fundamentals of mass spec
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8/6/2019 Fundamentals of Mass Spec
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Ion Sources and Mass Analyzers
in Protein Characterization
Principles of MS and MS/MS
Matrix Assisted Laser Desorption Ionization (MALDI)
Electrospray Ionization (ESI), Nano-ESI
Time of FlightQuadrupole Mass Filter
Quadrupole Ion Trap
Fourier Transform Ion Cyclotron Resonance
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Mass Spectrometry
Lenses and prisms focus and refract light.
Analogous systems can focus and deflect ions in a vacuum.
1. Get molecules into the gas phase & ionize them.
2. Give the ions a defined energy or velocity.
3. Separate or sort the ions on the basis of that defined property.4. Detect the ions & assign their masses.
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Online Separations with MS Detection
Sensitivity, Specificity, Transparency of Data
Differentiation of Co-eluting analytes
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Looking at MS Data: LC/MS Data is Three Dimensional
Mass spec data systems generate “total ion chromatograms” by integrating spectra
and plotting intensity versus time. It is analogous to that generated using a diode
array UV-detector on an HPLC system. The data is fundamentally 3-dimensional.
A “selected ion chromatogram” is the same graph of intensity over time for a
defined m/z. It is analogous to a UV chromatogram for a single wavelength.
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Looking at MS Data: Mass spectra show m/z, not mass
Mass spectrometers separate molecules on the basis of their mass to charge ratio, not
their mass. That means the x-axis is not necessarily reflective of M.
Mass spectra are normalized to the abundance (intensity) of the highest peak in a
given spectrum. The y-axis is always scaled from 0-100. Absolute intensity is alsooften shown in the corner of the spectrum as an arbitrary number unique to each
data system.
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Mass Resolution
dM
FWHM 25% valley
M1 M2Resolution is oftendefined as M/dM.
“Unit resolution”means that two adjacent
peaks are resolved from
one another.
In low resolution, dM may be 1 mass unit.
In high resolution, dM may be 0.010 mass unit.
However, the actual resolution depends on how one defines the
separation between the peaks (e.g. 50% vs 10% valley).
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Larger Peptides = More Complex Isotope Patterns
As ions grow larger, the “12C” peak is notnecessarily most abundant.
The mass resolution of analyzers may not
always be adequate to distinguish individual
peaks. In this case, average masses are used.
It is important to be aware of the capabilities
of the mass analyzer one is using.
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Average mass:The mass of an ion for a given empirical
formula calculated using the relative average
atomic mass of each element,
e.g. C = 12.01115, H = 1.00797, O = 15.9994.
Monoisotopic mass:
The mass of an ion for a given empirical formulacalculated using the exact mass of the most
abundant isotope of each element,
e.g., C = 12.000000, H = 1.007825,O = 15.994915.
Analyzer Resolution: Average vs. Monoisotopic Masses
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Mass Spectrometer
Tandem Mass Spectrometer
Tandem MS permits
selection and
isolation of specificions for subsequent
analysis.
Tandem instruments
have multiple mass
analyzers.
Tandem Mass Spectrometry (MS/MS)
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Magnetic Sector and Double Focusing Instruments
Quadrupole Mass Filters
Quadrupole Ion Traps
Fourier Transform Ion Cyclotron Resonance
Time of Flight
Mass Analyzers
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Mass Analyzers: The Quadrupole Mass Filter
A potential of ~100-1000 V is applied alternately to the opposing pairs of rods at a frequency of a few MHz. At a
specific combination of DC & RF, an m/z has a stable trajectory through the rods, and all other m/z are lost. Themass range is scanned as the voltages are swept from min to max, but at constant DC/RF ratio.
Faster Scanning than sector instruments (but not as fast as ion traps or TOF).
Mass Range generally m/z 0-2000 or 0-4000.
Facile MS/MS using Triple Quadrupole (Q-q-Q) analyzer.Exquisitely sensitive in selected ion monitoring (both analyzers parked at one m/z).
Largely replaced by the ion trap and hybrid Q-q-TOF for biopolymer analysis.
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MS/MS in a Triple Quadrupole (Q-q-Q) Mass Spectrometer
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Facile MSn
High resolution over narrow ranges
Extremely Sensitive
Fast ScanningSmall
Inexpensive
Mass Analyzers: The Quadrupole Ion Trap
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Mass Analyzers:
Fourier Transform Ion Cyclotron Resonance
Ions in a magnetic field move in circular
orbits characteristic of their m/z values. If
energy is provided at a frequency equal to
their precession frequency, and in a
direction perpendicular to their plane of
precession, the ions will absorb the energy,
enabling them to be detected.
Extremely High Resolution
MSn capability
Must Operate at very good
vacuum
Superconducting Magnet
Difficult to operate
Becoming increasingly reliable
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Mass Analyzers:
Fourier Transform Ion Cyclotron Resonance
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Linear TOF
Reflectron TOF
Constant Kinetic EnergyzeV = ½ mv2 v = (2zeV/m)½
Mass Analyzers: Time of Flight (TOF)
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Ion Sources
Gas Phase Ionization:Electron Impact (EI)
Chemical Ionization (CI)
Desorption Ionization:252Cf Plasma Desorption (PDMS)
Fast Atom Bombardment (FAB) / Secondary Ion MS (SIMS)
Laser Desorption (LDMS)Matrix Assisted Laser Desorption (MALDI)
Spray Ionization:Thermospray (TSP)
Atmospheric Pressure Chemical Ionization (APCI)
Electrospray (atmospheric pressure ionization) (ESI, API)
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http://www.nobel.se/chemistry/laureates/2002/index.htmlJohn B. Fenn – Nobel Lecture
"Electrospray Wings for Molecular Elephants"
http://www.nobel.se/chemistry/laureates/2002/fenn-lecture.html
John B. Fenn
electrospray ionization for MS
Koichi Tanaka
soft laser desorption ionization for MS
Kurt Wuthrich
solution NMR for protein structures
The Nobel Prize in Chemistry 2002
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MALDI-TOFMS
Analyte:
10 – 1000 fmol1 – 500 kDa
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MALDI-TOFMS
the three most commonly
used matrices
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Some Characteristics of MALDI-TOFMS
Ions are easy to generate
Buffers, salts, some detergents easily tolerated
Excellent sensitivity (< 20 fmol for digests)
High resolution at low mass with time lag focusing
Resolution drops off at higher mass (>20 kDa)
Protein or peptide mixtures can show suppression effects
Different matrices yield different results
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A MALDI Target with Digest Samples Spotted on
Nitrocellulose Films
R. G. Davis, GlaxoSmithKline
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MALDI-TOFMS
Constant Kinetic EnergyzeV = ½ mv2 v = (2zeV/m)½
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Ion Sources: Electrospray
Very gentle and efficient way of getting gas phase ions from solutions.
A fine spray of charged droplets is generated in an electric field.
Droplets evaporate - analyte molecules are left carrying charges.
Multiply Charged Ions are the rule.
Concentration dependent – High sensitivity at very low flow rates (<< 1 ul/min).
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Electrospray is a concentration-dependent technique.
Lower flow rates are favored significantly.
Smith et al, Acc. Chem. Res. 2004
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Quasimolecular ions, [M+nH], from myoglobin, Mr= 16,951.5 Da.
Using adjacent pairs of ions, the molecular mass of the myoglobin can be calculated
very accurately.
+21+12
m1 = (M+n)/n
m2 = (M+n+1)/(n+1)
Electrospray Mass Spectrum of Myoglobin
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Mass Spectrometer
Tandem Mass Spectrometer
Tandem MS permits
selection and
isolation of specificions for subsequent
analysis.
Tandem instruments
have multiple mass
analyzers.
Tandem Mass Spectrometry (MS/MS)
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Tandem Mass Spectrometry : Product Ion Scan
Q1 Q2 Q3MASS FILTER RF ONLY MASS FILTER
PRECURSOR IONSELECTION
NEUTRAL GASCOLLISIONS
PRODUCT IONDETECTION
ION SOURCEDETECTOR
1. “Parent” Ions are selected and isolated
2. Collision-Induced-Dissociation Results in fragmentation3. “Daughter” Ions are characterized with the second mass analyzer
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T d M S t t N t l L S
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Tandem Mass Spectrometry: Neutral Loss Scan
Q1 Q2 Q3MASS FILTER RF ONLY MASS FILTER
PRECURSOR IONSELECTION
NEUTRAL GASCOLLISIONS
PRODUCT IONDETECTION
ION SOURCEDETECTOR
1. The mass of a functional group whose loss is to be detected is selected.
2. Both Q1 and Q3 are scanned simultaneously, offset by the selected “neutral loss” mass.
3. Collision-Induced-Dissociation Results in fragmentation4. Daughter” Ions are detected only when the specified loss occurs in Q2,
indicating the presence of the moiety of interest.
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MS/MS f A i i III
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Micromass “Back to Basics” http://www.micromass.co.uk/basics/index.html
MS/MS of Angiotensin III:
selection and fragmentation of the (M+H)+ molecular ion at m/z932
532 669 784
400
Another way tolabel an MS/MS
spectrum is to
draw lines
through the
structure, withpointers
indicating which
part of molecule
is being detected
followingfragmentation.
These markers
may be labeled
with masses.
MaxEnt 3TM for Sequencing
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100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
m/z0
100
%
Y;136.1
L
86.1
y''10 2+
643.4
y''9 2+
578.8a1
197.1
558.3
y''7 2+
457.8293.1
y''11 2+
724.9
643.9
z5
644.4
z7
897.5757.9
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
mass0
100
%
y''11
y''10
1285.7
Y
136.1
L
86.1
y''9
1156.7
a1
197.1y''7
914.6265.1
b2
388.2y''4
505.3 643.3 781.4
y''8
1042.7
z10
1268.69
z11
1431.81672.9a11
1513.8
1655.8
MaxEnt-3TM
Raw data
1448.8
MaxEnt-3TM for Sequencing
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Links to Information on Mass Spectrometry
Information on FTICR at the national high magnetic field lab
http://www.nhmfl.gov/science/cimar/icr/
Introduction to mass spectrometry at SciMedia.com
http://www.rmsb.u-bordeaux2.fr/rmsb/ms/IntroMS.html
The Thermo Finnigan homepage
http://www.thermo.com/eThermo/CDA/BU_Home/BU_Homepage/0,12482,113,00.html
The Micromass homepage, Mass Spec Back to Basics course
http://www.micromass.co.uk/basics/default.asp
Mass Spec Glossary
http://www.genomicglossaries.com/content/mass_spectrometry.asp
The I-mass homepage
http://www.i-mass.com/
I-mass tutorials
http://www.i-mass.com/guide/tutorial.html
American Society for Mass Spectrometry: What is Mass Spectrometry
http://www.asms.org/whatisms/
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