5

qz

Operating Line

=1.0qz=.908

Stablez & r

az

0.2

0.0

-0.2

-0.4

-0.6

0.4

+

+

+

-

-

“Matthieu eqn”

az = 8eU/mω2r2

qz = 4eV/mω2r2

A± = U ± Vsin(ωt)

Figure 12. Mathieu stability diagram with four stability points marked. Typical corresponding ion trajectories are shown on the right.

0.5 1.0 qz

az0.2

0.0

-0.2

-0.4

-0.6

0.0

z stable

r stable

r and z stable

qz = 0.908

A

A B

C D

B

C

Daz = 0.02, qz = 0.7 az = 0.05, qz = 0.1

az = -0.2, qz = 0.2 az = -0.04, qz = 0.2

Ejection Frequency

QITMS: Theory of MS/MS

• Isolation waveform is applied to mass select precursor ion

• A dipolar resonant excitation amplitude is applied to the endcaps

• The selected ion gains energy and undergoes collisions with He atoms and dissociates via CID

• The fragment ions with stable trajectories are trapped and mass analyzed

qz isolation = 0.80

qz excitation = 0.25

Time

IonInjection

Isolation

Excitation

m/z analysis

RF Amplitude

TailoredWaveform

ResonanceExcitation /

EjectionAmplitude

Scan Function for MS/MS on QIT

Beir, M.E. and Schwatz, J.C. in Electrospray Ionization Mass Spectrometry. 1997 259.

Ion trap isolation

• Resonant ejection of particular ions (or ranges) is the standard method of isolation.

• The resonant pulse can be created in many ways.• Resonant ejection of one ion usually involves

simultaneous ejection of other (lower m/z) ions.

• Isolation resolution can be as high as 103, but is rarely used above 102 – or a 3-10 Da window.

• Ion recovery efficiency after resolution is the highest possible with mass spectrometry.

Excitation/Isolation methods in FTICR

Stored-Waveform Inverse Fourier Transform

Marshall, A. G., T.-C. L. Wang, et al. (1985). "Tailored Excitation for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry." J. Amer. Chem. Soc. 107: 7893-7897.

Correlated Harmonic Excitation Frequency (CHEF)

High resolution ion isolation

O'Connor, P. B. and F. W. McLafferty (1995). "High Resolution Ion Isolation with the Capacitively Coupled open cell." J. Am. Soc. Mass Spectrom. 6(6): 533-535.

Isolation of single isotopes of ubiquitin (8.6 kDa) and carbonic anhydrase (29 kDa) was demonstrated.

FTICR ion isolation• Resonant ejection of particular ions (or ranges) is the

standard method of isolation.• The resonant pulse can be created in many ways –

sweep, SWIFT, FNF, CHEF, etc.• Resonant ejection frequencies are largely independent

• Isolation resolution can be as high as 105, but is rarely used above 103 –1 Da window.

• Most of these isolation methods result in off-resonant ion excitation which can lead to fragmentation or poor performance due to magnetron expansion

Fragmentation Methods

• Collisional Activation (CAD or CID)

• Photodissociation (IRMPD and UVPD)

• Surface Induced Dissociation (SID)

•Electron ion reactions – ECD, ETD, EID, EDD, AI-ECD, …

•Metastable Atom dissociation (MAD)

Breaking up a molecule requires putting energy into it's vibrational modes or causing a reaction that breaks a bond.

Collisionally Activated Dissociationalso called Collision Induced Dissociation (CID)

+

N2N2

N2

N2 N2 N2

N2 N2

+

0

• Ion’s smack into neutral gas molecules and break up

• Energy of the collision is controlled by changing the kinetic energy of the ion.

• Fragments scatter radially

• By far the most common MS/MS technique

• slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation.

•SORI-CAD, ITMSn, Triple quad, TOF/TOF, etcetera

Figure 14. Quadrupole Time-of-Flight Hybrid Vr ≈ Vp

Laser

V

D (field

free drift region)

Source

S

Oscilloscope

++

+

Pusher (Vp)

+

+

Delay Generator

Q0 Q1 Q2

(RF-only) (mass filter) (RF-only)

+ +

Focusing

++++

++

+

+

Collision Cell

++

+

Collisional Activation in a QIT

• qz V/m• qz fion • az U/m

z stability

r stability0.5 1.0 1.5

qz

Operating Line

=1.0qz=.908

Stablez & r

az

0.2

0.0

-0.2

-0.4

-0.6

0.4

+

+

+

-

-

“Matthieu eqn”

az = 8eU/mω2r2

qz = 4eV/mω2r2

A± = U ± Vsin(ωt)

Collisional Activation inside

an FTICR

Gauthier, J. W., T. R. Trautman, et al. (1991). "Sustained off-resonance irradiation for CAD involving FTMS. CAD technique that emulates infrared multiphoton dissociation." Anal. Chim. Acta 246: 211-225.

Mirgorodskaya, E., P. B. O'Connor, et al. (2002). "A General Method for Precalculation of Parameters for Sustained Off Resonance Irradiation/Collision-Induced Dissociation." Journal of the American Society for Mass Spectrometry 13: 318-324.

Photo-Dissociation

++

0

• Ion absorbs photon(s) and break

• Energy of the fragmentation is controlled by changing the photon’s wavelength.

• No scattering, except for multiply charged ions

•slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation (depends on wavelength).

•IRMPD, UVPD, BIRD

+*

hυ

UV photodissociation

• High energy environment, cleaves the backbone yielding a- and x- radical cationic species which further dissociate

• If too much energy or wrong wavelength (193 nm), only immonium ions are observed.

157 nm photodissociation

IRMPD

• Ions are heated using a CO2 laser until they dissociate.

• Fragments follow lowest energy pathways which means preferential cleavages

• Fragments remain in the laser beam and continue to absorb resulting in secondary fragments.

Infrared Multiphoton Dissociation

Surface induced dissociation

+

+0

• Ion smack into a surface, break, and rebound

• Energy of the fragmentation is controlled by changing the ion kinetic energy.

• Fragments scatter radially

•slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation.

•Ions are lost by neutralization at the surface (much better with perfluorinated surfaces)

SID in an FTICR

Surface induced dissociation

Electron Capture Dissociation

n+

• Multiply charged ions capture a slow electron

• Energy of the fragmentation is determined by coulombic recombination.

• no scattering, but if both fragments are charged, coulombic repulsion will occur

•Fast fragmentation method involving a radical rearrangement in the region of the backbone carbonyl (for proteins)

•Generates very predicable and very even sequence ladder

•Nobody knows how it works on things other than proteins

e- (n-1)+*+m+

0

Odd vs. Even Electron Fragmentation

• Even electron = proton rearrangements• Odd electron = radical rearrangements

• Non-ergodic fragmentation = FAST!!

ECD Spectrum

Substance P ECDRPKPQQFFGLM-NH2

300 600 900 1200 1400400 500 700 800 1000 1100 1300

2

c4

*

c5

M2+

c6

a7

c7

c8

z9

c9

c10

[M+2H]+•

m/z

400 600 800 1000 1200 1400

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

C5•/C5

C4•/C4

C6•/C6

C7•/C7

C8•/C8

C9

Z9•/Z9

C10

674.8 = [M+2H]2+

1348.7 = [M+2H]+•

RPKPQQFFGLM

Self Assessment

• How do you isolate ions in a TOF instrument? An Ion Trap? An FTICR?

• Isolation and fragmentation of ions in an ion trap (using CAD) results in losses of ions below ~30% of the precursor mass. Why?

• For proteins/peptides, CAD results in what two main fragment types?

• For proteins/peptides, ECD results in what two main fragment types?

Fini…

CH908: Mass spectrometryLecture 1

Magnetic-Sector Mass Spectrometry

Magnetic-Sector Mass Spectrometry

THEORY:

The ion source accelerates ions to a kinetic energy given by:

KE = ½ mv2 = qV

Where m is the mass of the ion, v is its velocity, q is the charge on the ion, and V is the applied voltage of the ion optics.

Magnetic-Sector Mass Spectrometry

•The ions enter the flight tube and are deflected by the magnetic field, B.

•Only ions of mass-to-charge ratio that have equal centripetal and centrifugal forces pass through the flight tube:

mv2 /r = BqV, where r is the radius of curvature

Magnetic-Sector Mass Spectrometry

mv2 /r = BqV

•By rearranging the equation and eliminating the velocity term using the previous equations, r = mv/qB = 1/B(2Vm/q)1/2

•Therefore, m/q = B2r2/(2V)

•This equation shows that the m/q ratio of the ions that reach the detector can be varied by changing either the magnetic field (B) or the applied voltage of the ion optics (V).

THEORY:

•KE=qV when electrons are accelerated through an electric field

•KE of ion is ½mv2, so qV= ½mv2 and velocity is inversely proportional to mass

•Transit time (t) is L/v, where L is drift tube length and v is velocity

•So t=L/(2V/m/q)½ can be solved for charge-mass ratio

Time of Flight Mass Spectrometry (TOF-MS)

Time of Flight Mass Spectrometry (TOF-MS)

HOW IT’S DONE:

• Reflectron is series of rings or grids that serves to focus ions to improve resolution

• Exact values of L and V do not need to be known if two or more ions of known mass are used as mass calibration points

• Produces a mass spectrum as a function of time (can be measured every 10 nsec)

Time of Flight Mass Spectrometry (TOF-MS)

ADVANTAGES:

•Good for kinetic studies of fast reactions and for use with gas chromatography to analyze peaks from chromatograph

•Can register molecular ions that decompose in the flight tube

Outline: Isolation Methods• Sectors – slits• TOF – timed ion selector• Orbitrap – not possible – why?• Quadrupoles – matthieu stability diagram• Ion traps

– Resonant ejection – frequencies?– sweeps– Swift– Filtered noise field

• FTICR– Resonant ejection– Sweeps– SWIFT– Filtered noise field– CHEF, multi-CHEF

In each case:

Isolation resolution

Limitations

Requirements

Selectivity