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 Flight
Quadrupole Mass Filter
Quadrupole Ion Trap
Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry
Lenses and prisms focus and refract light.
Analogous systems can focus and deflect ions in a vacuum.
1.
2.
3.
4.
Get molecules into the gas phase & ionize them.
Give the ions a defined energy or velocity.
Separate or sort the ions on the basis of that defined property.
Detect the ions & assign their masses.
A very simple mass spectrum of Carbon Dioxide
Copyright ASMS: http://www.asms.org/whatisms/p5.html
Online Separations with MS Detection
Sensitivity, Specificity, Transparency of Data
Differentiation of Co-eluting analytes
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.
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 also
often shown in the corner of the spectrum as an arbitrary number unique to each
data system.
Mass Resolution
M1
M2
dM
FWHM
Resolution is often
defined as M/dM.
25% valley
“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).
Larger Peptides = More Complex Isotope Patterns
As ions grow larger, the “12C” peak is not
necessarily 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.
Analyzer Resolution: Average vs. Monoisotopic Masses
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 formula
calculated using the exact mass of the most
abundant isotope of each element,
e.g., C = 12.000000, H = 1.007825,O = 15.994915.
Tandem Mass Spectrometry (MS/MS)
Mass Spectrometer
Tandem MS permits
selection and
isolation of specific
ions for subsequent
analysis.
Tandem Mass Spectrometer
Tandem instruments
have multiple mass
analyzers.
Mass Analyzers
Magnetic Sector and Double Focusing Instruments
Quadrupole Mass Filters
Quadrupole Ion Traps
Fourier Transform Ion Cyclotron Resonance
Time of Flight
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. The
mass 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.
MS/MS in a Triple Quadrupole (Q-q-Q) Mass Spectrometer
Mass Analyzers: The Quadrupole Ion Trap
Facile MSn
High resolution over narrow ranges
Extremely Sensitive
Fast Scanning
Small
Inexpensive
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
Mass Analyzers:
Fourier Transform Ion Cyclotron Resonance
Mass Analyzers: Time of Flight (TOF)
Constant Kinetic Energy
v = (2zeV/m)½
zeV = ½ mv2
Linear TOF
Reflectron TOF
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)
The Nobel Prize in Chemistry 2002
John B. Fenn
electrospray ionization for MS
Koichi Tanaka
soft laser desorption ionization for MS
Kurt Wuthrich
solution NMR for protein structures
http://www.nobel.se/chemistry/laureates/2002/index.html
John B. Fenn – Nobel Lecture
"Electrospray Wings for Molecular Elephants"
http://www.nobel.se/chemistry/laureates/2002/fenn-lecture.html
MALDI-TOFMS
Analyte:
10 – 1000 fmol
1 – 500 kDa
MALDI-TOFMS
the three most commonly
used matrices
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
A MALDI Target with Digest Samples Spotted on
Nitrocellulose Films
R. G. Davis, GlaxoSmithKline
MALDI-TOFMS
Constant Kinetic Energy
zeV = ½ mv2
v = (2zeV/m)½
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).
Electrospray is a concentration-dependent technique.
Lower flow rates are favored significantly.
Smith et al, Acc. Chem. Res. 2004
Electrospray Mass Spectrum of Myoglobin
m1 = (M+n)/n
m2 = (M+n+1)/(n+1)
+21
+12
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.
Tandem Mass Spectrometry (MS/MS) is the Method
of Choice for Sequence Analysis of Peptides
Speed
Sensitivity
Tolerance for Amino-terminal Blocking Groups
High Specificity for Protein Identification
Tandem Mass Spectrometry (MS/MS)
Mass Spectrometer
Tandem MS permits
selection and
isolation of specific
ions for subsequent
analysis.
Tandem Mass Spectrometer
Tandem instruments
have multiple mass
analyzers.
Tandem Mass Spectrometry : Product Ion Scan
1. “Parent” Ions are selected and isolated
2. Collision-Induced-Dissociation Results in fragmentation
3. “Daughter” Ions are characterized with the second mass analyzer
Q1
MASS FILTER
PRECURSOR ION
SELECTION
ION SOURCE
Q2
RF ONLY
NEUTRAL GAS
COLLISIONS
Q3
MASS FILTER
PRODUCT ION
DETECTION
DETECTOR
Tandem Mass Spectrometry: Precursor Ion Scan
1.
2.
3.
“Product” Ion is selected and Q3 is parked
Q1 is scanned normally
Only precursors which fragment to produce selected product ion are detected.
Q1
MASS FILTER
PRECURSOR ION
SELECTION
ION SOURCE
Q2
RF ONLY
NEUTRAL GAS
COLLISIONS
Q3
MASS FILTER
PRODUCT ION
DETECTION
DETECTOR
Tandem Mass Spectrometry: Neutral Loss Scan
1.
2.
3.
4.
The mass of a functional group whose loss is to be detected is selected.
Both Q1 and Q3 are scanned simultaneously, offset by the selected “neutral loss” mass.
Collision-Induced-Dissociation Results in fragmentation
Daughter” Ions are detected only when the specified loss occurs in Q2,
indicating the presence of the moiety of interest.
Q1
MASS FILTER
PRECURSOR ION
SELECTION
ION SOURCE
Q2
RF ONLY
NEUTRAL GAS
COLLISIONS
Q3
MASS FILTER
PRODUCT ION
DETECTION
DETECTOR
MS/MS of Angiotensin III:
selection and fragmentation of the (M+H)+ molecular ion at m/z932
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
Another way to
label an MS/MS
spectrum is to
draw lines
through the
structure, with
pointers
indicating which
part of molecule
is being detected
following
fragmentation.
These markers
may be labeled
with masses.
532 669 784
400
Micromass “Back to Basics” http://www.micromass.co.uk/basics/index.html
MaxEnt-3TM for Sequencing
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200
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800
900
1000
1100
1200
1300
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1600
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500
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800
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Raw data
1000
1100
1200
1300
1400
1500
1600
m/z
1700
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/