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(TOF) Mass Spectrometry

Matrix assisted laser desorption ionization (MALDI) is a process that enables the transfer of compounds in a solid, crystalline phase to the gas phase in an ionized state thus allowing their measurement by mass spectrometry(34-36). The process involves mixing the analyte of interest with a strongly ultra-violet absorbing organic compound, applying the mixture to a surface and then allowing it to dry. Examples of typical organic matrix compounds used are 2,5- dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxy-trans-cinnamic acis (sinapic or sinapinic acid), and α-cyano-4-hydroxy-trans-cinnamic acid (α-CHCA). There are many techniques described for applying the matrix to the target such as the “dried droplet” method(36,37), and the “thin film” method(38). The dried crystalline mixture “film” or “spot” is then irradiated with a nitrogen laser (337 nm), or an Nd-YAG laser (266 nm). The strongly UV absorbing species accepts energy from the laser and desorbs from the surface carrying along any analyte that is mixed with it. The analyte is cationized in the plume above the crystalline surface with, in the positive mode, either a hydrogen proton or a metal cation such as sodium.

The desorbed, ionized compounds from the matrix assisted laser desorption ionization process are then introduced into a mass spectrometer for analysis. The most common mass spectrometer that is coupled to MALDI is the time-of-flight mass spectrometer (TOF MS)(39,40). The TOF mass spectrometer separates compounds according to their mass to charge ratios (m/z) through a direct relationship between a compounds drift time through a predetermined drift pathlength and its mass to charge ratio (m/z). Initially, all the ions have similar kinetic energies imparted to them from the draw-out pulse (representing time zero) which accelerates them into the flight tube. Because the compounds have different masses their velocities will be different

according to the relationship between kinetic energy and mass represented by: KE = eV = 1/2mv2. From this expression, the mass to charge ratio is related to the ions flight time by the following expression: m/z = 2vt2/L2. There is a high transmission efficiency of the ions into and through the drift tube which equates to very low levels of detection limits which are in the femtamole to atamole ranges. Theoretically, the mass range of the spectrometer is unlimited due to the relationship of drift time for mass measurement. In practice though, the sensitivity needed to detect a very slow moving large molecular weight compound limits the TOF-MS to 1 to 2 million Daltons or so.

The early designs of time of flight mass spectrometers suffered from poor resolution which is the ability of the mass spectrometer to separate ions. This is usually expressed as resolution = m/∆m where m is the mass of the peak of interest and ∆m is the difference between this mass and the next closest peak. The poor resolution was due to nonuniform initial spatial and energy distributions of the formed ions in the mass spectrometer’s ionization source. Due to the distribution spreads the mass resolution was directly dependant upon the initial velocity of the formed ions. Early work reported by Wiley et al.(41) reported upon the correction for initial velocity distributions using a technique they described as “time-lag energy focusing” where the ions were produced in a field-free region, then after a preset time a pulse was applied to the region to extract the formed ions. They also reported upon a way to correct for initial special distributions through a two field pulsed ion source. They demonstrated though that correction can only be made for one of the distributions at a time. In MALDI, the spatial distribution spread is not a significant problem, therefore “delayed extraction” of the ions from the field free source region should correct for the initial energy distribution spread of the formed ions(42). With this correction, the resolution should be directly dependent upon the total flight time ratioed to the

error in the time measurement, thus an increase in the length of the flight path should equate to a higher resolution. The resolution enhancement due to the delayed extraction initial energy focusing and flight path was demonstrated in a study reported by Vestal et al.(42) using a MALDI-TOF mass spectrometer.

A second modification of time of flight mass spectrometers involves time focusing of the ions while they are in flight through the use of an electrostatic mirror at the end of the flight tube(43,44). The electrostatic mirror focusing the ions with the same mass to charge ratio but slightly different kinetic energies by allowing a slightly longer path for the higher KE containing ion compared to a slightly lower KE ion thus allowing the ions to catch up to one another. The electrostatic mirror also increases the flight path length of the mass spectrometer, thus providing a double focusing effect. All recent TOF-mass spectrometers employ both delayed extraction and the reflectron electrostatic mirror for analysis of compounds below 10 kDa. For compounds greater than 10 kDa a linear mode is typically used where the electrostatic mirror is turned off.