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
Sample
+
_
Ionizer
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
+TOF MS: Experiment 1, 0.16...
DOUBLY
a=3.57102922947957410e-00...
SINGLY CHARGED
Max. 707.0 counts.
+TOF MS: 78 MCA scans from...
a=3.56297358347516750e-004...
1
500
400
300
1440.737
200
100
0
482.276
1439.734
1438
1440
m/z, amu
1442
2500
2000
482.777
1500
Intensity, cps
In te n s ity , c o u n ts
Intensity, cps
3000
1438.736
In te n s ity , c o u n ts
662
600
Max. 6140.0 counts.
CHARGED
1000
0.5
500
0
482.0
483.281
482.5
483.0
m/z, amu
483.5
484.0
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
Peaks in an Isotope Series have Identical Structural
Formulas but Different Isotope Components
895
800
Intensity, counts
In te n s ity , c o u n ts
+TOF MS: Experiment 1, 46.580 min from keyxx001_perez061_fxn19_queb2_1004.wiff
a=3.56649482550429800e-004, t0=4.09124284567078580e+001, Smoothed
Max. 2754.0 counts.
696.70
696.36
600
697.03
400
200
697.36
0
12C, 14N, 16O,
etc – containing ions
696
698 m/z
13C, 15N, 18O, etc – containing ions
m/z, amu
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Mass Spectrometry: Resolution
Monoisotopic vs. Average m/z
“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
Reference: http://cws.msi.umn.edu/mascot/help/mass_accuracy_help.html
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
1000
1000.3
tolerance
1
accuracy
1000 ppm
0.1
100 ppm
+TOF MS: 38 MCA scans from russeth0611a_5S_fs.wiff
a=3.56274449646042090e-004, t0=4.13301967471197710e+001, Smoothed
RESOLUTION = 8000
1 ppm
995.568
155
I n t e n s it y , c o u n t s
0.001
Absolute Intensity
1000.345
Max. 996.0 counts.
140
120
996.566
100
80
60
997.555
40
20
0
992
994
996
998
m/z, amu
1000
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
1002
Mass Accuracy Calculations
Accuracy in ppm (parts per million):
∆
theoretical value
x 106 = ppm error
Accuracy in Percent
∆
theoretical value
x 100 = % error
∆ = experimental/observed value (mass spec data) – theoretical value
300 ppm is equivalent to 0.03 %
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Comparison of Resolving Power: 645.7 m/z
(Fourier Transform-Ion Cyclotron Resonance)
Absolute Intensity
FT-ICR MS
645.793
646.791
645
646
m/z
647
648
+TOF MS: Experiment 1, 46.580 min from keyxx001_perez061_fxn19_queb2_1004.wiff
a=3.56649482550429800e-004, t0=4.09124284567078580e+001, Smoothed
Quadrupole-TOF MS
(Time of Flight)
In te n sity, co u n ts
247
645.70
Max. 2754.0 counts.
646.04
200
150
646.37
100
50
0
645.0
646.70
646.0
647.0
m/z, amu
648.0
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
1600
Absolute Intensity
1400
1200
1000
800
600
400
200
800
1000
1200
1400
1600
1800
2000
m/z
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)
750
Relative Abundance
100
637
75
50
x50
185
566
376
789
438
25
864
298
157
369
253
497
0
100
200
300
400
500
701
600
700
800
900
m/z
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
MALDI-TOF MS of Intact Protein:
Unoxidized Calmodulin
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
MALDI-TOF MS of Intact Protein:
Mixture of Calmodulin with Multiply Oxidized Methionines
a. i .
16832, 7 ox-Met
16817
6 ox-Met
200
16849, 8 ox-Met
150
100
16800
5 ox-Met
E. Balog, Univ MN, BMBB
50
0
16600
16800
/ i =/ m
ar 01/ cl 0306a6/ 1SLi n/ pdat a/ 1
17000
Adm
i ni st r at or
m
/z
Sun M
ar 11 23: 46: 53 2001
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Intact Protein ESI-MS: Cytochrome C
+12
1031.0
+13
951.8
+14
884.0
50
1) Raw MS data
+11
1124.6
+8
1545.7
+10
1236.9
+9
1374.2
+15
825.0
+17
727.5
0
200
Mathematical
deconvolution
2) Protein MW
(after mathematical deconvolution)
+7
1766.6
+16
772.4
1000
m/z
2000
100
Relative Abundance
Relative Abundance
100
12359 +/- 2
50
0
10000
12500
mass
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
15000
Mass Spectrometry Principles
Sample
+
_
Ionizer
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
337 nm UV laser
Liquid Analyte
+
_
analyte + matrix
needle
MALDI
ESI
• 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
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
MALDI with
Time-of-Flight (TOF) MS
velocity of the ion in the flight tube depends on the mass-to-charge ratio
N2 Laser
337 nm
Time of Flight tube
Ion sensitive
Detector
ion source
fast
slow
+
+
+
vacuum: 10-7 Torr
Target: Analyte Ions + “matrix” crystals
Apply Accelerating Potential (19kV)
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
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
Time (ms)
t
m
x
2 zeU ex
RP
0
m/z (z=1)
m
m
t
2 t
10,000
D.C. Muddiman Chemistry 727
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
QUADRUPOLE Mass Filter
Reference: http://www.chm.bris.ac.uk/ms/theory/quad-massspec.html
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
Comparison of Linear Quadrupole to
3-D Quadrupole (Ion Trap)
Figures from: http://www.abrf.org/ABRFNews/1996/September1996/sep96iontrap.html
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
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
ION TRAP MASS ANALYZER SCHEMATIC
Figure from: http://www.matrixscience.com/help/ion_trap_main_help.html
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
HYBRID QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETER
Reference: Chernushevich IV et al. J. Mass Spectrom. 2001; 36:
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
LTQ Orbitrap Velos Schematic
linear ion
trap
multipole
Page 1-3 LTQ Orbitrap Velos Hardware Manual, Thermo Fisher
Scientific
http://en.wikipedia.org/wiki/Orbitrap
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279