In Microbiology, it is being used as a rapid, accurate and cost-effective method for the identification of microorganism bacteria, fungi and viruses. A typical experiment consists of growth of the organism e. Matrix is a small organic molecule used to facilitate ionization process by absorption of UV light. The sample molecules vaporize into the vacuum while being ionized at the same time without fragmenting or decomposing.
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General schematic for TOF analyzer. Provided that all the ions start their journey at the same time or at least within a sufficiently short time interval, the lighter ones will arrive earlier at the detector than the heavier ones. To correct this problem, a reflection is often applied to the end of the drift zone.
The reflectron consists of a series of ring electrodes with high voltage, which can repulse the ions back along the flight tube usually at a slightly displaced angle. Ions of different kinetic energy penetrate the reflectron to different depths before they get expelled from the reflectron into the opposite direction. Faster ions carrying more kinetic energy will travel a longer path than slower ones, and thus spend more time within the reflectron than slower ions carrying less energetic.
In that way, the detector receives ions of the same mass at about the same time. Thereby, this design for TOF mass analyzer has increased their resolution significantly. However, reflectron TOF analyzer is not suitable for analytes that are not stable enough to survive the electric field. The solution of the analyte is then admixed to that of the matrix. For optimized MALDI spectra, the molar matrix-to-analyte ratio is normally adjusted as to fall into the range from 1 to , 1.
And then the mixture is then spotted onto a metal target plate for analysis. After drying, the mixture of the sample and matrix co-crystallizes and forms a solid deposit of sample embedded into the matrix. Application of MALDI-TOF mass spectrometry Intact Mass determination The intact mass determination is basic and important for protein characterization, due to the correct molecular weight of a protein can indicate the intact structure.
MALDI, a soft ionization technique, is suitable for proteins which tend to be fragile and fragment when ionized by other ionization methods. Therefore, it has widespread uses in proteomics to identify proteins from simple mixtures by a method called peptide mass fingerprinting, which are often used with two-dimensional gel electrophoresis 2-DE. In this approach, peptides are generated by digesting proteins of interest with a sequence-specific enzyme like trypsin.
The experimental masses are compared against a database containing theoretical peptide masses from a given organism with the same sequence-specific protease. Such ions are generally referred to as metastable ions, and the process of decomposition in the field free region between the ion source and the reflectron is commonly referred to as PSD. PSD fragment ions are formed within the field free region before entering the reflectron.
PSD fragment ions can be separated, collected, and recorded on the detector by continuously changing the reflector voltage to form a PSD mass spectrum that provides very rich and effective structural information for the primary structure of peptides and proteins. In the proteomics study, some 2DE-separated protein samples cannot be identified by PMF or the results of identification are not clear.
The PSD sequencing function can be applied to the identification of these proteins. Using PSD spectroscopy, combined with a database search, proteins can be identified quickly and with high specificity.
Oligonucleotides analysis With the development of molecular biology techniques and antisense nucleic acid drug technologies, more and more oligonucleotide fragments have been synthesized to be used as primers, probes and antisense drugs. It is entirely necessary to quickly detect these fragments to determine whether the synthesis is complete and whether the synthesized sequence is correct. It provides specific information about the local molecular composition, relative abundance and spatial distribution of peptides and proteins in the analyzed section.
MALDI-IMS can analyze multiple unknown compounds in biological tissue sections simultaneously through a single measurement that can obtain molecule imaging of the tissue while maintaining the integrity of cells and molecules in tissues. MALDI-TOF mass spectrometry can analyze a wide variety of biomolecules, such as peptides, proteins, carbohydrate, oligonucleotide, and so on. Due to the fact that formed ions have low internal energy, a great advantage of MALDI-TOF is that the process of soft-ionization enables observation of ionized molecules with little to fragmentation of analytes, allowing the molecular ions of analytes to be identified, even within mixtures.
And it is easy to use and maintain with fast data acquisition.
Principles of MALDI-TOF Mass Spectrometry
De afkorting MALDI wordt vaak gebruikt in combinatie met andere afkortingen uit de massaspectrometrie. Vervolgens worden de matrix-kristallen met daarin dus de analieten bestraald met een laser. De geladen ionen komen door het heersende spanningsveld - dat kan oplopen tot 30 kV - in een massaspectrometer terecht, alwaar de massa bepaald wordt. Het is een zachte ionisatietechniek, hetgeen impliceert dat de moleculen niet uiteenvallen tijdens die ionisatie.
MALDI-TOF Mass Spectrometry: Principle and Applications in Microbiology