Presentation is loading. Please wait.

Presentation is loading. Please wait.

CH 908: Mass Spectrometry Lecture 7 Tandem mass spectrometry

Similar presentations


Presentation on theme: "CH 908: Mass Spectrometry Lecture 7 Tandem mass spectrometry"— Presentation transcript:

1 CH 908: Mass Spectrometry Lecture 7 Tandem mass spectrometry
Prof. Peter B. O’Connor

2 Tandem Mass Spectrometry or MS/MS
Isolation Fragmentation Isolation Fragmentation Benefits: Extremely high specificity More structural information Limitations: Isolation window Fragmentation efficiency Ion Losses MS/MS/MS, or MS3 MS/MS

3 Tandem Mass Spectrometry
“Tandem in Time” – FTMS, QITMS “Tandem in Space” – Triple quad, TOF/TOF, sector

4 OUTLINE Tandem in space instruments Tandem in time 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

5 Magnetic sector instruments
Ions are deflected and accelerated down a curved path to the detector.

6

7 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.

8 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.

9

10 Figure 6. MALDI tandem time-of-flight mass spectrometer.
Laser Source deflector Collision Cell (Vc) Vr ≈ Vs + + + + + + + first field free drift region + + Vs + Delay Generator + + + second field free drift region Detector Oscilloscope Figure 6. MALDI tandem time-of-flight mass spectrometer.

11

12 Aerosol mass spectrometer

13 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.

14 Triple quadrupole

15 MS3

16 MS/MS scan modes

17 Ions in an Oscillating Electric Field
A± = U ± Vsin(ωt) “Matthieu eqn” az = 8eU/mω2r2 qz = 4eV/mω2r2 z stability r stability 0.5 1.0 1.5 qz Operating Line b=1.0 qz=.908 Stable z & r az 0.2 0.0 -0.2 -0.4 -0.6 0.4 + - qz a V/m qz a fion az a U/m

18 z stable r stable az qz qz = 0.908 A B B A r and z stable D
0.5 1.0 qz az 0.2 0.0 -0.2 -0.4 -0.6 z stable B A r and z stable D az = 0.02, qz = 0.7 az = 0.05, qz = 0.1 C C D r stable az = -0.2, qz = 0.2 az = -0.04, qz = 0.2 Figure 12. Mathieu stability diagram with four stability points marked. Typical corresponding ion trajectories are shown on the right.

19 Ejection Frequency

20 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

21 Scan Function for MS/MS on QIT
Time Ion Injection Isolation Excitation m/z analysis RF Amplitude Tailored Waveform Resonance Excitation / Ejection Amplitude Beir, M.E. and Schwatz, J.C. in Electrospray Ionization Mass Spectrometry

22 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.

23 Excitation/Isolation methods in FTICR

24 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:

25 Correlated Harmonic Excitation Frequency (CHEF)

26 High resolution ion isolation
Isolation of single isotopes of ubiquitin (8.6 kDa) and carbonic anhydrase (29 kDa) was demonstrated. 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):

27 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

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

29 Collisionally Activated Dissociation also called Collision Induced Dissociation (CID)
+ N2 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

30 Figure 14. Quadrupole Time-of-Flight Hybrid
Oscilloscope Laser Delay Generator Source S Pusher (Vp) + + + + + + + + + Q0 Q1 Q2 V (RF-only) (mass filter) (RF-only) + + + Focusing Collision Cell D (field free drift region) + + + Vr ≈ Vp Figure 14. Quadrupole Time-of-Flight Hybrid

31 Collisional Activation in a QIT
A± = U ± Vsin(ωt) “Matthieu eqn” az = 8eU/mω2r2 qz = 4eV/mω2r2 z stability r stability 0.5 1.0 1.5 qz Operating Line b=1.0 qz=.908 Stable z & r az 0.2 0.0 -0.2 -0.4 -0.6 0.4 + - qz a V/m qz a fion az a U/m

32 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: 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:

33

34 Photo-Dissociation + + +* hυ Ion absorbs photon(s) and break
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

35 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.

36 157 nm photodissociation

37

38 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.

39 Infrared Multiphoton Dissociation

40 Surface induced dissociation
+ + 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)

41 SID in an FTICR

42 Surface induced dissociation

43 Electron Capture Dissociation
m+ n+ (n-1)+* + e- 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

44 Odd vs. Even Electron Fragmentation
Even electron = proton rearrangements Odd electron = radical rearrangements Non-ergodic fragmentation = FAST!!

45 ECD Spectrum

46 Substance P ECD RPKPQQFFGLM-NH2 c5 c10 c7 c6 c4 c8 c9 * a7 z9 w2 300
[M+2H]+• c6 c4 c8 c9 * a7 z9 w2 300 600 900 1200 1400 400 500 700 800 1000 1100 1300 m/z

47 RPKPQQFFGLM 674.8 = [M+2H]2+ 1348.7 = [M+2H]+• Z9•/Z9 C7•/C7 C9 C10

48 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?

49 CH908: Mass spectrometry Lecture 1 Fini…

50

51 Magnetic-Sector Mass Spectrometry

52 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.

53 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

54 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).

55 Time of Flight Mass Spectrometry (TOF-MS)
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

56 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)

57 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

58 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 CHEF, multi-CHEF In each case: Isolation resolution Limitations Requirements Selectivity


Download ppt "CH 908: Mass Spectrometry Lecture 7 Tandem mass spectrometry"

Similar presentations


Ads by Google