CH 908: Mass SpectrometryLecture 5

Ionization sources, EI, MALDI, and ESI

Prof. Peter B. O’Connor

Objectives for this lecture

• Ionizing molecules

• The many attempts to ionize biomolecules– Direct Chemical Ionization– Plasma desorption– FAB– LDI

• ESI and MALDI

MS Block Diagram

Ion Source

Mass Separator

Detector

Chromatography

Inlet

Sample Cleanup

Computer

DataReduction

Bioinformatics

FragmentationMethod

Mass Spectrometers do not measure mass, they measure mass/charge ratio.

A simple, laser desorption ion source.

Sample

Laser

Ionization• Photons

• Electrons

• Fast Neutral Molecules

• Other Ions.

• Heat

• Combination methods

Because most molecules prefer to be electrically neutral, ionization requires addition or removal of one or more charges particles (usually electrons or protons). This can be done by hitting the sample with one of the following.

Exceptions: samples that naturally prefer to be ions or are somehow stabilized as ions.

EI Mass Spectrum of an acetylated and reduced peptide

Field ionization/ field desorption MS Field ionization/ field desorption MS (FI/FDMS) (FI/FDMS) H. D. Beckey, H. D. Beckey, IJMSIPIJMSIP, 1969, 1969

H. R. Schulten, Int. J. Mass Spectrom. Ion Phys., 32, 97-283, 1979

Emitters for field ionization/ field Emitters for field ionization/ field desorption MS (FI/FDMS)desorption MS (FI/FDMS)

C. E. Costello, electron microscopy courtesy of JEOL , 1979

Field desorption MS of bradykininField desorption MS of bradykinin

S. Asante-Poku, W. G. Wood, and D. E. Schmidt, Jr. Biomed. Mass Spectrom, 2, 121, 1975

Field desorption MS of Vitamin BField desorption MS of Vitamin B1212

H.-R. Schulten and H. M. Schiebel Naturwissenschaften, 65, 223, 1978

Methods for “soft” ionizationMethods for “soft” ionization

• Direct chemical ionization MS (DCIMS)• Field ionization/ field desorption MS (FI/FDMS)• Secondary ionization MS (SIMS)• 252Cf Plasma desorption (PDMS)• Fast atom bombardment (FABMS)• Liquid secondary ionization MS(LSIMS)• Laser desorption MS (LDMS)• Electrospray ionization MS (ESIMS)Electrospray ionization MS (ESIMS)• Matrix-assisted laser desorption/ionization MS Matrix-assisted laser desorption/ionization MS

(MALDI MS)(MALDI MS)

[Direct] chemical ionization MS (DCIMS)[Direct] chemical ionization MS (DCIMS)

Frank H. Field and Burnaby MunsonASMS Distinguished Contribution, 1996

252Cf Plasma desorption (PDMS)

ASMS Distinguished Contribution, 1990

Ronald D. Macfarlane

R. D. Macfarlane, Anal. Chem., 55, 1247A-1264A, 1983

252Cf Plasma desorption (PDMS) of porcine trypsin (23,463 Da)

Peter Roepstorff

B. Sundquist, P. Roepstorff, et al., Science, 226, 696 – 698, 1984

Per Håkansson

Michael BarberASMS Distinguished Contribution, 1991

Fast atom bombardment (FABMS)Fast atom bombardment (FABMS)

M. Barber, R. S. Bordoli, G. J. Elliott, R. D. Sedgwick and A. N. Tyler, Anal. Chem., 54, 645A-657A, 1982

Fast atom bombardment mass spectra of (a) Fast atom bombardment mass spectra of (a) vitamin Bvitamin B1212 and (b) the coenzyme of vitamin B and (b) the coenzyme of vitamin B1212

M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Nature, 293, 270-275, 1981

(a) (b)

Fast atom bombardment mass spectra: Fast atom bombardment mass spectra: (a) human insulin and (b) proinsulin(a) human insulin and (b) proinsulin

M. Barber, R. S. Bordoli, G. J. Elliott, N. J. Horoch and B. N. Green, Biochem. Biophys. Res. Commun, 110, 753-757, 1983

A. Dell and H. R. Morris, Biochem. Biophys. Res. Commun, 106, 1456-1462, 1982

(a) (b)

Laser

Sample

Target

Figure 2. Laser Desorption plus Electron Impact Ionization

+

+

+

+ +

+

cathode

+ -Anode

e- e-e-

Two Special Cases• Matrix Assisted Laser Desorption/Ionization (MALDI)1,2

• Analyte is mixed with a "Matrix" and dried down on a surface• Laser hits the spot and desorbs matrix and analyte together• some of the analyte ends up charged

• Electrospray Ionization (ESI)3,4

•Solution containing the sample is sprayed out a needle into a large electric field. Droplets are formed and are sucked down into the mass spectrometer. They evaporate and leave charges on the analytes.

1. Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T., Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Biopolymers Anal. Chem. 1991, 63, 1193A-1203A.

2. Karas, M. I.; Bachmann, D.; Bahr, U.; Hillenkamp, F., Matrix-Assisted Ultraviolet Laser Desorption of Non-Volatile Compounds Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68.

3. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray ionization for mass spectrometry of large biomolecules Science 1989, 246, 64-71.

4. Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B., Electrospray interface for LCMS Anal. Chem. 1985, 57, 675-679.

ESI and MALDI

Prof. John Fenn

Developed Electrospray

Prof. Dr. Ing. Franz HillenkampProf. Dr. Michael Karas

Developed MALDI

Nobel prize, 2002

+++

++

+ ++ + + + + ++ + +

+ + + ++ +++++

+ + +++

Robert A. Millikan (Nobel prize in physics, 1923)

Oil Drop Experiment

A simple ESI ion source.

Electrospray Ionization

• Electrospray Ionization (ESI)•Solution containing the sample is sprayed out a needle into a large electric field. Droplets are formed and are sucked down into the mass spectrometer. They evaporate and leave charges on the analytes. •Sample is a flowing liquid - compatible with LC•flow rates typically in the <1 μl/min range•sample cleanup is required!•sensitivity (Best case <1 amol, normal case 10-1000 fmol)•Multiple charging•upper mass range not really limited, but big masses get confusing if you don't have sufficient mass resolution•extremely soft ionization method - allows ionization of non-covalently bound species (e.g. protein complexes)

Generic Atmospheric Pressure Ionization Source/Mass Spectrometer System

Vacuum system with multiple stages:1st stage - free-jet expansion, molecular-beam stage2nd stage - ion optics3rd stage - mass analyzer

ElectrosprayMajor Components and Processes in ES-MS

ElectrosprayEmitter

AtmosphericPressure Region

(The Gap)

Atmosphereto VacuumInterface

MassAnalyzer

Žshrinkage/subdivisonof droplets

Ž gas-phase ionproduction

Ž gas-phasereactions

- ionization- charge permutation

Ž finaldesolvation

Ž fragmentation

Ž chargepermutation

Ž production ofcharged dropletsat emitter tip

Taylor cone

Spray ~1 microliter/min

In “nanospray”, flow rates of ~1 nl/min are used. The taylor cone and plume become invisible because the droplets are in the 100 nm diameter range, and sensitivity goes up due to greatly reduced space charge and improved capture efficiency.

A few examples of electrospray mass spectra…

~80 Da difference~25 Da wide

Bad mass spectrometer?

Peroxynitrite

MI=21284.4032 (21284.5511)

1000 1200 1400 1600 1800Mass/Charge (m/z)

16+

17+

18+

15+

14+

MI=21284.4080 (21284.5511)

1185 1230Mass/Charge (m/z)

1000 1200 1400 1600 1800Mass/Charge (m/z)

21291 21303 21291 21307

21+20+

800 900 1000 1100 1200 1300 1400 1500Mass/Charge (m/z)

22+23+24+

25+

19+ 18+

17+

16+

MI=21282.3880 (21282.53543)

21291 21303

21290 21304

MI=21282.3422 (21282.53543)

p21RasNormal distribution

DTT treated distribution

Zhao, C.; Sethuraman, M.; Clavreul, N.; Kaur, P.; Cohen, R. A.; O'Connor, P. B. A Detailed Map of Oxidative Post-translational Modifications of Human p21ras using Fourier Transform Mass Spectrometry Anal. Chem. 2006, 78, 5134-5142.

Calmodulin (Entire protein)

Micro-TOF

884.7075

933.7842

988.6485

1050.3689

1120.3351

1200.2786

1292.5220

1400.1262

1527.3098

1680.0067

+MS, 0.3-1.5min #(16-91)

0.0

0.5

1.0

1.5

4x10Intens.

800 900 1000 1100 1200 1300 1400 1500 1600 1700 m/z

Carbonic anhydrase (Entire protein Micro-TOF

429.2474

543.4134593.1336

659.8101

880.5196

1075.9647

1117.3119

1210.3295

1262.9047

1320.2672

1383.1014

1452.1785

1528.59651613.4771

1747.1915

908.0058

937.2675

968.4701

1001.8412

1037.5745

1161.9558

830.2614

807.2280

785.4459

764.7936

714.6845

854.6555

+MS, 0.4-1.2min #(23-70)

0

1000

2000

3000

4000

5000

6000

Intens.

400 600 800 1000 1200 1400 1600 1800 m/z

819.8634

956.4497

1147.5278

1433.7548

+MS, 0.0-0.3min #(3-16)

0

2

4

6

4x10Intens.

600 800 1000 1200 1400 1600 1800 m/z

Insulin (Entire protein)

Micro-TOF

659.8

714.7

779.5

857.4

952.6

1071.5

+MS, 0.1-1.3min #(7-79)

0.0

0.2

0.4

0.6

0.8

1.0

5x10Intens.

500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Ubiquitin (Entire protein)

Micro-TOF

MALDI

• Matrix Assisted Laser Desorption/Ionization (MALDI)• Analyte is mixed with a "Matrix" and dried down on a surface• Laser hits the spot and desorbs matrix and analyte together• some of the analyte ends up charged "Lucky Survivor Model"• pulsed beam - nicely couples with pulsed mass analyzers• sensitivity (best case ~1 amol, normal case 10-1000 fmol)• more tolerant of salts than ESI, less cleanup required• upper mass limit defined by

1) metastable decay (PSD)2) detector

• relatively soft, but generally not soft enough for non-covalent complexes

MALDI mass spectrometry

Laser

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+

Most commonly, Laser is a <10 nsec pulse at 337 nm (N2 laser) or 355 (frequency tripled Nd:YAG). 50 nsec pulse at 2.94 (Er:YAG) is also used.

ESI and MALDI

Prof. John Fenn

Developed Electrospray

Prof. Dr. Ing. Franz HillenkampProf. Dr. Michael Karas

Developed MALDI

Nobel prize, 2002Nobel prize, 2002

193 nm 200 nm 300 nm 400 nm 800 nm 1 µm 3µm 10µm wavelength

6.3 eV 6,1 eV 4 eV 3 eV 1.5 eV 0.4 eV 0.01 eV photon energy h

ultraviolet visible infrared

Excimer lasers ND:YAG laser OPO-lasers

ArF KrF XeCl ER:YAG laser CO2 laser 193 nm 248 nm 308 nm 2.94 µm

4xf 3xf Nitrogen laser 1xf 266 nm 353 nm 337 nm 1.06 µm 9.1-10.6 µm

Figure 1. Wavelength and photon energy of frequently used desorption lasers

2D Sample stage.

Figure 3. Laser Microprobe for Mass Analysis (LaMMA) configuration

Illumination

Laser

Camera

Ocular system

Ion Optics

TOF Analyzer

1

2

f1 f2 f3

Laser

Targetd1

d2

Figure 2. Laser focusing optics for adjustment of the spot size on the target

Infinite Conjugation

A.

B.

Figure 4. Two of the first MALDI spectra. A) the matrix effect, desorbing Alanine mixed with Tryptophan allowed observation of the Alanine peaks at 10x lower laser fluence than possible with pure Alanine. B) the first MALDI of a protein, showing that desorption/ionization could occur with no observable fragmentation.

Karas, M.; Bachmann, D.; Hillenkamp, F. Influence of the wavelength in high-irradiance ultraviolet laser desorption mass spectrometry of organic molecules Anal Chem 1985, 57, 2935-2939.

Karas, M. I.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Matrix-assisted ultraviolet laser desorption of non-volatile compounds Int J Mass Spectrom Ion Processes 1987, 78, 53-68.

Peptide digests

Laser

Temperature

Sample

Target

Figure 1. Laser Desorption/Ionization

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+

+

+ +

+

PEG (entire compound)

MALDI

MALDI of a 0.6 kDa Polymer

MALDI of a 1.6 kDa Polymer1581.914

1405.8121758.019

1361.785 1802.049

1846.0741317.760

1890.0981273.735

1934.1241229.709

1978.1431185.686

2022.1691141.659

2066.1871097.626 2110.212

1053.5862198.245

0

2000

4000

6000

8000Inte

ns.

[a.u

.]

1000 1200 1400 1600 1800 2000 2200m/z

MALDI of a 13 kDa polymer

0

250

500

750

1000

1250

Inte

ns.

[a.u

.]

10000 11000 12000 13000 14000 15000 16000m/z

Calmodulin (Entire protein)

MALDI

16794.350

0

500

1000

1500

2000

Inte

ns.

[a.u

.]

10000 12000 14000 16000 18000 20000 22000 24000 26000

m/z

Other laser wavelengths

In-gel digest experiment

In-gel digest experiment

Self Assessment

1. list 3 methods that people used to try to ionize proteins before 1985.

2. What two methods were discovered in 1985-1987 that revolutionized the study of biomolecules via mass spectrometry? How do they work?

3. What’s a good matrix for proteins/peptides in MALDI? What’s the structure? What laser wavelengths are used for MALDI?

4. How do you determine the mass of a protein from an ESI mass spectrum? 2 methods (at least).

Fini…

CH908: Mass spectrometryLecture 5 – Ionization sources.

Typical MALDI-TOF mass spectra of small proteins

First MALDI-TOF mass spectrum of a glycoprotein.

ElectronsThe oldest, most controllable method of ionizing most species is to smash electrons into them.

A few important ionization techniques using this method• Electron Impact

• >10 eV electrons • Gas phase samples only• Typically used for GC/MS• Deposits lots of energy into the molecule - causing extensive fragmentation

• Electron capture ionization •< 5 eV electrons get captured by the molecule• relatively gentle, but only works for some species with high electron affinity (like PCB's)

PhotonsUsing light is the next most reliable method. Some commonly used techniques are:

• Photoionization•Uses high energy photons (UV typically) to knock an electron off the molecule.• Very wavelength dependent - every molecule has particular wavelengths for ionization• Resonance Enhanced Multiphoton Ionization (REMPI)• Only used for gas phase samples• Very controllable energy deposition

• Laser Desorption/Ionization (LD/I)• Absorbs laser energy into electronic and vibrational modes of the molecule blasting it off of a surface (typically steel)• Not very controllable in energy deposition• Large suppression effects• Very small fraction of the desorbed molecules are ionized.

C60 IR-LDI

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Mass/Charge (m/z)

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Mass/Charge (m/z)

Neutral Molecules

High velocity neutral molecules can be used to generate ions.

• Fast Atom Bombarment (FAB) • Cesium ion gun (with deflector)• Used heavily in the early 80's• high energy deposition, very good for lipids

Other Ions

• Secondary Ion Mass Spectrometry (SIMS)• Cesium ion gun (with no deflector)• Not generally used for liquid/gaseous samples• Commonly used for imaging of solids• Deposits lots of energy into the molecule - causing extensive fragmentation

• Massive Cluster Bombardment• clusters (droplets) of glycerol ions• lower energy deposition• not used much, mostly in fundamentals work

Heat

• Thermal Desorption•Solid samples placed directly on a surface that is rapidly ramped up in temperature until the samples leap off the surface.•Mostly an experimental technique that got dropped with the advent of ESI/MALDI methods.

• Pyrolysis•determining species desorbed as a function of temperature.

Combined Methods

• Chemical Ionization (CI)•EI is used to ionize a background gas, and the background gas then undergoes a charge-transfer reaction to the analyte. •e.g. CH4 CH4

+· CH5+ + CH3

· A+H+ + CH4

• "somekind of" Desorption with "somekind of" postionization• for example, laser desorption with EI post ionization. • these are mostly fundamental methods used in physical chemistry experiments.

• ICP-MS, plasma desorption,

Electrospray is a soft ionization method.

Protein folding and protein complexes are accessible

Myoglobin natural theoretical isotope distribution

17,07017,06017,05017,040

100

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20

10

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Monoisotopic peak

C769 H1215 N209 O221 S4

13C = 1.1%2H=0.015%15N = 0.36%17O=0.04%18O=0.2%33S=0.76%34S=4.2%

ESI-FTMS of a tryptic digest

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8291137

15561332

2274

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Buda v1.2 C:\IONSPEC\FTDATA\PO000808.002Electrospray : aldolase 107 eap digested with trypsin

Inte

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arb

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Mass/Charge (m/z)

670 680 690 700

ESI-FTMS of a tryptic digest can give extremely high mass accuracy