1

Mass Analyzers

Árpád Somogyi

Campus Chemical Instrument Center

Ohio State University

Mass Spec and Proteomics Workshop

July 10, 2018

What is Mass Spectrometry?

• Mass spectrometry is a powerful tool in analytical/bioanalytical chemistry that provides– detailed structural information for a

– wide variety of compounds (MW: 1-106 daltons) by using

– a small amount of sample (ug, picomol, femtomol)

– often and easily coupled with separation techniques (GC, HPLC) – ideal for mixture analysis

2

Ionization Source Mass Analyzer Detector Computer

Sample IntroductionDirect probe/infusion

GCHPLC

Sample Preparation

Vacuum System

Electron impact (EI)Chemical ionization (CI)Atmospheric pressure (API)Electrospray (ESI)Matrix assisted laser Desorption/ionization (MALDI)Surface enhanced LDI (SELDI)Fast atom bombardment (FAB)

Electrostatic (ESA)Magnet (B)Time-of-flight (TOF)Quadrupole (Q)Ion Traps (2D & 3D IT)Ion-CyclotronResonance (ICR)Orbitrap (OT)

Rough, Turbomolecular, andCryo pumps

Electron MultiplierPhotomultiplierFaraday capArray DetectorsMultichanel plate

Want to do MS or MS/MS ?Need a Mass Spectrometer

inlet all ions

Ionizationsource

Massanalyzer

sortedions

Detector

Datasystem

3

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

4

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

Detectors

w/ TOF

5

Image Current Detectors

w/ FTICR w/ Orbitrap

B

Ion Detection Plates

Ion Detection Plates

RF-excitation

v

image current

D D

Ubiquitinmultiply charged statesno selection

image current

Ubiquitin+11 charged stateQ selection

image current

6

• 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

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 ___________

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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)

• 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

8

Example of ultrahigh resolution in an FTICR

J. Throck Watson “Introduction to Mass Spectrometry” p. 103

M

∆M

R=M/∆M

Resolution defined at 10% valley

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• 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

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

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Desirable mass analyzers characteristics

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 analyzers characteristics

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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

• 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

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TOF Quadrupole

Linear Ion Trap

OrbitrapFTICR

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)– orbitrap

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MS:

IonizationIonization AnalysisAnalysis

MS/MS:

Ionization

MS/MS:

Ionization AnalysisAnalysisSelectionSelection Activation

Collide withtarget to producefragments

Activation

Collide with

fragments

MS and MS/MS revisited

Simulation of Two-Step Processhttp://monte.chem.ttu.edu/index.html/animations/html/ani-digli-5.html

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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

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Disclaimers:

We are not intentionally favoring any single manufacturer.

We are more familiar with the operation of instruments we use.

Some of our slides involve simplifications. Listeners could likely find an exception

for almost any statement we make.

TOF Quadrupole

Linear Ion Trap

OrbitrapFTICR

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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

r

Time of Flight

Consider MALDI

Matrix

Analyte+

h

Targ

et

Analyte ion may have (1) Kinetic Energy distribution or (2) Spatial distribution

How does the ion generation step in TOF influence m/z analysis?

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Hasslightlygreater KE

Effect is broadpeaks

How will KE spread influence the spectrum?

c/o Cotter

Ions of samem/z

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?

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• Increased resolution by compensating for KE spread from the source

http://www.jic.bbsrc.ac.uk/services/proteomics/tof.htm

Time-of-Flight Reflectron

xx

KE = ½ mv2

x

x x

Ion Source(MALDI)

Detector

Mass Analyzer(TOF)

Reflectron

High resolution

Reflectron TOF

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x

KE = ½ mv2

Ion Source(MALDI)

Detector

Mass Analyzer(TOF)

Reflectron

High resolution

Reflectron TOF

http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html

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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)?

Low mass (below 40 k Da)

High resolution

10 KV

+

+

10 KV

+

+

7 KV

++

++

+

+

Delayed Extraction

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Continuous TOF

Resolution = 700 (FWHM)

Delayed Extraction

Resolution = 6000 (FWHM)

Continuous vs. Delayed Extraction

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

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• 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

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

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Can an MS/MS instrument be constructed if the only analyzer

type available is TOF?

http://docs.appliedbiosystems.com/pebiodocs/00106293.pdf

TOF-TOF

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High-energy (keV) Collisions with Gas in a TOF-TOF

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

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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

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

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TOF Quadrupole

Linear Ion Trap

OrbitrapFTICR

Quadrupole (Q)

http://www.files.chem.vt.edu/chem-ed/ms/quadrupo.html

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• 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

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+

+

--

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

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http://www.youtube.com/watch?v=pjCun7QF19U

Quadrupole animation

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Initial kinetic energy affects ion motion in quad

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.

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• 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

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

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What MS/MS instruments can be produced from Q?

What MS/MS instruments can be produced from Q and TOF?

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

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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.

http://www.waters.com/WatersDivision/waters_website/products/micromass/ms_top.asp (outdated)

Quadrupole Time of Flight (Q-TOF)

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Low-energy (eV) Collisions with Gas

Shevchenko, A., et al., Rapid. Commum. Mass Spectrom., 1997, 11: 1015-1024

80 fmol BSA digest

Q-TOF

QQQ

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QTOF with Ion Mobility

36

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Ion Mobility Process – Sample Introduction & Ionization

E

Shutter

Eiceman, G.A.; UMIST, 2002.

73

Ion Mobility Process – Injection into Drift Tube

E

Eiceman, G.A.; Karpas, Z. Ion Mobility Spectrometry.  New York: Taylor & Francis, 2005.

74

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Ion Mobility Process –Separation

Shutter

Eiceman, G.A.; Karpas, Z. Ion Mobility Spectrometry.  New York: Taylor & Francis, 2005.

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Ion Mobility Process – Detection

Shutter

Eiceman, G.A.; Karpas, Z. Ion Mobility Spectrometry.  New York: Taylor & Francis, 2005.

76

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Mobility Spectrum

Drift Time (ms)0 20

Eiceman, G.A.; Karpas, Z. Ion Mobility Spectrometry.  New York: Taylor & Francis, 2005.

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Principles of Mobility Separation

• K – mobility, cm2/V-s

• K reported as reduced mobility, Ko

• K related to collisional cross section, Ω

• Low E/N only

Borsdorf, H.; Eiceman, G.A. Appl. Spectrosc. Rev. 41, 2004.

KEvt

L

12

16

3 21

TkN

qK

B

Indicative of size and shape – compare to computationally calculated cross sections 

78

40

QTOF with Ion Mobility

Selection of [M+2H]+2 at m/z 246.1

100

80

60

40

20

0Rel

ativ

e In

ten

isty

76543210Drift Time (ms)

600

500

400

300

200

100

m/

z

SDGRGGRGDS

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Transfer CID of [M+2H]+2 at m/z 246.1

100

50

0

Rel

ativ

e A

bu

nd

ance

500400300200100m/z

100

50

0

3.0

2.5

2.0

Dri

ft T

ime

(ms)

dt = 2.49-2.68 ms

dt = 2.68-2.83 ms

GRGDS

SDGRG

b4

b3b2

y1

R

R

Si

Si

y4y3

TOF Quadrupole

Linear Ion Trap

OrbitrapFTICR

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What is small, inexpensive and still gives great MSMS ?????

Quadrupole Ion TrapsMiniature IT: Cooks, R. G. and coworkersAnal. Chem. 2002, 74, 6145-6153; 2000, 72, 3291-3297.

http://www.chem.wm.edu/dept/faculty/jcpout/faculty.html

Ion path in a trap

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RF ION TRAP ELECTRODE STRUCTURES

LCQ-Type 3DQuadrupole Trap

LTQ-Type (2D)Linear Quadrupole Trap

ASMS Fall Workshop2006 JEPS

2D vs. 3D Ion Traps

Tube Lens

Skimmer

Ion Transfer

Tube

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Linear Trap Demo

Tube Lens

Skimmer

Ion Transfer

Tube

Overall Performance Gains

•Trapping efficiency

•Detection efficiency

•Trapping capacity

LTQ 3D Traps

~ 50-100% ~50% ~ 1-2x

~ 55-70% ~5% ~ 11-14x

Increase

~ 20,000 ions ~500 ions ~ 40x

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9 Protein Mixture

0

10

20

30

40

50

60

70

80

Nu

mb

er o

f Pe

ptid

es

Deca XP plus

LTQ

10min 20min 30min 60min

Gradient (min)

12

21 24

3135

42

61

73

• Increased Trapping Efficiency• Increased Trapping Capacity

Motivating Factors Realized

• Increased Sensitivity• Increased Inherent Dynamic Range

• Increased S/N for full Scan MS• Practical MSn

•Faster Scan Times - no μscans (only one)

Which means....

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How are 3-D traps and linear ion traps used in MS/MS?

Stand alone

3-D traps

LTQ

Front or back end of tandem-in-space

LT-TOF

LT-FT, LT-Orbitrap,

QTrap

Activation:

CIDLow-energy (eV) Collisions

with Gas

IRMPD (Infrared Multiphoton Dissociation)

ETD(electron transfer dissociation)

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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

• Many instruments are complementary

• More Activation methods, the better

• Assess your needs

• Assess your budget

• @ Research level, may want to build your own

General Conclusions

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Learning Check

Draw the appearance of the mass spectrum in which both singly and doubly charged YGGFLR (molecular weight 711.3782) are produced and analyzed with a quadrupole, TOF and orbitrap (Hint: peaks widths important)

712 in Quad, TOF, ICR

m/z

m/z

m/z

Inte

nsity

Inte

nsity

Inte

nsity

QUAD

TOF

ICR

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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.

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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.

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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.