MS is a technology that measures the mass-to-charge ratio (m/z) of molecules. Mass spectrometers consist of three key components. An ion source that converts molecules into gas-phase ions, a mass analyzer that separates charged molecules according to their m/z and a detector that records the number of ions at each m/z
25 value. Various ionization sources and analyzers can be combined to facilitate proteomic research.
1.2.3.1 Ionization
Two soft ionization methods are used in proteomics due to their ability to produce intact ions from peptides and proteins, matrix-assisted laser desorption/ionization (MALDI) [141] and electrospray ionization (ESI) [142].
In MALDI, matrix (aromatic acids) is used to protect the analytes from being destroyed by laser light and to assist vaporization and ionization. The analytes are embedded into a crystalline matrix on a metal „target‟ plate. The target is then placed in the vacuum of a MALDI source and pulses of laser light (typically a nitrogen laser) are directed at the matrix. The matrix absorbs the laser energy and transfers its charge to the analyte molecules, as the matrix evaporates, analytes are liberated and ionized. The observed ion that contains a neutral molecule [M] is protonated to form a singly charged quasimolecule [M+H] + [117, 141].
In ESI, the sample is presented in a liquid form and thus can be easily associated with online liquid chromatography. The typical solvents are prepared with water, volatile organic compounds (e.g. methanol, ACN) and acetic acid which increases the conductivity. The solution containing the analytes flows into a capillary that is subject to a high voltage (2-3 kV) which forms the solution into a fine spray of highly charged droplets. The flow of droplets is then directed through a counter- current flow of heated gas, causing the solvent to evaporate and the charge concentration of the surface of the droplets to increase. It then reaches a critical unstable state, known as the Rayleigh limit; the droplets deform into smaller and lower charged particles in a process known as Rayleigh fission [143]. The Rayleigh
26 fission is repeated until individually charged analyte molecules remain. ESI generally produces a mixture of singly and multiply charged ions [M+nH] n+ [117, 142].
1.2.3.2 Mass analysis
Four types of mass analysers are commonly used in proteomic research: quadrupole (Q), ion trap, time-of-flight (TOF), Fourier-transform ion cyclotron resonance (FTICR).
Quadrupole mass analysers separate ions based on the stability of their trajectories in the oscillating electric fields. The quadrupole consist of four parallel metal rods, each opposing rod pair is connected together electrically with a radio frequency (RF) potential applied. A direct current voltage is superimposed on the RF potential to make ions travel along the central axis of the rod. Only ions of a certain m/z will reach the detector for a given ratio of potentials while other ions with unstable trajectories will collide with the rods. A range of m/z values can be scanned by continuously varying the voltages [117].
Ion trap mass analysers trap charged molecules using electric or magnetic fields. The Quadrupole ion trap (QIT) is most often used and includes the 3D ion trap (Paul ion trap) and the linear ion trap. In the 3D ion trap, ions are trapped by electric fields produced by a ring-shaped electrode (RF potential) and two end-cap electrodes (dc potential). Ions enter the trap from one of the end-cap electrodes and oscillate at the frequencies that related to their m/z values. By changing the voltages applied to electrodes, ions of certain m/z become excited and are ejected from the opposite end cap [144, 145]. The linear ion trap is similar to the 3D ion trap except that the electromagnetic signals are designed to trap ions in a rectangular-shaped space. Ions
27 are confined radially by a set of quadrupole rods with RF potentials and axially by a static electrical potential on end electrodes. Linear ion trap MS provides increased ion storage capacity (10 times compared to the 3D ion trap) and faster scanning speeds [117, 145, 146].
Orbitrap is a new type of ion trap mass analyser invented by Makarov [147]. It provides high mass accuracy and high-resolution capabilities which has the potential to be useful for proteomic research [148, 149]. It consists of an outer barrel-like electrode and a coaxial inner spindle-like electrode. Ions are trapped and orbit around an inner spindle-like electrode, and oscillate harmonically along its axis. The frequency of these harmonic oscillations is independent of the energy and spatial spread of ions and is inversely proportional to the square root of the m/z. These oscillations are detected using image current detection and are transformed into mass spectra using Fourier transform similar to FITCR [116, 147, 149].
In a TOF mass analyser, ions that are accelerated in an electrical field, then travel through a field-free vacuum tube towards an ion detector. All ions with the same charge receive the same amount of kinetic energy in the source, while the velocity of the ion depends on their m/z. A reflectron with a constant electrostatic field can be used to reverse the path of ions towards the detector. Given the tube length and the measured times of flight, the mass-to-charge ratio of the ion can be calculated. A delayed extraction device can be used to equilibrate the ions which allows the initial velocity of ions to be standardised prior to the entrance of the TOF analyser [117]. FTICR mass analysers provide the greatest capability for mass resolution and mass measurement accuracy. It determines the m/z of ions based on the cyclotron frequency of the ions in a fixed magnetic field. FT mass spectrometers consist of a
28 cubic cell inside a strong magnetic field. Injected ions rotate around the magnetic field with a frequency according to their m/z. By varying the electric fields, changes in the ion frequency of rotation can be measured and converted to m/z by performing a Fourier transform [117, 150].