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Since its introduction more than one hundred years ago, MS has been widely used as an analytical technique, offering excellent sensitivity and selectivity, in addition to providing the molecular weight or structural information of a compound or peptide in a very short time period (Canas et al., 2006). The overall aim of the mass spectrometer is to produce, and subsequently separate ions according to their mass-to-charge ratio (m/z). In order to make separations possible, an electromagnetic field must be generated inside the instrument, making ion movement inversely proportional to the overall mass of the ion and directly proportional to its electrical charge. A mass spectrum is then produced displaying the m/z ratio alongside the relative abundance of each ion. Every MS instrument consists of an ion source, for production of ions from the sample; at least one mass analyser, to separate ions according to their m/z ratio; a detector, to register the number of emerging

43 ions from the protein sample; and finally a computer, to both process and produce mass spectrum of the resulting data (Lane, 2005, Canas et al., 2006, Aebersold and Mann, 2003) (Figure 12).

Figure 12: A simplified schematic of a MALDI-TOF MS arrangement.

Peptide ions are directed through the mass analyser and separated according to their m/z ratio. The detector then measures the number of emerging ions from the sample and relays the information to a computer where a mass spectrum is produced.

For peptides to be separated in an electromagnetic field, they must first be converted into ions and subsequently transferred into the gas phase by use of an ionisation source (Canas et al., 2006). The two most suited methods for the ionisation of peptides include electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI). Ion formation takes place at atmospheric pressure using ESI whilst ion generation using MALDI yields the best results under vacuum conditions (Canas et al., 2006).

MALDI, first developed in the 1980’s by Karas and Hillenkamp, is the ionisation method most commonly utilised when analysing differentially expressed protein spots identified from 2D-PAGE (Aebersold and Mann, 2003). Like ESI, it is a ‘soft ionisation’ technique, but unlike ESI, relies on the utilisation of a matrix solution to ionise the analyte using laser pulses. The most common matrices used in combination with MALDI protocols include α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). The peptide sample to be analysed is co-crystallised with an excess of matrix solution which in turn absorbs the energy from the laser. Typical lasers include nitrogen lasers (337 nm) (Lane, 2005, Mann et al., 2001, Lin et al., 2003) and neodymium:yttrium aluminium garnet (Nd:YAG) lasers. Recently Bruker Daltonics have introduced the SmartbeamTM laser, which incorporates the better attributes of the nitrogen and Nd:YAG lasers, ultimately

44 leading to improved peak intensity. Irradiation of the matrix by any one of the above lasers, results in rapid heating and sublimation of the matrix crystals. Subsequent expansion of the matrix into the gas phase takes with it intact analyte molecules ultimately leading to ionisation of the sample (Lane, 2005).

As ions exit the ion source, they pass through a mass analyser. The mass analyser functions to separate ions according to their m/z ratio, the key parameters of which include mass accuracy, mass range, resolution, sensitivity and the capability of performing tandem MS (section 3.3.2.9) (Lane, 2005). Ultimately, the information obtained from a specific experiment is determined by the performance of the mass analyser. Several different mass analysers exist, each being different in design and performance. The four most common include the ion trap, time-of-flight (TOF), quadrupole and Fourier transform ion cyclotron (FT-MS) analysers (Aebersold and Mann, 2003).

The TOF mass analyser is most commonly coupled to the MALDI ionisation source, to generate peptide mass fingerprint (PMF) information on specific proteins. This analyser is well suited to the pulsed nature of MALDI, and with a high frequency laser, can produce high sample throughput with sensitivity extending to femtomole levels. Essentially the TOF mass analyser consists of a flight tube in high vacuum to ensure collisions do not occur before ions reach the detector. The ions generated from the peptide sample are accelerated by a strong electric field (typically 20 kV) (Canas et al., 2006). Ions of different mass are subsequently separated based on the time it takes to transverse the length of the flight tube and strike the detector. Ions of lower mass reach the detector before those of higher mass. The resulting TOF spectrum is a recording of the signal produced by the detector upon impact of each ion. A typical mass spectrum is achieved by incorporating the relationship between the time it takes to arrive at the detector (t) with the square root of the

m/z ratio value of the ion (Canas et al., 2006). However, MALDI can result in decreased

resolution by broadening peak width. This is caused by differences in energy distribution, by ions of the same mass. If ions of the same mass arrive at the detector at different times, due to differences in kinetic energy, it results in peak broadening and hence decreased resolution. To combat this problem two techniques were introduced. Firstly, delayed pulse extraction (or pulsed ion extraction). This allows for differences in kinetic energy between ions of similar m/z values to be corrected by enabling ions to expand in the field free region in the source, before a voltage pulse is applied. By using this method, ions with higher

45 initial energy (that would move faster through the flight tube) are exposed to less electric potential, whilst ions with lower initial energy (move slower through the flight tube) are exposed to more electric potential, hence enabling ions of the same mass to arrive at the detector together therefore increasing resolution by narrowing peak width. Secondly, resolution was increased further by the incorporation of an ion reflector at the end of the flight tube. The ion reflector is essentially a mirror that creates a retarding field to deflect ions, sending them back along the flight tube. Highly energetic ions penetrate the retarding field more deeply, enabling them to travel a longer flight path, and subsequently arrive at the detector at the same time as ions of the same mass, but with less energy (Lane, 2005). Once ions collide with the detector a PMF spectrum is produced (Figure 13).

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Figure 13: A schematic of a reflectron TOF mass analyser

The TOF mass analyser separates ions of different mass based on the time taken to transverse the flight tube and strike the detector. Mass resolution using the TOF mass analyser can be increased by (1) delayed pulse extraction, which corrects for differences in kinetic energy between ions of the same m/z value, by exposing them to different electric potentials and (2) the presence of an ion reflector, which creates a retarding field, and hence a longer flight path for ions of higher energy, subsequently enabling them to arrive at the detector at the same time as ions of similar mass, but with lower energy.