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 TrapFourier Transform Ion Cyclotron Resonance
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.
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 DataDifferentiation 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
dM
FWHM 25% valley
M1 M2 Resolution is often defined 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).
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.
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.
Analyzer Resolution: Average vs. Monoisotopic Masses
Mass Spectrometer
Tandem Mass Spectrometer
Tandem MS permits selection and isolation of specific ions for subsequent analysis.
Tandem instruments have multiple mass analyzers.
Tandem Mass Spectrometry (MS/MS)
Magnetic Sector and Double Focusing Instruments
Quadrupole Mass Filters
Quadrupole Ion Traps
Fourier Transform Ion Cyclotron Resonance
Time of Flight
Mass Analyzers
Mass Analyzers: The Quadrupole Mass FilterA 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
Facile MSn
High resolution over narrow rangesExtremely SensitiveFast ScanningSmallInexpensive
Mass Analyzers: The Quadrupole Ion Trap
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
Linear TOF
Reflectron TOF
Constant Kinetic EnergyzeV = ½ mv2 v = (2zeV/m)½
Mass Analyzers: Time of Flight (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)
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. Fennelectrospray ionization for MS
Koichi Tanakasoft laser desorption ionization for MS
Kurt Wuthrichsolution NMR for protein structures
The Nobel Prize in Chemistry 2002
MALDI-TOFMS
Analyte:10 – 1000 fmol1 – 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 EnergyzeV = ½ mv2 v = (2zeV/m)½
Ion Sources: ElectrosprayVery 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
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)/nm2 = (M+n+1)/(n+1)
Electrospray Mass Spectrum of Myoglobin
Tandem Mass Spectrometry (MS/MS) is the Method of Choice for Sequence Analysis of Peptides
SpeedSensitivity
Tolerance for Amino-terminal Blocking GroupsHigh Specificity for Protein Identification
Mass Spectrometer
Tandem Mass Spectrometer
Tandem MS permits selection and isolation of specific ions for subsequent analysis.
Tandem instruments have multiple mass analyzers.
Tandem Mass Spectrometry (MS/MS)
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 isolated2. Collision-Induced-Dissociation Results in fragmentation3. “Daughter” Ions are characterized with the second mass analyzer
Tandem Mass Spectrometry: Precursor Ion Scan
Q1 Q2 Q3MASS FILTER RF ONLY MASS FILTER
PRECURSOR IONSELECTION
NEUTRAL GASCOLLISIONS
PRODUCT IONDETECTION
ION SOURCEDETECTOR
1. “Product” Ion is selected and Q3 is parked2. Q1 is scanned normally3. Only precursors which fragment to produce selected product ion are detected.
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.
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
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 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.
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z
0
100
%
Y;136.1
L86.1
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
z5644.4
z7897.5757.9
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700mass0
100
%
y''11
y''10
1285.7
Y136.1
L86.1
y''9
1156.7
a1197.1
y''7
914.6265.1
b2388.2
y''4505.3 643.3 781.4
y''8
1042.7
z101268.69
z111431.8 1672.9a11
1513.81655.8
MaxEnt-3TM
Raw data
1448.8
MaxEnt-3TM for Sequencing
Links to Information on Mass Spectrometry
Information on FTICR at the national high magnetic field labhttp://www.nhmfl.gov/science/cimar/icr/
Introduction to mass spectrometry at SciMedia.comhttp://www.rmsb.u-bordeaux2.fr/rmsb/ms/IntroMS.html
The Thermo Finnigan homepagehttp://www.thermo.com/eThermo/CDA/BU_Home/BU_Homepage/0,12482,113,00.html
The Micromass homepage, Mass Spec Back to Basics coursehttp://www.micromass.co.uk/basics/default.asp
Mass Spec Glossaryhttp://www.genomicglossaries.com/content/mass_spectrometry.asp
The I-mass homepagehttp://www.i-mass.com/
I-mass tutorialshttp://www.i-mass.com/guide/tutorial.html
American Society for Mass Spectrometry: What is Mass Spectrometryhttp://www.asms.org/whatisms/