Technology - MALDI-TOF MS principles

Matrix-Assisted Laser Desorption Ioniziation (MALDI-TOF) MS is a relatively recent analytical method developed in the 1980’s (Karas & Hillenkamp, 1988) and since then it has been used in many different fields for peptide, protein and nucleic acid analysis.
In all mass spectrometrical techniques three basic components are essential: ionization, separation, and detection. The ionization of an analytic molecule is the prerequisite for separation in an electromagnetic field and for the detection in an ion detector.

MALDI is one of many vaporization and ionization methods that has been developed. MALDI actually accomplishes both vaporization and ionization in a single step and its applicable for fragile biomolecules (proteins). In MALDI-TOF MS a dissolved sample is mixed with a solution of an appropriate matrix and allowed to co-crystallize directly on special sample plates, most simply by room-temperature evaporation. The ratio of analytic molecules to matrix molecules should be in the order of 1:103 to 105 to not to interfere with the formation of crystals into which analytic molecules have to be incorporated. Typical MALDI matrices are low molecular weight aromatic, organic acids like a-cyano-hydroxy cinnamic acid (HCA, 189.17 Da) or 2,5-dihydroxy-benzoic acid (DHB, 154.15 Da).  Due to the required UV-absorbing chromophore in matrix molecules a minimum molecular weight of some 130 Da is given and thus for analyses in the lower mass ranges other matrices have to be used. The sample plate with matrix-analyte crystals is then placed in the high-vacuum of the machine (p < 10-7 Torr) where its actual position can be controlled and monitored on a video screen. A laser beam of l=337 nm (nitrogen laser) is focused and hits the sample in pulses of 0.5-2 nsec duration. The absorbtion of the photonic energy of each laser pulse leads to the desorption of the crystal and the formation of a ‘cloud’ of partly ionized matrix and analyte molecules and the ionization of analyte molecules by charge transfer. The process of desorption and ionization is not fully understood and the optimation of analytcal protocols is mainly based on emipirical data (Karas et al., 2000). Ionized molecules are then accelerated in an electromagnetic field built up between the sample plate and a grid electrode at a distance of some few cm (U » 20 kV). For optimal performance, the acceleration startes with a delay of 10-20 msec to the ionization (delayed extraction).


The acceleration in the electromagnetic field is the start of the separation process that is based of the time-of-flight principle (in contrast to mass spectrometers working with quadrupoles). After passing the grid electrode the ions enter a field-free drift range where they are not accelerated further and thus travel with a speed they have reached at the moment when passing the electrode. This speed, in turn, depends on the mass of the ions with heavier molecules having a higher moment of inertia and thence a lower velocity. For the resolution of the mass spectral analyses the length, L, of the field-free drift range is essential and in modern machines it measures about one meter. Further increase in resolution can be reached in the reflector mode: after having passed a distance in the drift range the ions enter another electromagnetic field and are accelerated in a nearly reversed direction towards an ion detector. Ion detection is accomplished with a conversion dynode with secondary electron amplification. The output of the detector is a time-resolved plot for each single laser pulse.

The relationship of time-of-flight to molecular mass can be described in the equation

T = L (m/2 z e U)1/2

The TOF mass spectrometer operates in a pulsed mode.

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