MALDI (Matrix Assisted Laser Desorption Ionization)

The MALDI ion source is an analytical technique for soft ionization of molecules from solid state, thus non-volatile high molecular weight molecules are easily analyzed. This ion source was invented in 1984[1] by Karas and his colleagues[2]. In 2002 Tanaka and his colleagues received the Nobel Prize for their work about MALDI, demonstrating that a protein could be ionized by the laser using a suitable matrix. This Nobel Prize attribution have been much discussed, because most scientists in mass spectrometry field believe that the Nobel Prize should have been attributed to Karas for his work in development of MALDI. Before MALDI, several ionization techniques have been invented to analyze biomolecules without much success, either the source is too energetic or it is not sensitive enough. The invention of electrospray and MALDI ionization has radically changed the performance of mass spectrometry, particularly in the field of biology. The electrospray ion source is undoubtedly essential for mass spectrometry for application in biology, MALDI is also an essential and very effective tool in this field, along with electrospray ionization, is now among the most important ionization methods for high molecular weight compounds. Several methods using the laser to ionize molecules have been invented such as SALDI[3] or DIOS[4] but the sensitivity of these ionization methods is much lower compared to MALDI, which since its invention in 1988 is able to analyze a quantity about 1pmol then some femtomole a few years later. At present, the limit of detection around some attomole is often reached by the MALDI ion source.

Ionization Process

Despite years of research, the MALDI ionization process is still not fully understood, though the energy absorption of the matrix molecules is easy to understand, but the ionization of the analysts and the matrix is ​​still poorly understood. One of the major reasons why it is difficult to understand MALDI ionization process is that, very probably, no single mechanism can explain all ions observed. In a MALDI spectrum, mainly monocharged precursor ions, some multicharged ions, cationized pseudo-molecular ions, radical matrix ions and a little fragment ion are observed. The MALDI spectrum is quite simple because it displays mainly monocharged ions, but the presence of other ions gives rise to a large number of hypotheses of the MALDI ionization process, but no hypothesis is capable to explain the formation of all the ions.

We will first see the absorption of laser energy by the matrix, each laser pulse brings a significant amount of photon, they are elementary energy parks so each photon has an amount of energy equivalent to hf, in which h is the constant of Plank and f is the frequency which is inversely proportional to the laser wavelength (equation 1).

f = 1/ʎ  (equation 1)

ʎ : wavelength.

According to equation 1, the shorter the laser wavelength, the more the photon is energetic. Each matrix molecule absorbs the amount of energy differently according to their absorption coefficient α which is the inverse of the penetration depth of the laser. This depth is about 20nm to 200nm and the laser fluence (laser efficiency) is reduced about 30% compared to the surface. This depth is more or less equal to the depth of a laser shot. If the matrix/analyst deposit has a thickness of about 100 μm, it can be analyzed in the same place a few dozen times, but in practice, the laser should not be at the same location to avoid inhomogeneity. Each laser shot has a duration of a few nanoseconds (UV laser) to a few microseconds (Laser IR), bringing a significant amount of photon. The amount of energy per photon depends on the laser wavelength, for example, for the N2 laser whose wavelength is 337 nm, each photon carries 3.7 eV. In MALDI, the N2 laser is often used and considered standard because of its low cost, but other lasers are also used, as well as the far-infrared laser, as the case of the CO2 laser (10.6 μm). Surprisingly, MALDI UV laser spectra are similar to MALDI spectra with IR laser, although the amount of energy yielding by the UV laser photon is much greater than that with IR laser. By absorbing a very localized amount of energy, a large part of energy transforms into thermal energy causing the sublimation of the matrix driving the analysts into the gas phase. The first step of the ionization process concerning energy absorption is well known and controlled, but the second step where the ions are formed is not yet clear. We can find a lot of hypothesis to explain the formation of ions, below we will see the assumptions that are most cited.

Multiphoton ionization

The best-known hypothesis of MALDI ionization is the multiphoton absorption[5], in which the matrix absorbs one photon and come into an energetic transition state, then absorbs another photon giving rise to the charged radical matrix, which explains the radical matrix ions observed in the MALDI spectra. However, the absorption of two photons does not bring enough energy to ionize the matrix molecule, since the ionization potential for matrix molecules in solid crystal (which is not yet determined) is estimated between 9 and 10 eV. With only 2 photons from the N2 laser the total energy amount is 7.4 eV is not enough, and the absorption of more than 2 photons by a matrix molecule is very unlikely, because in general, the number of photons is less than the number of matrix molecules. In addition, the MALDI mass spectra also show the fragment ions, which are probably derived from the precursor ions by having the surplus internal energy after their ionization. This hypothesis is even less probable for the IR laser, because the energy provided by the photon is too low, it is 1 to 2 orders of magnitude lower than that in UV. Although the duration of the IR laser pulse is much longer than the UV one, the possibility of absorbing multiple photons for sufficient energy is very improbable.

Following this hypothesis, the matrix molecule absorbs a photon and switches to the excitation state and then interacts with the analyst or other matrix molecule, resulting in a deprotonated matrix and the protonated analyst or protonated matrix[6]. To go further in this hypothesis, one can imagine a couple matrix/analyst that shares their proton. The excitation by the laser giving rise to the separation of the molecules, and luckily, the analyst has this proton (or one lose the proton for the negative ions) just before the separation.

This hypothesis explains for the positive ions but also true for the negative ions, but it cannot explain the presence of the radical ions, as well as the few differences between MALDI spectra recorded with UV laser and MALDI with IR laser. Moreover the local environment of MALDI which is almost unknown is also a factor that prevents advancing in this hypothesis (equation 2).

Ionization process hypothesis(equation 2)

Solid phase charge residue

Karas and his colleagues[7] have proposed that proteins that carry charges (protonated, or deprotonated) in solution, and the charges remain on the protein when the solvent is evaporated. In their model, they propose that the counter-ions are anion matrices, or trifluroacetates, this hypothesis is based on the pH of the matrix crystal, which is not a neutral pH. In MALDI, the matrix/analyte ratio is very high (about 2-3 orders of magnitude), an analyte is surrounded by several matrix molecules, so the Karas model may be possible because in the solid phase, is the matrix that solvates the ions and separates the counter-ions (equation 3). In the next step, the model proposes that the matrix/analyte crystal desorption results in the neutralization of the ions, but some monocharged ions survive because the neutralization process is not yet complete. In other words, the separation of the matrix and the analyte takes place before the neutralization.

The monocharged ion is therefore a lucky survivor of the neutralization process. This model can be used to explain the formation of positive ions, and it is also true for negative ions. However, the cluster {(M + nH)n+ + (n-1)A}1+ has never been observed in the spectra with the standard solutions. The Karas model is also incomplete, it cannot explain the presence of radical ions, or why the neutralization stops at the state of monocharged, in this case the charge state higher than 1 must have more or less the same probability.

charge residue ionization process maldi

Equation 3

Excited-State Proton Transfer

Excited-state proton transfer (ESPT) is one of the most frequently proposed MALDI ionization model. In this model, only one photon is required for MALDI ionization, when a matrix molecule is excited by absorbing a photon, it is presumed much more acidic or basic than analyte, so that it may give up or accept one proton from nearby analyte (equation 4).

M + hv → M*

M* + A → (M – H) + AH+

M* + M → (M + H)+ + MH+

Equation 4

However the near-complete lack of knowledge regarding the local environment in a MALDI sample is a major limitation in evaluating or implementing this mechanism.

Finally, no model can completely explain the formation of all the ions in MALDI, but one can imagine that there can be several phenomena that go into the formation of ions, and the models coexist.

Matrix MALDI

The choice of the matrix is ​​a very important step for the MALDI analyzes, there is no matrix that fits for all analytes. However to be a good matrix in general, it is necessary that the molecule answers a few criteria, as being stabilized under vacuum, to have a strong laser absorbance, the matrix molecules are often acidic, therefore act as a proton donor to encourage ionization of the analyte … As the process of ionization in MALDI is not well known, there is no precise rule to choose the right matrix for an analyte, often one have to try several matrices with different matrix/analyte ratios in order to find the most suitable matrix. Table 1 shows the list of matrices that are the most widely used in MALDI ionization.

Tableau 1 : The most used matrix for MALDI
Compound Abbreviation Structure Analyte
α-cyano-4-hydroxycinnamique acid CHCA  chca PeptideHydrate de carbone
Sinapinic acid SA  acide sinapinique Protéine, dendrimères, fullerenes
2,4,6-trihydroxyacétophénone THAP  thap Oligonucléotide, hydrate de carbone, glycoprotéine
2,5-dihydroxybenzoïque acid DHB  dhb Polymère systhétique polaire, hydrate de carbone
Dithranol DIT  dit Polymère synthétique non polaire, lipide, dendrimères
2,5-dihydroxyacétophénone DHAP

 

Sample preparation

Like the choice of the matrix substance, there is also a choice of how to prepare the sample. Several methods of sample preparation are described in the literature, but three methods that are best known are the dried droplet, thin layer and sandwich methods. The dried droplet method is a method most used, because it’s very easy to implement. The matrix solution is mixed in equal volumes with the sample solution. The sample solution should be acidic, since basic conditions will neutralize the matrix. The mixture is then plotted on the metal plate (0.5 to 2 µl) and dried at ambient temperature. The preparation will yield relatively large crystals on the target surface as well as regions without matrix or analyte.

The thin layer method is suitable only for HCCA. The matrix is prepared on the target to form a thin layer of small and homogenous crystals. This is achieved by dissolving the matrix in Acetone. After spotting this solution on the target the acetone spreads on the target and evaporates very fast. This thin matrix layer remains on the surface of the target. The sample solution is applied on top of this thin layer. After the sample is dried, the analyte molecules remain on top of the matrix. Advantages of the thin layer method are the very homogenous size of the crystals. The methods yields high resolution spectra and the detection limit is better compared to the dried droplet method.

The sandwich method is more complicated, here a thin layer of matrix is prepared as described above, and on top of that a small quantity of sample solution is plotted. Finally a thin layer of matrix is applied. The difficulty of this method is the deposition of analyte solution on the matrix layer (or vice versa), the solvent of the solution deposited on the matrix layer locally solubilizes the matrix, and the mixture becomes inhomogeneous.

Applications

As MALDI is a soft and sensitive ionization method, which preserves the intact molecules of the ionization process, it is widely applied in organic chemistry, biology, pharmaceutical, clinical… By choosing right matrix and the appropriate ratio one can analyze almost any type of molecule. In addition, the rapid preparation on several deposits of the metal plate to analyze a large number of different samples in a very short time is an advantage of MALDI. However, MALDI is an ionization technique from solid phase, so it is not possible to couple in line with liquid or gas phase chromatography. MALDI also produces fragment ions, so it offers the possibility of doing tandem mass spectrometry (MS/MS). The ions produced by MALDI are in parquet, requiring an analyzer capable of analyzing all the ions at the same time, therefore, MALDI is often coupled with TOF analyzer.

A very interesting application of MALDI is MALDI imaging. This technique involves directly analyzing the sample by moving the laser across multiple locations in the sample. Most often, this technique is used to analyze peptides, lipids or proteins within tissues. The advantage of this technique is not to delocalize molecules allowing having images according to the molecules of a tissue. MALDI couple with TOF analyzer is an analytical method of microbial identification and characterization based on the fast and precise assessment of the mass of molecules in a large range of mass from 100 Da to 100 KDa. The possibility of MALDI-TOF to quickly characterize a wide variety of microorganisms including bacteria, fungi and viruses[8] increase it potential application in some areas of microbiological diagnosis. So that, this technology can be used for rapid microbial identification at a relatively low cost and it is an alternative for conventional laboratory diagnosis and molecular identification systems.

Reference

[1] M. Karas, D. Bachmann, F. Hillenkamp. Influence of the wavelength in highirradiance ultraviolet laser desorption mass spectrometry of organic molecules. Anal. Chem. 198557, 2935–2939.

[2] M. Karas, F. Hillenkamp. Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons. Anal. Chem. 1988, 60, 2299–2301.

[3] M. Schürenberg, K. Dreisewerd, F. Hillenkamp. Laser desorption/ionization mass spectrometry of peptides and proteins with particle suspension matrixes. Anal. Chem. 1999, 71, 221–229.

[4] J. Wei, J.M. Buriak, G. Siuzdak. Desorption–ionization mass spectrometry on porous silicon. Nature

[5] Liao, P.–C.; Allison, J. Enhanced detection of peptides in matrix-assisted laser desorption/ionization mass spectrometry through use of charge-localized derivatives. J Mass Spectrom 1995, 30, 511.

[6] Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Matrixassisted ultraviolet laser desorption of non-volatile compounds.

[7] R. Krueger, A. Pfenninger, I. Fournier, M. Glückmann, M. Karas. Analyte incorporation and ionization in matrixassisted laser desorption/ionization visualized by pH indicator molecular probes. Anal. Chem. 200173, 5812–5821.

[8] Giebel R, Worden C, Rust SM. Microbial fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications and challenges. Adv Appl Microbiol. 2010;71:149–84.

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