Metabolomics/Analytical Methods/Mass Spectrometry/MALDI-MS
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Introduction
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MALDI, or Matrix-assisted laser desorption/ionization, is a soft ionization technique used in mass spectrometry. Using this technique allows for the analysis of biomolecules (proteins, peptides, and sugars) and larger organic molecules (polymers and dendrimers). MALDI is used to ionize these molecules because they are very fragile and will usually fragment when ionized by other, more conventional ionization methods. A laser beam is used for ionization, and the biomolecule is protected by a matrix to prevent it from being destroyed, and to allow for optimal vaporization and ionization.
The Matrix
editThe matrix used in MALDI-MS is a mixture of crystallized molecules, with the most common three being 3,5-dimethoxy-4-hydroxycinnamic acid, alpha-cyano-4-hydroxycinnamic acid, and 2,5-dihydroxybenzoic acid (DHB). These are mixed into solution in purified water and an organic solvent such as ethanol or acetonitrile.
How it Works
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The laser is fired at the matrix, with the matrix absorbing the lasers energy, and ionizing the crystals. These crystals then transfer some of their charge to the compound being testing, thus protecting it from the laser's destructive energy, but still allowing it to be ionized. Ions observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na]+ in the case of an added sodium ion, or [M-H]- in the case of a removed proton. MALDI generally produces singly-charged ions, but multiply charged ions ([M+nH]n+) can also be observed, usually as a function of the matrix, the laser intensity and/or the voltage used. 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.
Mass Spectrometer
MALDI (Matrix-Assisted laser desorption/ionization) is usually used with the TOF mass spectrometer. The TOF spectrometer is ideal because of the wide range of masses it can operate with.
Experiments Done Using MALDI-MS
editEffect of Culture Conditions on Microorganism Identification by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Article Link Here
PNNL: Pacific Northwest National Laboratory, they are responsible for the making of the data base used to identify the unknown bacteria based on their spectral readings.
Sheep Blood Agar: Link:Blood Agar Types Used in this experiment because it is widely used in differentiating various strains of the Bacillus genus. Some strains exhibit hemolysis, while others do not. This is important to differentiate the form that causes anthrax from other less harmful types.
Spectral signature peaks: Link:Mass Spectrometry Data Representation Peaks in the spectrometry results, that will appear in the majority of a bacteria species, these peaks will represent some sort of protein that the bacteria are making for their own metabolism.
Automated Statistically Based Data Analysis Algorithms: These algorithms are used to compare the signatures of the unknown microorganisms, or in this case the known microorganisms grown on various media, to the extensive library of signatures already in place. The ions that are present in at least 70% of the spectra are included into a signature library for that microorganism.
MALDI-TOF MS is already being used to create a database of characteristic spectral signature peaks of different bacteria genera and species grown on controlled cultures. This group of researchers questioned whether or not bacteria grown on different sorts of media would cause the bacteria to switch on different proteins, and thus change the spectrometry results. According to various reports, use of the minimal medium chosen for this study has been shown to induce physiological changes in both E. Coli and Bacillus subtilis. However, the results have shown that despite being culture in the different media, whole-cell bacteria are still identifiable by MALDI-TOF MS and PNNL's (automated statistically based data analysis algorithms. This is due to the majority of the proteins in a bacteria working in all culture conditions i.e. housekeeping functions, and not involved in metabolizing the culture.
On-Probe Sample Pretreatment for Direct Analysis of Lipids in Gram-Positive Bacterial Cells by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Article Link Here
Peptidoglycan: A polymer consisting of sugars and amino acids that makes up bacterial cell walls Wiki Link
Trifluoroacetic acid (TFA): is the chemical compound with the formula CF3CO2H. It is a strong carboxylic acid widely used in organic synthesis. Wiki Link
In this experiment, the researchers were testing a new method to analyze the lipid components of bacterial cell walls without doing extensive pre-treatments. This new method is called on-probe sample pretreatment, and involves the transfer of growing bacterial colonies to the matrix surface where they are covered with a microvolume of matrix solution prior to being exposed to the laser. In previous experiments no signature peaks were shown using this method. In this experiment, they added a compound known as TFA to the on-probe treatment to enhance the ion formation of phospholipids in the bacterial cell wall. With this addition of TFA, the signature peaks appeared in the test done on B. subtilus. However, when this same procedure was done on a sample of E.Coli (a gram negative bacteria), the results were not as clear. It is suspected that this is due to TFA hydrolyzing the phospholipids in the thinner peptidoglycan layer of the gram negative bacterium.
Direct Tissue Analysis by MALDI MS: Application to Kidney Biology Article Link
Drug Nephrotoxicity: is a poisonous effect of some substances, both toxic chemicals and medication, on the kidney. Wiki Link
Glomeruli: is a capillary tuft surrounded by Bowman's capsule in nephrons of the vertebrate kidney. Combined with the Bowman's capsule it makes up the renal corpuscle, the basic functional unit of the kidney.
Glomerulosclerosis: Scarring of the glomeruli found in scattered regions of the kidney, it impairs kidney function. MALDI-MS can be used for tissue protein profiling in these diseased regions. Outside Link
This is a very general article describing how MALDI-MS is used to determine biomolecule concentration and location in a tissue sample (in this case, the kidney). MALDI-MS allows for the direct analysis of the molecules in the tissue without special regents specific for various molecules. MALDI-MS can be used to look for certain proteins, peptides, lipids, and drugs (important for the pharmaceutical industry). The ability to detect drug concentrations is key to determining medicinal levels in the kidney, giving the researchers an idea to the effect the drug may have had on the patients kidney. The researchers conclude that the applications that this technology can be used for have only just begun, and that this technology has extraordinary potential.