WHAT is MASS SPECTROMETRY? - University of Minnesota · WHAT is MASS SPECTROMETRY? Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds,

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

WHAT is MASS SPECTROMETRY? Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known compounds, and to elucidate the structure and chemical properties of molecules.

• Identify structures of biomolecules, such as carbohydrates, nucleic acids and steriods

• Sequence biopolymers such as proteins and oligosaccharides • Determine how drugs are used by the body • Perform forensic analyses such as conformation and quantitation of

drugs of abuse • Analyze for environmental pollutants • Determine the age and origins of specimens in geochemistry and

archaeology • Identify and quantitate compounds of complex organic mixtures • Perform ultrasensitive multielement inorganic analyses

(Ref: http://www.asms.org/whatisms)


Goals Attainable by MS

• Small molecule MW & structural composition

• Intact protein molecular weight

• Peptide mass

• Peptide sequence protein ID

• Identification and location of post-translational modifications of amino acids

• de novo Sequencing of unknown proteins

• Relative Quantification of Proteins


History

1803 John Dalton proposes theory that each element has distinct, measurable atomic weight*

1905 J. J. Thomson produces the first mass spectrum (e/m)

(Cambridge University)*

1919 Francis Aston discovers elements have stable isotopes (Cambridge University)*

2002 John Fenn (Virginia Commonwealth) and Koichi Tanaka

(Shimadzu Corp., Japan) joint Chemistry Nobel Prize winners for ionization techniques ES and MALD

* Source: Measuring Mass: from positive rays to proteins; Grayson MS, ed. Chemical Heritage Press 2002


How does a mass spectrometer work?

• Ion Source: Makes ions • Mass Analyzer: Separates Ions • Detector: Presents information as a Mass Spectrum So for a mass spectrometer to work, the molecules must be in the gas phase and ionized so they can be mass separated


Mass Spec Principles

Ionizer

Sample

+ _

Mass Analyzer Detector

Figure: Dr. David Wishart, University of Alberta


Electron Impact (EI) and Chemical Ionization (CI) Spectra of Ephedrine

Reference: asms.org


Mass Spectrometry Data: m/z

• Mass (m): • calibrate instrument with standard compound (molecule with a known MW)

• Charge (z): • Calculate CHARGE STATE OF AN ION from peak spacing in RESOLVED ISOTOPE

SERIES/ENVELOPE • MALDI mainly produces singly charged ions - easy to interpret

SINGLY CHARGED +TOF MS: 78 MCA scans from...a=3.56297358347516750e-004...

Max. 707.0 counts.

1438 1440 1442m/z, amu

0

100

200

300

400

500

600

662

Inte

ns

ity, c

ou

nts

1438.736

1439.734

1440.737

Inte

nsi

ty, c

ps 1

DOUBLY CHARGED +TOF MS: Experiment 1, 0.16...a=3.57102922947957410e-00...

Max. 6140.0 counts.

482.0 482.5 483.0 483.5 484.0m/z, amu

0

500

1000

1500

2000

2500

3000

Inte

ns

ity, c

ou

nts

482.276

482.777

483.281

Inte

nsi

ty, c

ps

0.5


http://www.ionsource.com/Card/Mass/mass.htm


+TOF MS: Experiment 1, 46.580 min from keyxx001_perez061_fxn19_queb2_1004.wiffa=3.56649482550429800e-004, t0=4.09124284567078580e+001, Smoothed

Max. 2754.0 counts.

696 698m/z, amu

0

200

400

600

800

895

Inte

ns

ity

, c

ou

nts

696.70

696.36

Peaks in an Isotope Series have Identical Structural Formulas but Different Isotope Components

697.03

697.36

12C, 14N, 16O, etc – containing ions 13C, 15N, 18O, etc – containing ions

m/z

Inte

nsi

ty, c

ou

nts


Mass Spectrometry: Resolution Monoisotopic vs. Average m/z

Reference: http://cws.msi.umn.edu/mascot/help/mass_accuracy_help.html

“Mass resolution is the dimensionless ratio of the mass of the peak divided by its width. Usually, the peak width is taken as the full width at half maximum intensity, (FWHM).”

Average peak Monoisotopic peak


The Resolving Power of a Mass Spectrometer Dictates the Accuracy of the m/z Values Produced

m/z Value tolerance accuracy 1000 1 1000 ppm 1000.3 0.1 100 ppm 1000.345 0.001 1 ppm

+TOF MS: 38 MCA scans from russeth0611a_5S_fs.wiffa=3.56274449646042090e-004, t0=4.13301967471197710e+001, Smoothed

Max. 996.0 counts.

992 994 996 998 1000 1002m/z, amu

0

20

40

60

80

100

120

140

155

In

te

ns

ity

, c

ou

nts

995.568

996.566

997.555A

bso

lute

In

tensity

RESOLUTION = 8000


Accuracy in ppm (parts per million): Accuracy in Percent ∆ = experimental/observed value (mass spec data) – theoretical value

300 ppm is equivalent to 0.03 %

Mass Accuracy Calculations

x 100 = % error ∆ theoretical value

x 106 = ppm error ∆ theoretical value


FT-ICR MS (Fourier Transform-Ion Cyclotron Resonance)

m/z 648 647 646 645

m/z 648 647 646 645

m/z 648 647 646 645

645.793

646.791

Absolu

te Inte

nsity

Quadrupole-TOF MS (Time of Flight)

Comparison of Resolving Power: 645.7 m/z

+TOF MS: Experiment 1, 46.580 min from keyxx001_perez061_fxn19_queb2_1004.wiffa=3.56649482550429800e-004, t0=4.09124284567078580e+001, Smoothed

Max. 2754.0 counts.

645.0 646.0 647.0 648.0m/z, amu

0

50

100

150

200

247

Inte

nsity

, cou

nts

645.70 646.04

646.37

646.70


MALDI-TOF: Peptide Mass Fingerprint In-Gel Tryptic Digest: GOAL = Protein Identification

800 1000 1200 1400 1600 1800 2000

m/z

200

400

600

800

1000

1200

1400

1600

Absolute Intensity


ESI-Ion Trap MS: Product Ion Spectrum

MS/MS Spectrum from [M + 2H+2], Precursor m/z = 467.8 (MW = 933)

100 200 300 400 500 600 700 800 9000

25

50

75

100x50

157

185

253

298

369

376

438

497

566

637

701

750

789864

m/z

Rela

tive A

bundance


MALDI-TOF MS of Intact Protein: Unoxidized Calmodulin


MALDI-TOF MS of Intact Protein: Mixture of Calmodulin with Multiply Oxidized Methionines

16600 16800 17000 m/ z

0

50

100

150

200

a. i .

/ i =/ mar01/ cl 0306a6/ 1SLi n/ pdat a/ 1 Admi ni st rat or Sun Mar 11 23: 46: 53 2001

16817

6 ox-Met

16832, 7 ox-Met

16849, 8 ox-Met

16800

E. Balog, Univ MN, BMBB

5 ox-Met


Intact Protein ESI-MS: Cytochrome C

200 1000 2000 m/z

+7

1766.6

+8 1545.7

+9 1374.2

+10

1236.9

+11

1124.6

+12

1031.0

+13 951.8

+14 884.0

+15 825.0

+16 772.4

+17 727.5

0

50

100

Rel

ativ

e A

bu

nd

ance

1) Raw MS data

10000

mass 12500 15000

0

50

100

Rel

ativ

e A

bu

nd

ance

12359 +/- 2

Mathematical

deconvolution

2) Protein MW

(after mathematical deconvolution)


Mass Spectrometry Principles

Ionizer

Sample

+

_

Mass Analyzer Detector

Figure: Dr. David Wishart, University of Alberta


IONIZATION

• Mass spectrometers can detect the presence of IONS (+ or –)

• Ions must be in the GAS PHASE

• Ions must be free from salts, solvents...

• Ionization from SOLID CRYSTALS

• Destroys non-covalent interactions

• MALDI (Matrix Assisted Laser Desorption Ionization); typically produces singly charged ions

• Ionization from LIQUID STATE:

• ESI (Electrospray Ionization); typically produces multiply charged ions

• Non-covalent interactions are retained

• Compatible with in-line HPLC

• solvents must be volatile- e.g. ammonium acetate, ACN, methanol

• Not all ionization modes are applicable to a given molecule


Electron Impact Ionization

Odd-electron ions (radical cations) are formed during Electron Impact


Ionization Techniques for MS

• Electron Impact (EI)

• Chemical Ionization (CI)

• Inductively Coupled Plasma (ICP)

• Field Desorption (FD)

• Fast Atom Bombardment (FAB)

• Thermospray

• Desorption Electrospray Ionization (DESI)

• Direct Analysis in Real Time (DART)

• Atmospheric Pressure Chemical Ionization (APCI)

• Secondary Ion Mass Spectrometry (SIMS)

• Matrix-assisted Laser Desorption (MALDI)

• Electrospray (ESI)


Technology Breakthroughs (1990’s) in Mass Spectrometry Accelerated Proteomics Studies

Ionization Methods suitable for Peptides/Proteins (i.e. methods for producing gas phase ions):

• Electrospray Ionization: John Fenn (Virginia

Commonwealth) 2002 Nobel prize Chemistry

• MALDI (matrix-assisted laser desorption

ionization): Koichi Tanaka (Shimadzu Corp) 2002 Nobel prize

Chemistry


Soft Ionization Methods

• Unlimited analyte mass

• Insensitive to moderate salts

• Excellent for mixtures

• Off-line sample prep

• Unlimited analyte mass

• Some sample cleanup

• Mixtures problematic

• On-line sample injection

337 nm UV laser

MALDI

analyte + matrix needle

Liquid Analyte

ESI

+ _

Adapted from Dr. David Wishart, University of Alberta




DESI Desorption Electrospray Ionization

Reference: Prosilia Omni Spray™ Ion Source manual


Instrumentation Multiple types of Mass Analyzers are used for a

variety of purposes


Target: Analyte Ions + “matrix” crystals

Apply Accelerating Potential (19kV)

N2 Laser

337 nm

vacuum: 10-7 Torr

Ion sensitive

Detector

+ +

fast

Time of Flight tube

ion source

+ slow

MALDI with Time-of-Flight (TOF) MS

velocity of the ion in the flight tube depends on the mass-to-charge ratio


Image source: http://www.anagnostec.de/index/modul/portal/kernwert/technology/


D.C. Muddiman Chemistry 727

The Time an Ion Spends in Flight Summary: Time of flight of the ion varies with the square root of its mass-to-charge ratio

m/z (z=1) 0 10,000

Tim

e (

ms)

2 ex

mt x

zeU

2

m tRP

m t


Reference: http://www.chm.bris.ac.uk/ms/theory/quad-massspec.html

QUADRUPOLE Mass Filter


Quadrupole Mass Analyzer • Uses a combination of RF and DC

voltages to operate as a mass filter • Has four parallel metal rods with

alternating positive and negative voltages

• Can scan through all masses or sit at one fixed mass


Figures from: http://www.abrf.org/ABRFNews/1996/September1996/sep96iontrap.html

Comparison of Linear Quadrupole to

3-D Quadrupole (Ion Trap)


3D Ion Trap The ion trap is an energy well. Ions with sufficient energy to enter the trap are retained by an energy barrier on the exit side of the trap. The advantage of the ion trap is that it accumulates selected ions prior to their analysis giving it high initial sensitivity. Ions can be fragmented by collision with helium gas and their product ions analyzed within the trap. Selected product ions can undergo further fragmentation, thus allowing MSn. The ion trap has a high efficiency of transfer of fragment ions to the next stage of fragmentation (unlike the triple quadrupole instrument). The 3D ion trap consists of two end cap electrodes and a center ring electrode


Figure from: http://www.matrixscience.com/help/ion_trap_main_help.html

ION TRAP MASS ANALYZER SCHEMATIC


Reference: Chernushevich IV et al. J. Mass Spectrom. 2001; 36:

HYBRID QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETER


LTQ Orbitrap Velos Schematic

Page 1-3 LTQ Orbitrap Velos Hardware Manual, Thermo Fisher

Scientific

multipole linear ion trap

http://en.wikipedia.org/wiki/Orbitrap

WHAT is MASS SPECTROMETRY? - University of Minnesota · WHAT is MASS SPECTROMETRY? Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds,

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