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Matrix assisted laser
desorption ionization mass spectrometry is carried out on an AppliedBiosystems Voyager DE-STR instrument equipped
with delayed extraction and reflector. A total sample volume of up to 2
µl is loaded onto MALDI sample plate in volatile solvents. In
reflector mode, masses of peptides (low molecular weight, 750-4,500) can
be determined on low fmol quantities with an
average mass accuracy lower than 10 ppm. Under
optimum conditions, the limit of sensitivity of tryptic
peptides (below 4,000) is in the high amol
range. Masses can potentially be obtained on numerous biopolymers
including oligosaccharides, nucleotides and proteins that range from ~600
to 750,000 Daltons.
Some MALDI-TOF theory
This MS
approach uses a nitrogen UV laser (337 nm) to generate ions from high
mass, non-volatile samples such as peptides and proteins. The key to this
technique is that in the presence of an aromatic matrix large molecules
like peptides ionize instead of decomposing. Although the mechanism
remains uncertain, it may involve absorption of UV light by the matrix
followed by transfer of this energy to the peptide - which then ionizes
into the gas phase as a result of the relatively large amount of energy
absorbed. To accelerate the resulting ions into a flight-tube in the mass
spectrometer they are subjected to a high electrical field.
Three different models have been proposed to explain desorption of the
matrix-sample material from the crystal surface: (1) quasithermal
evaporation as a result of increased molecular motion, (2) expulsion of
upper lattice layers, and (3) an increase in the hydrodynamic pressure
due to the rapidly expanding molecules in the crystal lattice. However,
there is no consensus yet as to how the sample molecules are ionized. The
widely accepted view is that, following there desorption as neutrals, the
sample molecules are ionized by acid-base proton transfer reactions with
the protonated matrix ions in a dense phase just above the surface of the
matrix. The protonated matrix molecules are generated by a series of
photochemical reactions.
The matrix performs two important functions: (1) it absorbs photon energy
from the laser beam and transfers it into excitation energy of the solid
system, and (2) it serves as a solvent for the analyte, so that the
intermolecular forces are reduced and aggregation of the analyte
molecules is held to a minimum. Some desirable characteristics of a
typical MALDI matrix are:
- A strong light absorption property at the
wavelength of the laser flux.
- The ability to form micro-crystals with the
sample.
- A low sublimation temperature, which
facilitates the formation of an instantaneous high-pressure plume of
matrix-sample material during the laser pulse duration.
- The participation in some kind of a
photochemical reaction so that the sample molecules can be ionized
with high yields.
Several matrix-laser combinations have been tested successfully. For
peptides and small molecular mass proteins (<10,000 Da), good results are obtained with
a-cyno-4-hydroxycinnamic acid (CHCA), whereas high-mass proteins are
analyzed with sinapinic acid. The use of
3-amino-4 hydroxybenzoic acid and 2,5-dihydroxybenzoic acid (DHB) has been recommended
for the analysis of oligosaccharides.
Our MALDI instrument can be used in either linear or reflector mode. In
linear mode the ions travel down a linear flight path and their
mass/charge (m/z) ratio (see below for an explanation of the difference
between mass and mass/charge ratio) is determined by the time it takes
for them to reach the detector. Hence, this instrument is called a time
of flight (TOF) instrument. The relationship that allows the m/z ratio to
be determined is E = ½ (m/z)v2.
In this equation, E is the energy imparted on the charged ions as a
result of the voltage that is applied by the instrument and v is the
velocity of the ions down the flight path. Because all of the ions are
exposed to the same electric field, all similarly charged ions will have
similar energies. Therefore, based on the above equation, ions that have
larger mass must have lower velocities and hence will require longer
times to reach the detector, thus forming the basis for m/z determination
by a mass spectrometer equipped with a time of flight detector.
A reflector MALDI has an ion mirror at its end which reflects the ions
back (at a slight angle) to a detector. Reflector mode have several major
advantages: (1) it permits limited mass spectrometric sequencing to be
carried out via a process called post source decay (PSD); (2) it permits
higher mass accuracy.
During high voltage extraction of the peptide ions produced by exposure
to UV light, there are slight differences in the amount of energy that is
actually acquired by similarly charged ions. In a linear instrument these
differences result in slight differences in times of flight which results
in broader peaks and lower mass accuracy. In terms of resolving fragment
ions, a reflector also compensates for similarly charged ions having
slightly different overall energies (the more energetic ions that have
slightly faster velocities will penetrate further into the ion mirror and
hence be slightly delayed relative to less energetic ions - thus both
will tend to reach the detector at the same time). As a result, the
reflector improves both resolution and mass accuracy. Although there is
always the possibility of observing fragmentation ions when using the
reflector (and mistaking these for contaminating peptide ions), by
adjusting the settings on the instrument it is possible to minimize the
possibility of seeing peptide fragmentation in the reflector mode.
Relevant
literature
- Chapman, J. R., Mass Spectrometry of
Proteins and Peptides, 2001,Humana Press
- Dass, C.,
Principles and practice of biological mass spectrometry, 2001, John
Wiley & Sons
- James, P., Proteome research: mass
spectrometry, 2001, Springer
- Kellner, R., F.
Lottspeich, and H. E. Meyer, Microcharacterization of Proteins, 2nd Ed, 1999,
Wiley-VCH.
- Kinter, M.,
and N. E. Sherman, Protein Sequencing and Identification Using
Tandem Mass Spectrometry, 2000, Wiley Interscience
- Siuzdak, G.,
Mass Spectrometry for Biotechnology, 1996, Academic Press
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