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MALDI-TOF Spectrometry

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MALDI-TOF Spectrometry

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    1. MALDI-TOF Spectrometry Stephen Gangemi November 2002

    2. Overview What is MALDI-TOF? How does it work? Why is it useful? What are its figures of merit? What is its future?

    3. What is MALDI-TOF? Matrix-assisted laser desorption/ionization time of flight mass spectrometry Ionizes molecules via laser pulses Separates molecules according to mass to charge ratio Mainly used for detection of large biomolecules

    4. Development of MALDI-TOF Developed in 1988 by Professor Hillenkamp Designed to enhance mass-spec. by solving two main problems Thermal instability and low volatility Large and heavy biomolecules Developed in 1988 by Professor Franz Hillenkamp and a group of his assistants at Germany University of Munster Mass spectrometry has been used almost exclusively for almost 50 years for the analysis of molecular molar masses. However, difficulties have been encountered with large biomolecules and synthetic polymers due to their thermal instability and low volatility. New techniques such as chemical ionization (CS), secondary-ion mass spectrometry (SIMS), and fast atom bombardment (FAB) have helped somewhat with these problems. However, all have difficulty with large and heavy biomolecules. MALDI has no problems with biomolecules over 200,000 Daltons by vaporization without degradation. Developed in 1988 by Professor Franz Hillenkamp and a group of his assistants at Germany University of Munster Mass spectrometry has been used almost exclusively for almost 50 years for the analysis of molecular molar masses. However, difficulties have been encountered with large biomolecules and synthetic polymers due to their thermal instability and low volatility. New techniques such as chemical ionization (CS), secondary-ion mass spectrometry (SIMS), and fast atom bombardment (FAB) have helped somewhat with these problems. However, all have difficulty with large and heavy biomolecules. MALDI has no problems with biomolecules over 200,000 Daltons by vaporization without degradation.

    5. The Principles of MALDI Dissolve compound and add matrix Evacuate air from sample chamber Laser shoots pulses of light at sample First, the polymer needs to be dissolved in a solvent. Proteins are usually dissolved in water, sometimes acetonitrile is added. For other biomolecules, other solvents may need to be found. Also, some MALDI specs do not need to have the sample in solution, it can be in a solid state. Next, after the compound is in solution, a matrix needs to be added. Similar to AA, different matrices work better with different compounds. However, all matrices need absorb UV radiation. Some common matrices are trans-cinnamic acid or 2,5-dihydroxybenzoic acid. Matrix is generally added in an amount that is greater than 10^4 times the sample, ensuring that the matrix absorbs a majority of the radiation rather than the sample. This will prevent unwanted sample fragmentation. Another important aspect of the matrix is that it serves to isolate polymers from one another. Finally, the matrix serves as a source of protons for the sample to ionize. After the sample has been prepared, the solution is loaded into the sample chamber, which is then vacuum pumped to evacuate all of the air in the chamber. At this time, the solvent evaporates, leaving the sample in a dispersed compound containing both the matrix and the sample. At this point, the laser shoots short pulses of light, in the 330-360 nm range, at the sample, causing it to essentially explode. The matrix is vaporized, and the sample is ionized into the +1 or –1 state. No one is really sure of the mechanism of this ionization, but for some reason it occurs, and the +/- 1 state is found. It is also at this point that the polymers do something unusual. They evaporate. Usually, the polymers are too big and heavy to evaporate, but at these high temperatures and low pressures, evaporation or desorption occurs. Thus, we are left with ionized polymers in the gaseous phase.First, the polymer needs to be dissolved in a solvent. Proteins are usually dissolved in water, sometimes acetonitrile is added. For other biomolecules, other solvents may need to be found. Also, some MALDI specs do not need to have the sample in solution, it can be in a solid state. Next, after the compound is in solution, a matrix needs to be added. Similar to AA, different matrices work better with different compounds. However, all matrices need absorb UV radiation. Some common matrices are trans-cinnamic acid or 2,5-dihydroxybenzoic acid. Matrix is generally added in an amount that is greater than 10^4 times the sample, ensuring that the matrix absorbs a majority of the radiation rather than the sample. This will prevent unwanted sample fragmentation. Another important aspect of the matrix is that it serves to isolate polymers from one another. Finally, the matrix serves as a source of protons for the sample to ionize. After the sample has been prepared, the solution is loaded into the sample chamber, which is then vacuum pumped to evacuate all of the air in the chamber. At this time, the solvent evaporates, leaving the sample in a dispersed compound containing both the matrix and the sample. At this point, the laser shoots short pulses of light, in the 330-360 nm range, at the sample, causing it to essentially explode. The matrix is vaporized, and the sample is ionized into the +1 or –1 state. No one is really sure of the mechanism of this ionization, but for some reason it occurs, and the +/- 1 state is found. It is also at this point that the polymers do something unusual. They evaporate. Usually, the polymers are too big and heavy to evaporate, but at these high temperatures and low pressures, evaporation or desorption occurs. Thus, we are left with ionized polymers in the gaseous phase.

    6. TOF Spectrometer Ions are accelerated via charge Separated by time it takes to reach detector Ions are accelerated by electrodes at opposite end of tunnel of a known length. If it is a negative ion, it accelerates to the cathode, positive ions accelerate towards the anode. The charge depends on the type of molecule being analyzed as well as the matrix used. This electrical force is used to accelerate the particles down tunnel towards the detector at the far end. Since all of the ions have the same charge, their acceleration, and thus the time it takes to reach the detector are completely dependent upon the mass of the fragment. Once the molecules reach the detector, a peak is registered. These peaks are recorded via a high speed recording mechanism. The size of the peak is proportional to the number of molecules that reach the detector at a given time interval.Ions are accelerated by electrodes at opposite end of tunnel of a known length. If it is a negative ion, it accelerates to the cathode, positive ions accelerate towards the anode. The charge depends on the type of molecule being analyzed as well as the matrix used. This electrical force is used to accelerate the particles down tunnel towards the detector at the far end. Since all of the ions have the same charge, their acceleration, and thus the time it takes to reach the detector are completely dependent upon the mass of the fragment. Once the molecules reach the detector, a peak is registered. These peaks are recorded via a high speed recording mechanism. The size of the peak is proportional to the number of molecules that reach the detector at a given time interval.

    7. TOF Analysis… the Math Behind It All Equate accelerating potential to Kinetic energy zV=1/2mv2 Substitute distance / time for v zV= 1/2m(x/t)2 m/z=(2Vx2)/t2 For more precision use calibration standards m/z = A(B-t)2, where A and B are calibration constants Accelerating potential gives the ion an energy of zV. This can be set equal to kinetic energy, which is equal to 1/2mv^2. After substituting distance/time in for velocity, we arrive at the next step. Rewriting the equation gives us the equation in terms of a mass to charge ratio. We know that the charge is going to be one, thus we can solve for the mass. It is often difficult to measure the distance and the V accurately, so calibration standards are usually run. In these standards, the m/z ratio is known, so A and be can be experimentally determined. Thus, the precision is increased.Accelerating potential gives the ion an energy of zV. This can be set equal to kinetic energy, which is equal to 1/2mv^2. After substituting distance/time in for velocity, we arrive at the next step. Rewriting the equation gives us the equation in terms of a mass to charge ratio. We know that the charge is going to be one, thus we can solve for the mass. It is often difficult to measure the distance and the V accurately, so calibration standards are usually run. In these standards, the m/z ratio is known, so A and be can be experimentally determined. Thus, the precision is increased.

    8. Factors Affecting Quality of Output Size of crystals Energy of laser Detector voltage Accelerator voltage Extraction time Number of laser shots Evaporation rate Sample concentration Baseline calculations Scanning settings

    9. Uses of MALDI-TOF Used to characterize and identify large molecules Used in pharmaceutical for QC, monitoring of enzyme reactions Used in DNA sequencing for forensics Used to identify different strains of viruses to help develop vaccines

    10. Figures of Merit

    11. Advantages / Disadvantages Small sample needed Works well with large biomolecules Gives absolute mass measurements Finding matrices to work with samples Ions may collisionally relax Sensitive to contaminants, such as salts Advantages 1. A very small sample size is needed. As little as 1 pmol can give a very resolved spectra. 2. It is often difficult to get large biomolecules/polymers into the gas phase to use in mass spec. Using the low temperatures and high pressure, it is possible to get large molecules into the gas phase with fragmenting them. Unlike SEC (Size Exclusion Chromatography), samples aren’t compared to any other standards, they are absolute measurements of mass. Disadvantages It is often difficult to find suitable matrices to isolate specific polymers. The matrix not only has to isolate the molecule, but also must absorb large amounts of UV radiation so that the sample is not fragmented, but instead vaporized. Being that this is a “soft” ionization technique, a majority of the energy is used volatizing the matrix rather than exciting the ions. Thus, it is possible for ions to relax from the excited state after some collisions. Due to the extreme sensitivity, contaminants can easily interfere with the produced spectra. For example, trace amounts of buffer salts will produce such intense peaks that they will effectively drain out the peaks of the sample. Thus, when these contaminants can be removed, the sample should be prepared at a very high concentration.Advantages 1. A very small sample size is needed. As little as 1 pmol can give a very resolved spectra. 2. It is often difficult to get large biomolecules/polymers into the gas phase to use in mass spec. Using the low temperatures and high pressure, it is possible to get large molecules into the gas phase with fragmenting them. Unlike SEC (Size Exclusion Chromatography), samples aren’t compared to any other standards, they are absolute measurements of mass. Disadvantages It is often difficult to find suitable matrices to isolate specific polymers. The matrix not only has to isolate the molecule, but also must absorb large amounts of UV radiation so that the sample is not fragmented, but instead vaporized. Being that this is a “soft” ionization technique, a majority of the energy is used volatizing the matrix rather than exciting the ions. Thus, it is possible for ions to relax from the excited state after some collisions. Due to the extreme sensitivity, contaminants can easily interfere with the produced spectra. For example, trace amounts of buffer salts will produce such intense peaks that they will effectively drain out the peaks of the sample. Thus, when these contaminants can be removed, the sample should be prepared at a very high concentration.

    12. What’s its future? Will help revolutionize the medical world and will help lead to treatments for many diseases Will be useful for DNA sequencing, thus can be useful for forensic investigations

    13. Sources

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