MALDI-ToF
A basic description is provided bellow. For a comprehensive,
well written and easy to read article by John Lennon, click
the link . The article was Published by the ABRF News group
MALDI-Lennon-ABRF-document
-- Matrix-assisted laser desorption/ionization-time
of flight (MALDI TOF) mass spectrometry has become a poweful
tool for large bio-molecule analyses due to the fact that
it is relatively cheap, fast, efficient and widely used in
proteomics. MALDI TOF is the method of choice for quality
control of multiple types of sample including oligo and peptide
synthesis. The detection limit currently lies somewhere between
femtomoles (10-15 moles) and picomoles (10-12 moles). The
classic proteomics role has been protein identification by
peptide mass fingerprinting.
-- Initially a peptide/protein
sample is combined with a matrix material such as a-cyano-4-hydroxycinnamic
acid which is commonly used for peptides and some proteins.
The matrix is able to absorb laser energy and helps to provide
a "soft" ionization of the analyte (prevents total
destruction of the peptide). The mixture is crystallized (dried)
on a stainlees steel target (100-well) plate which is placed
inside the MS. A nitrogen UV laser is used to shoot any of
the 100 samples and ions are generated in the gas phase. MALDI
is well known for its tendancy to generate singly protonated
species of peptide which makes mass spectra very easy to interpret.
-- A laser shot produces packets
of ions with very similar kinetic energies. After ionization
the ions are accelerated by a very breif potential field that
introduces into a high-vacuum, field-free drift tube. The
peptide's (ions) kinetic energies are a function of only their
mass and velocity and therefore the kinetic energy of all
ions is the same. This means that lighter ions will be traveling
faster than heavier ones and beacuse the drift tube is a constant
and known length and the initial accelerating potential started
a "timer" the masses (or m/z) of the ions can be
determined.
Peptides may either be analyzed on an individual basis or
as a more complex mixture from proteolytic digestions. When
a pure protein is digested by a specific protease such as
trypsin, a characteristic family of peptides is generated
which may be used to identify the original protein. MALDI-TOF
analysis of this digest yields a spectrum containing a group
of peaks which is a representative 'fingerprint' for the original
protein. If a theoretical, in silico, trypsin digestion is
performed on a database of proteins, this database of peptide
fingerprints is then searched using the MALDI-TOF spectrum
of peptides to find the best match. No other soft MS ionisation
technique is as efficient as MALDI at generating singly charged,
intact protein ions.
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