Desorption (Phase Transition)

Several theories have been developed to explain the desorption of large molecules by MALDI. The thermal-spike model proposes that the matrix molecules sublime from the surface as a result of local heating at low laser influence. Desorption in MALDI is the phase transition of samples from the condensed phase to the gas phase. It is a prerequisite for molecules to be detected by MALDI mass spectrometry. Desorption processes, including thermal desorption occurring at low and moderate laser energy density and phase explosion happening at high laser energy density, were studied quantitatively on both experimental and theoretical bases. Results of these studies suggest that factors affecting the desorption efficiency can be classified into two categories: excitation parameters (Hornefer et al., 1999) and intrinsic properties of samples (Jaskolla et al., 2009; Mowry and Jhonston, 1994; Schulz et al., 2006). Generally, desorption efficiency increases when MALDI crystal size decreases and the system temperature increases (Schulz et al., 2006). On the basis of factors influencing desorption efficiency, mathematical equations as well as computational methods, such as molecular dynamics (MD) simulations, have been used to quantitatively describe the desorption process (Zhigilei et al., 2003).

Mass Analyser – Time of Flight (TOF)

There are three types of mass analysers typically used with the MALDI ionization source: a linear time-of-flight (TOF), a TOF reflectron, and a Fourier transform mass analyser. The linear TOF mass analyser is the simplest of the three devices and has enjoyed a renaissance with the invention of MALDI. TOF analysis is based on accelerating a set of ions to a detector where all of the ions are given the same amount of energy. Because the ions have the same energy, but a different mass, the ions reach the detector at different times. The smaller ions reach the detector first because of their greater velocity while the larger ions take longer owing to their larger mass. Hence, the analyser is called TOF because the mass is determined from the ions’ time of flight. The arrival time at the detector is dependent upon the mass, charge, and kinetic energy (KE) of the ion. Since KE is equal to 1/2 mv2 or velocity (v) = (2KE/m)1/2. Ions will travel a given distance, d, within a time, t, where t is dependent upon their mass-to-charge ratio (mlz).

The ions then accelerate in a TOF analyser because they are subject to an equal electric field. TOF is a field-free flight tube. The ions travel in a straight and linear direction to the detector. The mass to charge ratio (mlz) of the sample ions can be calculated using the Equation 3.5.

Cl and C2 are instrumental constants, which can be determined with compounds of known mass.

The TOF reflectron combines TOF technology with an electrostatic analyser, the reflectron. The reflectron serves to increase the amount of time, t, and ions need to reach the detector while reducing their KE distribution, thereby reducing the temporal distribution 1 It. Since resolution is defined by the mass of a peak divided by the width of a peak or m/lm (or t = since m is related to t), increasing t and decreasing 11 results in higher resolution. This increased resolution, however, often comes at the expense of sensitivity and a relatively low mass range, typically <10,000 mlz. One innovation that has had a dramatic effect on increasing the resolving power of MALDTTOF instruments is delayed extraction (DE). DE is a relatively simple means of cooling the ions (and possibly focusing them) immediately after the MALDI ionization event. In traditional MALDI instruments the ions are accelerated out of the ionization source as soon as they are formed. However, with delayed extraction the ions are allowed to “cool” for 150 nano-seconds before being accelerated to the analyser. This cooling period generates a set of ions with a much lower KE distribution, ultimately reducing the temporal spread of ions once they enter the TOF analyser. Overall, this results in increased resolution and accuracy.

Fragmentation

Fragmentation techniques are essential methods to deposit energy into a targeted ion to cause reproducible bond cleavages. Cleavages yield diagnostic and interpretable fragment ions that reveal structural or sequence information about the molecule of interest. Commonly used fragmentation techniques in MALDI mass spectrometry include laser-induced promptly in-source decay (ISD), post-source decay (PSD, a unimolecular dissociation of metastable ions), and collision-induced dissociation (CID). ISD ions are promptly produced by thermal activation and/or chemical reactions during desorption/ ionization processes in the source region, such as hydrogen radical-mediated and/or free electron-mediated reactions. More energy input and higher energy-coupling efficiency between matrices and analytes enhance the ISD process. Because ISD ions receive the full acceleration energy in the ion source, they can be well-resolved in the mass spectra. On the other hand, PSD ions are typically generated on a time scale of micro- to milliseconds in the field-free region. Owing to having the same velocity as their precursor ions, PSD ions cannot be resolved in linear TOF instruments. The energy liberated by gas-phase proton transfer reactions can greatly influence fragmentation. For oligonucleotides, Zhu et al. (1995) have proposed that the loss of protonated base, followed by backbone fragmentation and disproportionation, ultimately leads to anionic fragments. Consequently, the difference in proton affinity between the analyte and matrix should determine the energy available. For reducing the fragmentation of oligo-deoxynucleo- tides, matrices with relatively high proton affinities should, therefore, be chosen (Zenobi and Knochenmuss, 1998).

 
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