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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.
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.
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 25% valley 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).
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
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.
Extremely High Resolution MSn capability Must Operate at very good vacuum Superconducting Magnet Difficult to operate Becoming increasingly reliable
Mass Analyzers: Time of Flight (TOF) Constant Kinetic Energy v = (2zeV/m) zeV = 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)
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
MALDI-TOFMS
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
R. G. Davis, GlaxoSmithKline
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.
Q1
MASS FILTER
Q2
RF ONLY
Q3
MASS FILTER
Q1
MASS FILTER
Q2
RF ONLY
Q3
MASS FILTER
Q1
MASS FILTER
Q2
RF ONLY
Q3
MASS FILTER
MS/MS of Angiotensin III: selection and fragmentation of the (M+H)+ molecular ion at m/z932
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
MaxEnt-3TM
y''10 1285.7
Y 136.1 a1 L 86.1 265.1 197.1 y''7 b2 388.2 400 y''4 505.3 500 914.6 643.3 600 700 781.4 800 900 1000 1100 y''8 1042.7
y''9 1156.7
z10 1268.69
100 Y;136.1
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mass 1700
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y''10 2+ 643.4
y''11 2+ 724.9
643.9
z5 644.4 z7 897.5
293.1 0
Raw data
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