Matrix-assisted laser desorption/ionization

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Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing, among other things, the ionization of biomolecules (biopolymers such as proteins, peptides and sugars) which tend to be more fragile and quickly lose structure when ionized by more conventional ionization methods. It is most similar in character to electrospray ionization both in relative softness and ions produced.

The ionization is triggered by a laser beam (normally a nitrogen-laser). A matrix is used to protect the biomolecule from being destroyed by direct laser beam.

1 Matrix
2 Laser
3 AP-MALDI
4 Mass spectrometer
5 History
6 Use
7 Problems
8 See also
9 Reference

[edit] Matrix

The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), $α$ -cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). A solution of one of these molecules is made, in a mixture of highly purified water and another organic compound (normally acetonitrile (ACN) or ethanol). Normally some trifluoroacetic acid (TFA) is also added. A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN:water:TFA (50:50:0.1).

The matrix-solution is then mixed with the analyte molecule (e.g. protein-sample) which you wish to investigate. The organic compound ACN allows for the hydrophobic proteins in the sample to dissolve into the solution, while the water allows for water-soluble (hydrophilic) proteins to do the same. This solution is spotted onto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with proteins spread throughout the crystals. The matrix and the analyte are said to be co-crystalized in a MALDI spot.

[edit] Laser

The laser is fired at the crystals in the MALDI spot. The spot absorbs the laser energy and it is thought that primarily the matrix is ionized by this event. The matrix is then thought to transfer part of its charge to the analyte molecules (e.g. protein), thus ionizing them while still protecting them from the disruptive energy of the laser. Ions observed after this process are quasimolecular ions that are ionized by the addition of a proton to [M+H]+, or other cation such as sodium ion [M+Na]+, or the removal of a proton [M-H]- for example. MALDI generally produces singly-charged ions, but doubly-charged ions such as [M+2H]2+ have been observed as well. Note that these are all even-electron species. Ion signals of radical cations can be observed eg. in case of matrix molecules and other stable molecules.

[edit] AP-MALDI

Atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an ionization technique (ion source) that in contrast to vacuum MALDI operates at normal atmospheric environment. The main difference between vacuum MALDI and AP-MALDI is the pressure in which the ions are created. In vacuum MALDI, ions are typically produced at 10mTorr or less while in AP-MALDI ions are formed in atmospheric (760 Torr) pressure. Disadvantage of the AP MALDI source is the limited sensitivity observed and the limited mass range.

AP-MALDI is used in mass spectrometry (MS) in a variety of applications ranging from proteomics to drug discovery fields. Popular topics that are addressed by AP-MALDI mass spectrometry include: proteomics, DNA/RNA/PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules and synthetic polymers, similar applications as available also for vacuum MALDI instruments.

The AP-MALDI ion source is easily coupled to an ion trap spectrometer or any other MS system equipped with ESI (electrospray ionization) or nanoESI source. AP-MALDI ion sources are produced by MassTech, Inc. [1] (Columbia, Maryland).

[edit] Mass spectrometer

The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range.

MALDI-TOF instruments are typically equipped with an "ion mirror", deflecting ions with an electric field, thereby doubling the ion flight path and increasing the resolution.

Commercial Reflector TOF instruments reach today a resolving power m/Δm of well above 20'000 FWHM (full-width half-maximum, Δm defined as the peak width at 50% of peak height).

[edit] History

The term matrix-assisted laser desorption ionization (MALDI) was coined in 1985 by Franz Hillenkamp, Michael Karas and their colleagues (Karas et al, 1985). These researchers found that the amino acid alanine could be ionized more easily if it was mixed with the amino acid tryptophan and irradiated with a pulsed 266 nm laser. The tryptophan was absorbing the laser energy and helping to ionize the non-absorbing alanine. Peptides up to the 2843 Da peptide melittin could be ionized when mixed with this kind of “matrix” (Karas et al, 1987). The breakthrough for large molecule laser desorption ionization came in 1987 when Koichi Tanaka of Shimadzu Corp. and his co-workers used what they called the “ultra fine metal plus liquid matrix method” that combined 30 nm cobalt particles in glycerol with a 337 nm nitrogen laser for ionization (Tanaka et. al. 1988). Using this laser and matrix combination, Tanaka was able to ionize biomolecules as large as the 34,472 Da protein carboxypeptidase-A. Tanaka received one-quarter of the 2002 Nobel Prize in Chemistry for demonstrating that, with the proper combination of laser wavelength and matrix, a protein can be ionized. Karas and Hillenkamp were subsequently able to ionize the 67 kDa protein albumin using a nicotinic acid matrix and a 266 nm laser (Karas & Hillenkamp, 1988). Further improvements were realized through the use of a 355 nm laser and the cinnamic acid derivatives ferulic acid, caffeic acid and sinapinic acid as the matrix (Beavis & Chait 1989). The availability of small and relatively inexpensive nitrogen lasers operating at 337 nm wavelength and the first commercial instruments introduced in the early 1990s brought MALDI to an increasing number of researchers (Karas & Bahr, 1990). Today, mostly organic matrices are used for MALDI mass spectrometry.

[edit] Use

In proteomics, MALDI is used for the identification of proteins isolated through gel electrophoresis: SDS-PAGE and two-dimensional gel electrophoresis. One method used is peptide mass fingerprinting by MALDI-MS, or with post ionisation decay or collision-induced dissociation (further use see mass spectrometry).

[edit] Problems

The sample preparation for MALDI is important for the result. Inorganic salts which are also part of protein extracts interfere with the ionization process. The salts are removed by solid phase extraction or washing the final target spots with water. Both methods can also remove other substances from the sample. The matrix protein mixture is not homogenous because the polarity difference leads to a separation of the two substances during crystallization. The spot diameter of the target is much larger than that of the laser, which makes it necessary to do several laser shots at different places of the target, to get the statistical average of the substance concentration within the target spot. The matrix composition, the addition of trifluoroacetic acid and formic acid, delay between laser pulses, delay time of the acceleration power, laser wavelength, energy density of the laser and the impact angle of the laser on the target are among others the critical values for the quality and reproducibility of the method.