WHAT is MASS SPECTROMETRY?...(Shimadzu Corp., Japan) joint Chemistry Nobel Prize winners for...
Transcript of WHAT is MASS SPECTROMETRY?...(Shimadzu Corp., Japan) joint Chemistry Nobel Prize winners for...
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)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Mass Spec Principles
Ionizer
Sample
+ _
Mass Analyzer Detector
Figure: Dr. David Wishart, University of Alberta
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Electron Impact (EI) and Chemical Ionization (CI) Spectra of Ephedrine
Reference: asms.org
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
http://www.ionsource.com/Card/Mass/mass.htm
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
+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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
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MALDI-TOF MS of Intact Protein: Unoxidized Calmodulin
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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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Mass Spectrometry Principles
Ionizer
Sample
+
_
Mass Analyzer Detector
Figure: Dr. David Wishart, University of Alberta
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Electron Impact Ionization
Odd-electron ions (radical cations) are formed during Electron Impact
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
DESI Desorption Electrospray Ionization
Reference: Prosilia Omni Spray™ Ion Source manual
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Instrumentation Multiple types of Mass Analyzers are used for a
variety of purposes
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Image source: http://www.anagnostec.de/index/modul/portal/kernwert/technology/
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Reference: http://www.chm.bris.ac.uk/ms/theory/quad-massspec.html
QUADRUPOLE Mass Filter
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Figures from: http://www.abrf.org/ABRFNews/1996/September1996/sep96iontrap.html
Comparison of Linear Quadrupole to
3-D Quadrupole (Ion Trap)
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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
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Figure from: http://www.matrixscience.com/help/ion_trap_main_help.html
ION TRAP MASS ANALYZER SCHEMATIC
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Reference: Chernushevich IV et al. J. Mass Spectrom. 2001; 36:
HYBRID QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETER
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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