1
Mass Analyzers
Árpád Somogyi
OSU, Summer Workshop
August 18, 2015
Want to do MS or MS/MS ?Need a Mass Spectrometer
inlet all ions
Ionizationsource
Massanalyzer
sortedions
Detector
Datasystem
2
inlet all ions
Ionizationsource
Massanalyzer
sortedions
Detector
Datasystem
Ion movement
• Biased metal plates (electrodes, lenses) used to move ions between ion source and analyzers
• Electrodes and physical slits used to shape and restrict ion beam
• Good sensitivity is dependent on good ion transmittance efficiency in this area
inlet all ions
Ionizationsource
Massanalyzer
sortedions
Detector
Datasystem
3
inlet all ions
Ionizationsource
Massanalyzer
sortedions
Detector
Datasystem
Ion detection
• Ions can be detected efficiently w/ high amplification by accelerating them into surfaces that eject electrons
• Kinetic energy of ions defined by
E = zeV = qV = ½ mv2
E = kinetic energym = massv = velocitye = electronic charge (1.60217e-19 C)z = nominal chargeV = accelerating voltage
Ion energy
4
Learning Check
Consider two electrodes,
one at 1000 V and one at ground (0 V)
1000 V 0 V
+ ion will travel with kinetic energy of ___________
Question: Consider an ion source block and an extraction lens. How would you bias the block and lens if you want the ions to be accelerated by
a) 8000 eV (appropriate for magnetic sector)
b) 5 eV (appropriate for entering a quadrupole)
c) 20,000 eV (appropriate for entering a TOF)
5
• Mass spectrometers separate ions with a defined resolution/resolving power
• Resolving power- the ability of a mass spectrometer to separate ions with different mass to charge (m/z) ratios.
Mass Resolution
M
∆M
R=M/∆M
Resolution defined at 10% valley
6
Example of ultrahigh resolution in an FTICR
J. Throck Watson “Introduction to Mass Spectrometry” p. 103
• Common features– Accelerated charged species (ions) interact
with/can be controlled by• Electrostatic field (ESA, OT)
• Magnetic field (B, ICR)
• Electromagnetic (rf) fields (Q, IT, LT)
– Or ions just fly (TOF)
General about mass spectrometers
7
For each of the following applications, choose the most appropriate mass analyzer from the following list.
orbitrap (OT) quadrupole (Q)time-of-flight (TOF) FTICR
Analyzer Purpose
______ synthetic organic chemist wants exact mass of compound
_______ biochemist wants protein molecular weight of relatively large protein (MW 300,000)
_______ EPA (Environmental Protection Agency) wants confirmation of benzene in extracts from 3000 soil samples
_______ Petroleum chemist wants to confirm the presence of 55 unique compounds at onenominal mass/charge value in a mass spectrum
Desirable mass spectrometer characteristics
8
1) They should sort ions by m/z
2) They should have good transmission (improves sensitivity)
3) They should have appropriate resolution (helps selectivity)
4) They should have appropriate upper m/z limit
5) They should be compatible with source output (pulsed or continuous)
Desirable mass spectrometer characteristics
Type of analyzer• electric sector• magnetic sector• quadrupole, ion trap• time-of-flight• FT-ion cyclotron resonance
Physical parameter used as basis for separation• kinetic energy/z• momentum/z• m/z• flight time• m/z (resonance frequencies)
m/z values are determined by actually measuring different physical parameters
Mass spectrometers record m/zvalues
9
• Magnetic (B) and/or Electrostatic (E) (HISTORIC/OLDEST)
• Time-of-flight (TOF)
• Quadrupole (Q)
• Quadrupole Ion Trap (IT)
• Linear Ion Trap (LT)
• Orbitrap
• Fourier Transform-Ion Cyclotron Resonance (ICR)
Performance Advantages / Disadvantages / $$$
Types of Mass Analyzers
TOF Quadrupole
Quadrupole Ion trap
FTICR
Orbitrap FTICR
10
Notice: accelerating voltages vary with analyzer(has consequences for MS/MS)
• High voltage (keV energy range)– magnet (B)– electrostatic (E)– time-of-flight (TOF)
• Low voltage (eV energy range) – quadrupole (Q)– ion trap (IT, LT)– ion cyclotron resonance (ICR)
MS:
IonizationIonization AnalysisAnalysis
MS/MS:
Ionization
MS/MS:
Ionization AnalysisAnalysisSelectionSelection Activation
Collide withtarget to producefragments
Activation
Collide with
fragments
MS and MS/MS revisited
11
Tandem in Space
QqQ
Q Trap
Q TOF
TOF TOF
LTQ-Orbi (& QExactive)
Tandem in Time
Ion Trap (2 or 3 D)
FT-ICR (FT)
Current popular MS/MS arrangements
Decision factors when choosing a mass spectrometer
• S p e e e e e e e e e e e e e d
• R e s o l u t i o n• Sensitivity
•Dynamic range
• Co$t
12
TOFQuadrupole
Quadrupole Ion trap
FTICROrbitrap
KE = zeV = ½mv2 m = mass V = velocity
v = D/t D = distance of flight t = time of flight
½m(D/t)2 = zeV KE = kinetic energy e = charge
D=t21
2zeVm
/
m/z
V
D
Det
ecto
rTime of Flight
13
Consider MALDI
Matrix
Analyte+
hTa
rget
Analyte ion may have (1) Kinetic Energy distribution or (2) Spatial distribution
How does the ion generation step in TOF influence m/z analysis?
Hasslightlygreater KE
Effect is broadpeaks
How will KE spread influence the spectrum?
c/o Cotter
Ions of samem/z
14
Solution to peak broadening caused bykinetic energy spread:
Reflectron (ion mirror)
Series of ring electrodes, typically with linear voltage gradient
How will KE spread influence the spectrum?
• Increased resolution by compensating for KE spread from the source
http://www.jic.bbsrc.ac.uk/services/proteomics/tof.htm
Time-of-Flight Reflectron
15
xx
KE = ½ mv2
x
x x
Ion Source(MALDI)
Detector
Mass Analyzer(TOF)
Reflectron
High resolution
Reflectron TOF
x
KE = ½ mv2
Ion Source(MALDI)
Detector
Mass Analyzer(TOF)
Reflectron
High resolution
Reflectron TOF
16
http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html
We talked about how to deal with kinetic energy spread.
How do we deal with ions formed at different locations in the source (spatial distribution)?
17
Low mass (below 40 k Da)
High resolution
10 KV
+
+
10 KV
+
+
7 KV
++
++
+
+
Delayed Extraction
Continuous TOF
Resolution = 700 (FWHM)
Delayed Extraction
Resolution = 6000 (FWHM)
Continuous vs. Delayed Extraction
18
3145.708
3177.722
* Pepmix\0_N6\1\1SRef
0
1000
2000
3000
4000
5000
6000
Inte
ns.
[a.u
.]
3140 3145 3150 3155 3160 3165 3170 3175 3180 3185m/z
Recent TOF designs: improved resolution, better sensitivity and mass accuracy (Ultraflex III MALDI TOF-TOF)
Resolution: 25,000S/N: 46
• m/z Range: unlimited u
• Resolution: ~20,000 (Reflectron)
• Mass Accuracy: ~ 3-10 ppm (300-4,000 u)
• Scan Speed: 106 u/s
• Vacuum: 10-7 Torr
• Quantification: low -medium
• Positive and Negative Ions
• Variations:
Linear, Reflectron,
• Tandem: w/ quads, TOFs and/or sectors
Typical TOF Specs
19
TOF Quad 3D TrapLinear Trap
FT‐ICR Orbitrap
mass range
Resolution
exact m/z
sensitivity
all ion detection
ion storage
speed
dynamic range
quantification
MS/MS types
sample introduction
simplicity
cost/performance
Can an MS/MS instrument be constructed if the only analyzer
type available is TOF?
20
http://docs.appliedbiosystems.com/pebiodocs/00106293.pdf
TOF-TOF
High-energy (keV) Collisions with Gas in a TOF-TOF
21
Medzihradszky, KF et al. Anal Chem. 2000, 72: 552-558
16O/18O-labeled DLEEGIQTLMGR
High (keV) collisions allow for w peptide ions and immonium ions
*Resolution
MALDI TOF-TOF fragmentation spectrum of a sodiated polymer
O
O CH2 CH2 *m
nO
3-OEB, m=1-3 (164, 44)Copolymer of 2-hydroxybenzoic acid and ethylene carbonate
328.574
981.346492.623
656.765186.600
821.038
120.694
146.669
284.552941.46890.771
721.026443.572 776.938612.695
0
1
2
3
4
5
6
4x10
Inte
ns.
[a.u
.]
200 400 600 800 1000m/z
22
Mass Spectrometer Advantages Disadvantages
TOF-TOF high resolution, high m/z fragment ions
Large size
keV CID
Not ideal for continuous
ionization source
easier de novo peptide sequencing
$$$
dn and wn to distinguish Ile/Leu
MALDI source
Advantages and Disadvantages
TOFQuadrupole
Quadrupole Ion trap
FTICR
Analyzers
Orbitrap
23
Quadrupole (Q)
http://www.files.chem.vt.edu/chem-ed/ms/quadrupo.html
24
• four parallel rods or poles
• fixed DC and alternating RF voltages
• only particular m/z will be focused on the detector, all the other ions will be deflected into the rods
• scan by varying the amplitude of the voltages – (AC/DC constant).
rf voltage+dc voltagerf voltage 180° out of phase-dc voltage
Quadrupole (Q)
http://www.kettering.edu/~drussell/Demos/MembraneCircle/Circle.html
Positive rod
Positive rodNegative rod
Negative rod
Quadrupole Field Animation
25
+
+
--
negative DC offset
positive DC offset
-DC
+DC
• + DC, ions focused to center
• - DC, ions defocused
• +DC w/ rf, light ions respond to rf, eliminated (high mass filter)
• -DC w/ rf, light ions respond to rf, focused to center (low mass filter)
Ion Motion in Quadrupoles- a qualitative understanding
26
http://www.youtube.com/watch?v=pjCun7QF19U
Quadrupole animation
Initial kinetic energy affects ion motion in quad
27
Figure 9. The a-q stability diagram: a) The shaded area represents those areas in a-q space which correspond to stable solutions of Mathieu’s differential equation. B) The one amu bandpass mass filter: Notice that only ions of m/e m+1 fall within the stability diagram.
Miller, P., Denton, M.B., J.Chem Ed., 63:7, pg 617, 1986.
• m/z Range: 2-4000 u• Resolution: Unit• Mass Accuracy: ca +/-
0.1 u• Scan Speed: 4000 u/s• Vacuum: 10-4 – 10-5
Torr• Low Voltages: RF ~6000 –10000 VDC ~500V-840VSource near ground
• Quantification: good choice
• Positive and Negative Ions
• Variations: SingleQ, TripleQ, Hybrids
Quadrupole Typical Specs
28
TOF Quad 3D TrapLinear Trap
FT‐ICR Orbitrap
mass range
Resolution
exact m/z
sensitivity
all ion detection
ion storage
speed
dynamic range
quantification
MS/MS types
sample introduction
simplicity
cost/performance
What MS/MS instruments can be produced from Q?
What MS/MS instruments can be produced from Q and TOF?
29
Triple Quadrupole- since 1970’s and still going strong!
S D
Q1 Q2 Q3
Attractive Features:
• Source near ground and operates at relatively high pressure
Couples well to source and to chromatography
•Multiple scan modes easy to implement
Source
HeatedCapillary
DetectionSystem
Q3
Q2
Q1Q0
c/o Thermo Finnigan Corp.
Dynode
Q3
Q2
Q1 Q0 Q00
Turbo
Triple Quadrupole (QQQ)
c/o Agilent Corp.
30
http://www.waters.com/WatersDivision/waters_website/products/micromass/ms_top.asp (outdated)
Quadrupole Time of Flight (Q-TOF)
Low-energy (eV) Collisions with Gas
31
Shevchenko, A., et al., Rapid. Commum. Mass Spectrom., 1997, 11: 1015-1024
80 fmol BSA digest
Q-TOF
QQQ
TOFQuadrupole
Quadrupole Ion trap
FTICR
Analyzers
Orbitrap
32
Linear Trap Demo
Tube Lens
Skimmer
Ion Transfer
Tube
TOF Quad 3D TrapLinear Trap
FT‐ICR Orbitrap
mass range
Resolution
exact m/z
sensitivity
all ion detection
ion storage
speed
dynamic range
quantification
MS/MS types
sample introduction
simplicity
cost/performance
33
Suggested Reading List
GENERAL MS AND MS/MS
Kinter, M. and Sherman, N. E., Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley and Sons, New York, NY, 2000.
De Hoffmann E., Mass Spectrometry – Principles and Applications (Second Edition), Wiley and Sons, New York, NY, 2002.
Busch, K.L., et al., Mass spectrometry/ mass spectrometry: techniques and applications of tandem mass spectrometry, VCH Publishing, New York, NY, 1988.
McLafferty, F.W. (Ed) Tandem Mass Spectrometry, Wiley and Sons, New York, NY, 1983.
McLafferty, F.W. and Turecek, F. Interpretation of Mass Spectra, University Science Books, New York, NY, 1993.
Siuzdak, G. Mass Spectrometry for Biotechnology, Academic Press, San Diego, CA, 1996.
Gygi, S.P. and Aebersold, R., Curr. Opin. Chem. Biol., 4 (5): 489, 2000.
Roepstorff, P., Curr. Opin. Biotech., 8: 6, 1997.
De Hoffmann, JMS, 31: 129, 1996.
Mann, M. et al., Annu. Rev. Biochem., 70: 437, 2001.
McLuckey, S and Wells, J.M., Chem. Rev., 101: 571, 2001.
Suggested Reading ListQ-TOF
Morris, H.R. et al., J. Protein Chem., 16: 469, 1997.
Shevchencko A., et al., Rapid Commun. Mass Spectrom., 11: 1015, 1997.
Shevchencko A., et al., Anal. Chem., 72: 2132, 2000.
Medzihradszky, K.F., et al., Anal. Chem., 72: 552, 2000.
Glish, G.L. and Goeringer, D.E., Anal. Chem., 56: 2291, 1984
Morris, H.R. et al., Rapid Commun. Mass Spectrom., 10: 889, 1996.
Wattenberg, A. et al., JASMS, 13 (7): 772, 2002.
Lododa, A.V. et al., Rapid Commun. Mass Spectrom., 14(12): 1047, 2000.
TOF
Bush, K., Spectroscopy, 12: 22, 1997.
Cotter, R.J., Time-of-Flight: Instrumentation and Applications in Biological Research, ACS, Washington, DC, 1994.
34
Suggested Reading ListTOF-TOF
Yergey, A.L. et al., JASMS, 13 (7): 784, 2002.
Go, E.P. et al., Anal. Chem., 75: 2504, 2003.
FTICR
Buchanan, M.V. (Ed), Fourier Transform MS, ACS, Washington, DC, 1987.
Comisarow, M.B. and Marshall, A. G., Chem. Phys. Lett., 25: 282, 1974.
McIver, R.T. et al., Int. J. Mass Spectrom. Ion Processes, 64: 67, 1985.
Kofel, P. et al., Int. J. Mass Spectrom. Ion Processes, 65: 97, 1985.
Williams, E. R. Anal. Chem., 70: 179A, 1998.
McLafferty, F. W. Acc. Chem. Res., 27: 379, 1994.
Fridriksson, E. K. et al., Biochemistry, 39: 3369, 2000.
Fridriksson, E. K. et al., Biochemistry, 38: 8056, 1999.
Kelleher, N. L.et al., Protein Science, 7: 1796, 1998.
McLafferty, F. W. et al., Int. J. Mass Spectrom., 165: 457, 1997. .
Suggested Reading ListGreen, M. K. and Lebrilla, C. B. Mass Spectrom. Rev., 16: 53, 1997.
Guan, S. and Marshall, A. G. Chem. Rev., 94: 2161, 1994
QIT
Marsh, R.E. et al., Quadrupole Storage Mass Spectrometry, vol.102, Wiley and Sons, New York, NY, 1989.
Cooks, R.G. et al., Ion trap mass spectrometry. Chem. Eng. News, 69(12): 26, 1991.
Jonscher, K.R. et al., Anal Biochem, 244(1): 1, 1997.
Louris, J.N. et al., Anal. Chem., 59(13): 1677, 1987.
Louris, J.N. et al., Int. J. Mass Spectrom. Ion Processes, 96(2): 117 1990.
Cooks, R.G. et al., Int. J. Mass Spectrom. Ion Processes, 118-119: 1 1992.
McLuckey, S. A. et al., Int. J. Mass Spectrom. Ion Processes, 106: 213, 1991.
Ngoka, L. C. M. and Gross, M. L. Int. J. Mass Spectrom. 194: 247, 2000.
35
Suggested Reading List
QQQ
Yost, R. A. and Enke, C. G. J. Am. Chem. Soc., 100: 2274, 1978. Arnott, D. in Proteome Research: Mass Spectrometry, james, P (Ed.), pp 11-31, Springer-Verlag, Germany, 2001.Steen H. et al., JMS, 36: 782, 2001Miller, P.E. and Denton, M.B., J. Chem. Educ., 7: 617, 1986.Yang, l. et al., Rapid Commun. Mass Spectrom., 16(21): 2060, 2002.Mohammed, S. et al., JMS, 36: 1260, 2001.Dongre, A.R. et al., JMS, 31: 339, 1996.
Orbitrap
Hu, Q, Noll, RJ, Li, H., Makarov, A., Hardman, M., Cooks, R.G. JMS, 430-443, 2005.Makarov A. Anal. Chem. 2000; 72(6):1156-1162.
Makarov A, Denisov E, Kholomeev A, Baischun W, Lange O, Strupat K, Anal. Chem. 2006; 78(7):2113-2120.
Makarov A, Denisov E, Lange O, Horning S. JASMS. 2006; 17(7):977-982.
Macek B, Waanders LF, Olsen JV, Mann M. Mol. Cell. Proteom. 2006; 5(5):949-958.
Perry RH, Cooks RG, Noll RJ. Mass Spectrom. Rev. 2008; 27(6):661-699.
Scigelova M, Makarov A. Orbitrap mass analyzer - Overview and applications in proteomics. Proteomics. 2006: 16-21.
Olsen JV, Macek B, Lange O, Makarov A, Horning S, Mann M. Nature Methods. 2007; 4(9):709-712.