MALDI MS and MALDI Imaging
MALDI is an acronym that stands for matrix-assisted laser desorption ionization. It is an ionization technique introduced in the early 1980s by F. Hillenkamp, M. Karas et.al. Earlier experiments in the 1960s and 1970s had demonstrated the concept of photoionization, where photons were used to directly ionize molecule that were able to absorb them. The introduction of a matrix (a small molecule mixed with the sample) capable of absorbing the laser energy results in ionization of molecules that are not directing interacting with the photons. The exact ionization mechanism is still not fully understood, and it is highly likely that more than one physical and chemical process is responsible for ionization of the analyte depending on the conditions used, the matrix selected and the analyte itself. The end result of most MALDI experiments conducted with acidic or basic matrixes (CHCA, DHB, 9-AA etc.) is the protonation or deprotonation of the analyte, although radical ions are also measured. In particular, utilization of aprotic matrixes such as DCTB mostly results in molecular ions that have either lost or acquired an electron.
MALDI samples are often solid samples that have been created by drying matrix and analyte together on a metal plate. It is also possible to premix the two in powder form, although this requires more material. This ionization technique is more tolerant toward classic contaminants (such as salts) and it is often couple to time of flight mass spectrometers. Therefore, MALDI is used for the characterization of a broad range of analytes, from peptides to protein to polymers.
As mentioned in the previous section, MALDI is a technique especially suited for analyzing solid samples. The laser of a MALDI instrument can be rastered over a surface where matrix has been uniformly sprayed, recoding a spectrum at regular intervals. The user can then construct heat-maps of the surface using mass values, displaying the intensity of a given mass across the surface. This process is called MALDI Imaging, and it can be conducted on several type of samples, from tissue sections to fingerprints. This approach is especially useful to interrogate the localization of molecules of interest. The MSF utilizes a Waters rapifleX TOF/TOF instrument that allows recording of spectra up to a rate of 40 Hz, making it the fastest MALDI instrument on the market. Imaging experiments can be completed in a matter of minutes, and targets of interests include small molecules, lipids, sugars, peptides, proteins etc.
Description: this type of analysis provides measurements of masses from mixtures or purified samples. It can be conducted on a mass range from 0 to 500,000 Da, and it provides a fast and simple way to measure unknowns and confirm the presence of target molecules. This experiment is often performed on HPLC fraction, reaction mixtures and standards. If the sample contains known molecules, these can be used to calibrate the acquired spectrum, achieving mass precision as low as 1 ppm.
Amount needed: generally 5-10 µL of a 1mg/mL solution or 1 mg of solid sample. Actual amounts depend on the type of matrix and analyte.
Type of samples: any ionizable molecule.
MSF protocol available: yes.
Degree of difficulty: low.
Notes: this method is ideal for quick screening projects. For example, one may want to test the purity of reagents before starting a reaction or monitor degradation of a product during a kinetic test. Training is available to any LSU user.
Description: this type of analysis specifically targets polymers, and it is aimed at verifying the actual composition of the polymer (repeating units and end units) as well values such as Mn, Mw and degree of polymerization. Execution is similar to the simple mixture analysis although it may require addition of a cationizing agent (e.g., sodium trifluoroacetate) to improve ionization.
Amount needed: generally 5-10 µL of a 1mg/mL solution or 1 mg of solid sample. Actual amounts depend on the type of matrix and analyte.
Type of samples: any ionizable polymer
MSF protocol available: yes.
Degree of difficulty: low.
Notes: this technique is complementary to other polymer analytical tools such as gel permeation chromatography. The MSF is working with the SLMBR to establish protocols that will help users perform both techniques to maximize the results and increase the interpretation confidence.
Description: this type of analysis is conducted when the sample is a surface, and the user wants to measure the spatial localization of one or multiple targets. Samples need to be mounted on metal surfaces or conductive microscope slides, the most common being indium tin oxide (ITO) coated slides. Samples can be prepared within the MSF using our HTX 3+ automated sprayer, which automates the entire matrix deposition process.
Amount needed: Any surface that can be mounted on a conductive support with a size of 1x3 inch.
Type of samples: Cell, tissue, organoids, biofilms etc.
MSF protocol available: yes.
Degree of difficulty: medium.
Notes: MALDI has the ability to measure analytes with very high molecular weight. At the same time, this capability is limited when performing an imaging experiment, mostly because of the low relative concentration of an analyte within the space (or pixel) irradiated by the laser to generate a spectrum. The larger the molecule, the harder it is for it to fly in the mass spectrometer and generate signal.
Data analysis can be conducted on Bruker software installed on the data acquisition and the data analysis computers. The main software used is flexAnalysis, which allows visualization and manipulation of the acquired data. In addition, user can also utilize utility apps such as Compass IsotopePattern, which provides a graphical representation of the theoretical isotopic pattern of a given structure.
Bruker provides tools for exporting raw data in alternative, open source, formats using a command line utility. A simple GUI that simplifies the sue of utility is provided by the Proteowizard team in the form of MSConvertGUI. This tool allows conversion of Bruker raw data to various formats, such as mzXML or mzML. The exported spectra can then be opened with tool such as mMass (downloadable from the MSF website).
MALDI imaging data analysis can be performed with two tools. The first is Bruker flexImaging, which is installed on the acquisition computer. This is the tool used to set up and run imaging experiment, and it useful for visualization of masses and validation of dataset quality.
The second tool is Bruker SciLS, which is a dedicated MS imaging software capable of analyzing and visualizing imaging data. Further details on SciLS are available on Bruker website.