FulmerResearchFinal

Matrix-Assisted Laser Desorption Ionization Mass Spectrometry
Bryan J. Fulmer
Drexel University, Department of Chemistry
Submitted December 6th, 2011

Abstract:
Since the 1980s matrix assisted laser desorption/ionization mass spectrometry has been continuously advancing. Originally MALDI MS was developed as a high molecular mass detection technique. While MALDI is still used for high mass analysis, MALDI can be used for much more. MALDI is capable of not only organic but inorganic analyses. Phamacological groups have been using MALDI to study drug metabolism. Proteomic divisions have been using MALDI for peptide and protein sequencing. As a tool for inorganic analysis, researchers have been confirming masses determined using techniques such as thin-layer chromatography (TLC). Inorganic researches have used MALDI for mass analysis of insoluble compounds. Finally, researchers are using MALDI for analysis and confirmation of metal oxidation states.

Introduction to MALDI:
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a relatively new and yet to be fully understood analytical technique. The technique of MALDI was first discovered and published by M. Karas and F. Hillenkamp in the 1980s and is continuously being researched in order to gain understanding of the multiple phenomena that occur. MALDI is primarily used as a high molecular mass, soft-ionization mass spectrometry technique. This is because of the ability of the technique to transfer solid material from a surface into a gas-like phase and ionize those molecules, which then allows for mass separation and detection.
In order to understand MALDI, one must understand the laser desorption that occurs. Laser desorption occurs when a focused beam of light energy is directed at solid state molecules on a surface. The surface molecules absorb the energy and the intermolecular forces between molecules are no longer able to keep all the molecules together, and some are ablated from the surface. This creates a pseudo gas phase of molecules. This plume of excited molecules then interact with each other and, in MALDI, excited matrix molecules. The interaction of the matrix will be discussed later. As for the types of lasers used for laser desorption, and MALDI, they are primarily ultraviolet wavelengths. This is due to the absorption spectrum of the target molecules. The target molecules must be able to sufficiently absorb the wavelength of the laser in order to be promoted to the excited state. The typical ultraviolet lasers that are used in MALDI are N2 (at 337 nm) and NdYAG (at 355 nm). In recent years, NdYAG has become standard over N2 lasers due to the improved lifespan and faster pulse rates that the NdYAG lasers are able to obtain. Current research is underway to determine if the difference between the two wavelengths is significant.
The matrix used has an equally pivotal role in MALDI MS. In MALDI, choosing a matrix can be quite a task in itself. One way to chose a matrix is based on the type of analyte molecules under study. In 1990, M. Karas et. al. discovered one of the most widely used matrices used today, 2,5-dihydroxybenzoic acid (DHB) [1] as a suitable matrix for MALDI. In a later study [02], they used DHB for bioorganic molecules such as proteins. It was found that the ability for this matrix to protonate the proteins without disrupting the structure or size of the proteins. This matrix was also used in polymeric analyses by Thompson et. al. in 1994 [3]. Currently, DHB is a common starting place and a point of reference for most organic based MALDI analyses. The ability of this molecule to absorb the UV radiation of the then used N2 lasers and the molecules low proton affinity allows the donation of the acidic proton to the analyte molecule. Detailed ionization information can be found in the work of R. Knochenmuss [4]. Other organic acids have been found to work as matrices such as vanillic acid, however, their uses are limited by their UV absorbance spectrum. Vanillic acid does not work with the typical 337 or 355 lasers, it will only work with in the region of 266 nm, and its absorbance drops to nearly zero at 320 nm, thereby requiring a non common laser. It should be noted, however, that not all variations of dihydroxybenzoic acid, such as 3,5-DHB, are suitable matrices. At this time, only 2,5-DHB has been shown to produce signals in MALDI MS.
MALDI samples are commonly prepared on a metal target plate, containing wells for sample to be applied. Various methods for sample deposition have been developed including electrospray deposition [5]. This technique provides a homogenous distribution of matrix and analyte to the MALDI sample target. This method has been found to produce highly reproducible data.
Finally, the ions produced by MALDI are introduced into a mass separation and detection device such as a time of flight (TOF) or Fourier transform (FT) mass spectrometer using electromagnetic fields and gradients. The operation of these sections of the instrument will not be covered and it is understood that the reader have a prior basic knowledge of the principles of TOF MS and FT MS.

Organic MALDI:
The ability for MALDI MS to analyze high mass non volatile biomolecules, polymers and organic molecules without the fragmentation of the molecular ion has lead to the ability of high mass accuracy of molecules once using only relative molecular masses.
Recently in the organic based MALDI experiments, researchers have been performing MALDI imaging. MALDI imaging is used to obtain mass spectra with spatial resolution. Prior to MALDI imaging, autoradiographic studies were conducted. These studies used drug molecules previously labeled with 14C or 3H. The host animal being used for the study was then analyzed to determine the metabolic nature of the drug. This method, while still in use today, has its limitations. One limitation can be spatial resolution. Most autoradiography studies are only able to obtain a spatial resolution of 50 to 100 um [6]. This resolution has been sufficient for decades, however, with appropriate sample preparation and instrumental settings, MALDI imaging is reporting spatial resolutions of 10 um[6]. The sample preparation for MALDI imaging on tissue samples, in short, is as follows. Tissue samples are mounted to a MALDI imaging target plate. These plates are not unlike the welled plates used for standard chemical analysis. The tissue sample is then coated in a matrix, dissolved in an appropriate solvent. As the solvent evaporates, the matrix co-crystalizes with the analytes of interest on the tissue surface. The MALDI imaging sample plate is then introduced into the instrument for analysis. There are both manual and automated settings for the instrument to obtain spectra. Once the spectra are collected, data analysis software packages compile the spectra into a user-friendly format. According to Caprioli et. al. MALDI imaging can be described as analogous to a digital photograph [7]. Each photograph (MALDI imaging experiment) is made up of orderly arranged pixels (individual spectra) containing color information (the masses detected) which when put together is all used to display an image.
MALDI has also been able to be used in the area of protein sequencing. A method called In-Source Decay (ISD) has been developed for doing just that. ISD is similar to the Edman degradation, however it is faster, cheaper and simpler to perform. The key to ISD is selecting an appropriate matrix. It has been discovered that 1,5-diaminonaphthalene (1,5-DAN) is sufficient [8]. The sample is prepared in similar fashion to other MALDI techniques. The difference comes in the data analysis of the collected spectra. Companies have created software that will automatically retrieve the data from the mass spectra, detect the differences in the peaks and assign associated amino acids with that mass loss. These software packages will display multiple possibilities and the error in each sequencing variation, still requiring the researcher to perform some level of manual interpretation of the data presented. There are currently attempts to combine both MALDI imaging and MALDI ISD into a single experiment. It was discovered that not all proteins would be simultaneously ionized. This is due to the ionization sensitivity of each protein, and those that ionize readily will do so and the abundance of signal from those proteins will likely suppress the little signal coming from other, less sensitive proteins[8]. Zimmerman et. al. believe this not to be a full detriment, in that those proteins that are sensitive may be enough to provide sufficient information depending on the study.


Inorganic MALDI:
Unlike the organic based MALDI analyses, the use of MALDI as an investigative technique on molecules containing transition metals is less popular. Techniques such as electrospray ionization (ESI) and fast-atom bombardment (FAB) have long been used to study masses of inorganic materials. When performing MALDI on metals, it is unlikely that the complexes will be able to protonated; therefore a different ionization method must be used. Commonly matrices are used as chelating agents in MALDI and ligand exchange can occur.
One method of using MALDI for inorganic species is the use of thin-layer chromatography (TLC) in conjunction with MALDI. Commercially available TLC plates can be used as desired for separation of complexes. In past, many compounds were and still are identified using retention factors (Rf) for TLC. This technique, while adequate, leaves room for error and misidentifications. By combining a TLC separation with a MALDI analysis, confirmation can be obtained that the compound believed to be at a specific Rf value, is indeed, that compound. Matsumoto et. al. separated Co(II) and Co(III) species using TLC then applied the matrix dithranol to the TLC plate after removal from the development tank. MALDI MS confirmed that all species were present at expected locations.[9]
Since MALDI was primarily introduces as a high molecular weight analysis technique, few have considered it for lower mass molecules. One area that has the possibility of use is the inorganic species that are insoluble in analysis solvents. One such analysis was performed by Matsumoto et. al. in 2001 [10]. Instead of using a solvent to dissolve the matrix and the analyte, both materials were kept in the solid phase. This author obtained Co(II) and Co(III) acetonates and Co3O4. The author mixed the samples with the matrix dithranol or 1-(2-pyridylazo)-2-naphthol (PAN). These samples were ground in a mortar and pestle and formed into discs for MALDI analysis. This method showed promise according to the author as a possible technique for insoluble metal complex identification via MALDI MS.
Spectroscopy has been long used to determine the oxidations states of transition metals. One new method currently being researched is using MALDI to determine metal oxidation states. Matsumoto et. al. was able to show the possibility of using MALDI MS for this purpose. This research group purchased known standards of copper and iron for analysis. Iron was chosen in order to create a standard set of spectra in order to possibly relate them to the detection of iron oxidation states in the naturally occurring mineral, pyrite.[11] The author was able to show, using suitable matrices that the oxidation states can be assigned based on the m/z values obtained on the spectra. The author stressed the ability to use methanol as the solvent and stated no complicated sample preparation was needed. The Matsumoto further expanded their research for assigning oxidation states of metals [12]. They expanded to using manganese, cobalt and chromium oxides. The solid samples were ground with chelating matrices by again using a mortar and pestle. These samples were these suspended in a solvent. It is important to note that these samples were not dissolved in the solution and the solution was turbid. This being said, the author reported discernable spectra with identifiable and assignable m/z values in order to assign oxidations states. The lack of homogeneity in the sample solution would cause problems in quantitative aspects of this technique. Therefore it is believed this was done for qualitative results only. Again, this group was able to show MALDI as an effective technique for determining the oxidation state of transition metals. Using MALDI would allow simple, fast and accurate detection of metals and their oxidation states in naturally occurring materials.

Conclusion:
Much research has been performed in the organic based area of MALDI MS. Currently in the organic section of MALDI uses, the technique is still limited to high mass detection. Proteomic research groups are currently using MALDI MS for imaging and protein sequencing, with the hope to combine the two techniques. As for the inorganic based MALDI MS experiments, there is minimal research in comparison to the organic studies. MALDI has shown promise for studies in the mass determination for otherwise insoluble compounds as well as the possibility for simultaneously assigning oxidation states to the associated metals under study.

References:

1. Karas, M., et. al. (1990). "Principles and applications of matrix-assisted UV-laser desorption/ionization mass spectrometry." Analytica Chimica Acta. 241, 175-185. Link

2. Strupat, K., et. al. (1991). "2,5-Dihydroxybenzoic acid: a new matrix for laser desperation-ionization mass spectrometry."International Journal of Mass Spectrometry and Ion Processes. 111, 89-102. Link

3. Thompson, B., et. al. (1996). "Characterisation of low molecular weight polymers using matrix assisted laser desorption time-of-flight mass spectrometry." European Polymer Journal. 32, 2, 239-256. Link

4. Knochenmuss, R. (2006). Ion formation mechanisms in UV-MALDI. Analyst. 131, 966-986. Link

5. Owens, K. et. al. (2011). ES Deposition. http://esdeposition.wikispaces.com/. Link

6. Castellino, S., et. al. (2011). MALDI imaging MS: bridging biology & chemistry in drug development. Bioanalysis. 3, 21, 2427-2441. Link

7. Caprioli, R., et. al. (1997). Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI-TOF MS. Analytical Chemistry. 69, 4571-4760. Link

8. Zimmerman, T., et. al. (2011). An Analytical Pipeline for MALDI In-Source Decay Mass Spectrometry Imaging. Analytical Chemistry. 83, 6090-6097. Link

9. Matsumoto, K., et. al. (2002). Application of TLC-MALDI-TOFMS to Identification of Co(II) and Co(III) Acetylacetonates. J. Mass Spectrom. Soc. Jpn. 50, 1, 15-17. Link

10. Matsumoto, K., et. al. (2002).Detection of solid cobalt species by matrix-assisted laser desorption/ionization mass spectrometry time-of-flight. Rapid Commun. Mass Spectrom. 16, 730-732. Link

11. Matsumoto, K., et. al. (2004). Detection of Metal Oxides by MALDI/TOF MS. J. Mass Spectrom. Soc. Jpn. 52, 6, 325-327. Link

12. Matsumoto, K., et. al. (2005). Identification of oxidation states of metal oxides by MALDI-TOF MS. Microchemical Journal. 81, 195-200. Link

13. Stump, M., et. al. (2002). Matrix-Assisted Laser Desorption Mass Spectrometry. Applied Specoscopy Reviews. 37, 3, 275-303. Link

14. Matsumoto, K., et. al. (2003). Detection of inorganic species by chemical reaction laser desorption ionization mass spectrometry. Rapid Commun. Mass Spectrom. 17, 678-684. Link

15. Wyatt, M., et. al. (2008). Analysis of transition-metal acetylacetonate complexes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 22, 11-18. Link

16. Zhang, J., et. al. (2002). Reduction of Cu(II) in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J Am Soc Mass Spectrom. 14, 42–50. Link