Laser Desorption-ionization assisted by matrix

The laser desorption-ionization assisted by matrix (in English Laser Matrix Assisted Desorption Ionization or MALDI) is a technique of soft Ionization used in Spectrometry mass, allowing the analysis of Biomolécule S (of the bio Polymère S like the Protéine S, the Peptide S and the sugars) and the grosses molecules organic (like the Polymère S, the Dendrimère S and others Macromolécule S) which tend to becoming fragile and to split up when they are ionized by more conventional methods. It is relatively similar to the ionization by électrospray carefully relative and in produced ions (although it creates less multichargés ions).

Ionization is caused by a laser beam (normally a Laser with the nitrogen). A matrix is used in order to protect the biomolécule from the destruction by a direct beam and to facilitate vaporization and ionization.

Stamp

The matrix consists of molecules Cristal smoothed, which are in general selected among the three following ones: the 3,5-dimethoxy-4-hydroxycinnamic Acid (Acid sinapinic), the α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) and the 2,5-dihydroxybenzoic Acid (DHB). A Solution of a type of its molecules is made, sometimes in a mixture of ultrapure Eau and organic Solvant (usually of the Acétonitrile (ACN) or ethanol). The trifluoroacetic Acide (TFA) can sometimes be added. An good example of solution-matrix is, for example, a mixture of 20 mg/ml of acid sinapinnic in the ACN: water: TFA (50: 50: 0,1).

The nature of the components for a reliable matrix is determined by groping with tests and errors, but it is based on specific considerations of molecular profiles . Its components:

is of low molecular mass (in order to facilitate vaporization), but rather large (with a rather large Steam pressure) in order not to evaporate during the preparation of the sample or during its introduction into the spectrometer.
is acid, or at least acting like source of proton (S) in order to support the ionization of the analyte.
has a strong absorption in the Ultraviolet, enabling them to absorb effectively and quickly the laser Irradiation.
is functionalized with polar groups, for a use in aqueous solutions.

The solution matrix is mixed with the analyte (the sample). The organic solvent allows the hydrophobic molecules to dissolve in the solution, whereas water allows the absorbent molecules to make in the same way. The solution is then deposited on a cup MALDI (usually out of metal conceived for this use). The solvent vaporizes, leaving only the recristallized matrix, but with now of the molecules of analyte distributed in all the crystal. The matrix and the analyte are known as then Co-crystallized in a spot MALDI.

Laser

The Laser is directed on the crystals of spot MALDI. The spot absorbs laser energy and it seems that the matrix is ionized in first by this fact, then would transfer part of its load to the molecules to be studied, ionizing them all while protecting them from a disruptive energy of the laser. The ions observed after this stage quasi-molecular, are ionized by the addition of a Proton in ⁺, or of another Cation like the ion Sodium ⁺, or by the subtraction of a proton ^- for example. Produced technique MALDI in a general way of the monochargés ions, but of the multichargés ions (ⁿ⁺) can also be observed, according to the matrix used and/or the intensity of the laser. These species have a whole an even number of electron S. Of the signals corresponding to ridicalizing cations can also be observed, for example in the case of the molecules of the matrix or other molecules stable.

MALDI with atmospheric pressure

The AP-MALDI ( Atmospheric presses matrix-assisted laser desorption/ionization ) is a technique of ionization (source of ions) which contrary to the vacuum MALDI operates in an environment with atmospheric pressure. The principal difference between the vacuum MALDI and AP MALDI is the pressure under which the ions are created. In the vacuum MALDI, the ions are produced under 10 m Torr or less then aue ions AP MALDI are formed under Atmospheric pressure. The disadvantage of source AP MALDI is the observation of a sensitivity limited like its interval of mass restreint.
Technique AP MALDI ets used in varying mass spectrometry for applications of the fields proteomic to discovered new drugs. Subjects more popular which addresses itself the technique are: the Protéomique, DNA/ARN/ANP, Lipid S, Oligosaccharide S, Phosphopeptide S, Bacterium S, small molecules and Polymeric S synthetic. Similar applications are available for the vacuum technique. Source AP MALDI can be easily coupled to a trap with ions of mass spectrometer or any other system of mass spectrometry equipped with a source ESI (ionization by électrospray) or nanoESI.

Mass spectrometry (MALDI-TOF)

History

The English expression of matrix-assisted laser desorption ionization (MALDI) was forged in 1985 by Franz Hillenkamp, Michael Karas and their colleagues. These researchers discovered that the Alanine, an Amino-acid , could be ionized more easily if it were mixed with the Tryptophane (other amino-acid) and were irradiated with a pulsated laser with 266 Nm. Tryptophan absorbed laser energy and helped to ionize alanine not absorbing. Peptides to peptide 2843 Da (Melittine) can be ionized when they are mixed with this type of stamp . The projection for laser desorption-ionization for a large molecule occurred in 1987 when Koichi Tanaka (of Shimadzu Corp.) and his/her colleagues used what they called method of the matrix metal ultra-end/liquid who combined particles of 30 Nm of Cobalt in glycerol with a Laser with nitrogen to 337 Nm for ionization. By using the combination of a laser and matrix, Tanaka was able to ionize biomolécules as important as the protein 34 472 Da carboxypeptidase-A. Tanaka accepted (jointly) the Nobel Prize of chemistry 2002 to have shown, that with an adequate combination of matrix and laser, that a protein could be ionized. Karas and Hillenkamp were then able to ionize the Albumine (protein 67 kDa) in utilisantune matrix of nicotinic acid and a laser with 266 Nm. Additional improvements were carried out by using a laser with 355 Nm and the derivative of the Cinnamic acid , the Ferulic acid , the Cafeic acid and the sinapinic Acide like matrix. The availability of lasers with nitrogen functioning with a wavelength of 337 small Nm and relatively inexpensive and the first commercial instruments introduced with the beginning of the year 1990 conduit of many researchers to use technique MALDI. Today, the mass spectrometry MALDI uses mainly organic matrices.

Use

In organic chemistry

Synthetic macromolecules, like the Catétane S and the Rotaxane S, the Dendrimère S, the Polymère S hyper-ramified as well as other molecular units, have molar masses about thousands or of tens of thousands of grams per moles, with the result that the majority of the techniques of ionization meet diffultés to produce molecular ions. The MALDI is a simple analytical method and rapid which makes it possible to the chemists to analyze the results of this kind of syntheses and to check their results.

In biochemistry

In Protéomique, the MALDI is used for the identification of proteins isolated by electrophoresis on freezing: SDS-PAGE and electrophoresis on freezing with two dimensions. A method used is the Empreinte of peptide mass by MALDI-MS, or Affaiblissement post-ionization or Dissociation induced by collision (see Spectrometry mass).

Problems

The preparation of sample for MALDI is important for the result. Inorganic salts which are also parts of the protein extracts interfere with the process of ionization. Salts are removed by an extraction in solid phase or washing the final target spots with water. The two methods can withdraw other substances of the sample. The mixture of proteins of the matrix is not homogeneous because of the difference in polarity which leads to the separation of the two substances during crystallization. The diameter of the spot of the target is more arge that of the laser, which makes necessary to carry out several laser shootings in various places of the target, in order to obtain the statistical average of the concentration of the substance in the target spot. The composition of the matrix, the addition of Acid trifluoroacetic and Acid formic, the time between the impulses, the latency for the power of acceleration, the wavelength of the laser, the density of energy of the laser and the angle of impact of the laser beam appear among the other critical data for the quality and the reproducibility of the method.

References

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