MALDI-TOF Technology

Background:

Matrix Assisted Laser Desorption/Ionizing mass spectroscopy is quickly becoming an important tool in the biological sciences. This technology holds the promise of insight into the molecular world around us. The matrix assisted technology relies on the ability of a growing crystal to incorporate analyte molecules into its matrix. The incorporation of the molecule depends upon analyte-solute and analyte-matrix interactions. MALDI generally uses an acidic acetonitrile solution saturated with an organic UV absorbing acid. A small amount of analyte solution (0.5-1ul) is placed on a conductive sample stage. This solution is then layered with 0.5-1uL of the matrix solution and allowed to dry. As the acetonitrile evaporates, the acid and analyte become increasingly concentrated. Almost immediately, the acid will start to crystallize out of solution. Under optimum conditions, the analyte will begin to bind to faces of the crystal were growth is occurring. Growth will eventual envelope the analyte producing a doped crystal which is adhered to the sample stage. Next the sample is placed in the instrument. A voltage potential of 0-30keV is then induced between the sample stage a an acceleration grid. A nitrogen laser is then used to bombard the doped crystal with UV radiation (337nm). Pi electrons within the acid absorb the radiation. Some of the energy is emitted as photons, some of which can be seen in the visible spectrum in the sample monitor. Transfer of energy occurs through excitation of valence electrons in the analyte, translational energy transfer and thermal transfer to produce analtye ions. The ionization products are accelerated by the potential field and directed to a 2m flight tube where they are allowed to travel in a vacuum with the kinetic energy obtained from the potential field. The speed of the molecules will depend on their mass because they all started with the same kinetic energy. This Time-of-Flight separation technique will obtain mass values with a 0.01% accuracy.

Sensitivity:
Using a matrix as a ionization vehicle produces some beneficial effects in analyzation of samples. Sensitivity of 100 fmol can be achieved. Inclusion of analytes within crystals can isolate them from usual contaminants found in the sample. When analyzing biomolecules, this is very beneficial due to chemicals such as salts and detergents that can render other analysis techniques useless. Therefore, there is very little sample preparation needed.

Resolution:
The BioPerSeptive Elite Research Station has three mechanisms at its disposal for increasing the resolution and subsequent accuracy. It is equipped with a Delayed Extraction technique which is the first mechanism charged molecules will encounter. This employs a delay time from laser bombardment to engagement of the potential field for acceleration. This gives a chance for the molecules in the gas plume to equilibrate producing a better energy distribution. This allows molecules that were desorbed last to be at the same level as the ones that desorbed first producing a closer grouping of the molecules as they hit the detector. Running down the center of the flight tube is a guide wire which has a variable potential on it. This is used as a focusing tool for the molecules in flight. This gives a better grouping of molecules of the same mass by removing extraneous flight trajectories. This also increases the sensitivity of the instrument from focusing molecules that would otherwise not reach the detector. At the end of the flight tube is the detector. This detector is actually two detectors in one. First, molecules can take a linear flight path to the detector producing the electric signal sent to the internal oscilloscope to be processed by the Grams soft ware. This primary detector plate can also be induced with a positive potential which will reflect the molecules back a distance of 1m to another detector plate which the molecules traveled past. Again this technique produces better resolution of the spectra. For a given group of same mass molecules, there is slight variation in their kinetic energy producing variations in their flight times. In this grouping, the faster molecules will travel further into the potential field before being reflected back. The slower molecules will not travel as far as the faster ones before being reflected. Consequently, the speed distribution of the molecules are canceled out producing a sharper spectrum peaks due to close grouping as they bombard the detector. One of the result of redirecting the flight path of the molecules is the induction of strain in the molecule. This can fragment the molecule into smaller components. The spectra are received with a greater count of signals below the mass range of the initial compound. These fragments are characteristic of its parent compound. As a result, this reflector application can also be used to obtain structural analysis and identify various compounds.

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