MS instruments

MS measures accurately the mass-to-charge ratio (m/z) of a molecule. A general mass spectrometer is composed of three elements [173]: an ion source ensuring that sample molecules are ionised and brought into the gas phase, a mass analyser(s) separating the gas- phase ions on the basis of mass-to-charge ratio using electromagnetic fields in a vacuum, and a detector responsible for recording the presence of ions (Figure 1. 10).

Ion sources

There are two well established ionisation sources commonly used in proteomics: matrix- assisted laser desorption/ionisation (MALDI) [177] and electrospray ionisation (ESI) [178]. MALDI sources are typically based on a pulsed nitrogen UV laser and generate singly charged peptide ions, while ESI sources typically heat a needle to between 40 °C and 100 °C to assist nebulisation and evaporation of samples, thereby generating singly and multiply charged peptide ions (2+, 3+, 4+ ). Both methods are sensitive, but MALDI is more tolerant than ESI to small amounts of contaminants, such as salts and detergents [5].

Mass analyser

The mass analyser is the core of MS technology. There are four commonly used types of analyser: quadrupole (Q) [179], ion trap (IT) [180], time-of-flight (ToF) [181] and Fourier transform ion cyclotron resonance (FT-ICR) [182-183]. Each of the analysers possesses distinct advantages and limitations due to their physical principles and performance standards, as well as their mode of operation. Depending on the purpose of the proteomics study, one can choose the appropriate analyser to support the chosen analytical strategies. Because no instrument can currently offer all capabilities simultaneously, the determination of which platform and strategy is preferred is restricted from the outset: the focus is either

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on identification or quantification. In the case of prioritising identification, resolving power (to obtain improved separation of the various components) and mass accuracy are important. On the other hand, sensitivity, dynamic range and multiple reaction monitoring (MRM) capability are the emphasis in the case of an experiment focusing on quantification. In ToF analysers, the velocity of the ion through a tube of specific length is related to the mass-to-charge ratio and therefore mass separation can be achieved by different time of flight of ions towards the detector [181]. ToF analysers can be operated with both MALDI and ESI ion sources and their performance has significantly improved in the last few years [5]. Several combinations of ToF with other analysers, such as Q-Q-ToF and ToF-ToF have been used to improve the sensitivity, resolution and mass accuracy [5, 184].

In IT analysers, effective trapping of ions in an oscillating electrical field leads to accumulation of these ions over time in a physical device [180]. Mass separation is acquired by tuning the oscillating field to eliminate only ions of a specific mass. IT possesses unrivalled sensitivity and fast data acquisition but is limited by low resolution and ion trapping capacity [185]. The dynamic range and overall sensitivity of IT instruments was improved by the development of the linear ion trap (LIT or LTQ), as this instrument offers a unique capability for analysis of PTMs [186].

In the quadrupole analyser, which consists of four rods forming two pairs running different current modes, ions are filtrated by high-pass mass between the first pair of rods and by low-pass mass between the other pair of rods. This filtration can be fine-tuned to overlap only in a specific mass-to-charge ratio interval. Ions with m/z values outside this interval will be excluded. Quadrupoles are usually applied in conjunction with other analysers (e.g.: Q-Q- ToF, Q-Q-Q, QQ-LIT) to expand either sensitivity or selectivity [5, 184].

In the FT-ICR analyser, the ions are trapped in orbits by a very strong magnetic field in a particle accelerator called a cyclotron, while being accelerated by an applied voltage [182- 183]. The cyclotron frequency is related to m/z and Fourier transformation is required to extract individual ion frequencies. This technique allows measurement to accuracies in the low ppm to sub-ppm range. High resolution leads to better quality data and boosts the peak capacity, therefore this instrument can detect more signals compared to low resolving power instruments.

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Figure 1. 10: Mass spectrometry instruments. The basic physical principles of each component of the mass spectrometer are shown. MALDI and ESI are ionisation sources and introduce sample into the analyser. Q analyser select by time-varying electric fields between four rods, which permit a stable trajectory only for ions of a particular desired m/z. In the IT, ions are trapped in an oscillating electrical field leading to accumulation of these ions over time. The FT-MS instrument also traps the ions, but does so with the help of strong magnetic fields. In Orbitrap, ions are captured and form an orbital harmonic oscillation along the axis of the field with a frequency characteristic of their m/z values. In TOF, the ions are accelerated to high kinetic energy and are separated along a flight tube as a result of their different velocities. At the detector (EM) ions are impinged on several Faraday cup dynodes, amplified and counted. Figure adapted from the lecture of Dr. Martens, EBI, Cambridge, UK.

Ion sources

Mass analysers and detectors

sample ion source mass analyser(s) detector digitiser Generalised mass spectrometer

MALDI ESI

laser irradiation

target surface

desorption proton transfer

analyte matrix molecule H+ +   h + + + + + + + + + + + + ++ + + + +++ + + + ++ Gas phase high vacuum 0 + 0 + 0 0 0 0 0 0 0 0 0 + + + + + + + + + + + + + + + + + + +₊ + needle barrier + 3-5 kV droplet evaporation and charge-driven fission or ion expulsion m/z analyzer inlet 0 0 0 0 0 0 0 0 0 0 0 0 evaporation only nebulisation N2 N2 sample ring electrode capping electrode capping electrode source detector DC/ACRF voltage Permitted m/z ejected m/z ejected m/z ) cos (UV t  ) cos (UV t  Q IT single ion in 106_{electrons out} 20V 60V 100V 40V 80V 120V extraction plate (30 kV) field-free tube (time-of-flight tube) > 1 meter high vacuum sample ions TOF EM

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In recent times, a new type of mass analyser, the Orbitrap [187], based on a new physical principle, was introduced to the field of proteomics [188]. It consists of an outer and inner coaxial electrode, which generate an electrostatic field. In the Orbitrap, ions are captured and form an orbital harmonic oscillation along the axis of the field with a frequency characteristic of their m/z values. This frequency is measured with very high precision and can then be calculated by Fourier transform. The performance of the Orbitrap is similar to the FT-ICR, but this instrument is cheaper (because it does not require an expensive superconducting magnet), much more robust and simpler to maintain [189-190]. Used in conjunction with a linear ion trap (LTQ), a new mass spectrometer named the LTQ-Orbitrap (introduced by Thermo Fisher Scientific [191]) demonstrated the combined advances of both techniques in robustness, sensitivity and MS/MS capability, possessing very high mass accuracy and high resolution capabilities, and has become a powerful tool in proteomics studies.

Detector

The most common type of detectors are electron multipliers (EM) [192], which are based on several Faraday cup dynodes with growing charges to generate an electron cascade based on a few incident ions.

MS mode of operation

Both MS mode and MS/MS mode are widely applied in proteomics. In MS mode, only one MS selection takes place and provides peptide mass spectra that allow subsequent identification of the parent proteins using peptide mass fingerprinting (PMF) [176, 193]. In contrast, MS/MS, also known as tandem-MS, employs two mass analysers in series (tandem) with some form of fragmentation taking place in between each MS selection [5]. The first mass analyser serves as an ion selector, by selectively passing through only ions of a given m/z. The second mass analyser positioned after fragmentation is triggered and exerted in its normal capacity as a mass analyser for the fragments. Tandem-MS allows determination of the amino acid sequence of a specific peptide, which can also be used for identification of parent proteins by PMF but with more confidence in the identification results. Various types of tandem-MS experiments are reviewed in several publications [5, 103, 175, 190, 194].

43 Fragmentation methods

Several fragmentation techniques are utilised in proteomics, such as collision-induced dissociation (CID) [195], post-source decay (PSD) [196], electron-capture dissociation (ECD) [197-198] and electron-transfer dissociation (ETD) [199]. CID is the most widely used method and relies on multiple collisions events of a gas-phase peptide with rare gas atoms to provide the peptide precursor with sufficient energy to fragment. CID typically causes backbone fragmentation [195]. On the other hand, PSD is based on a single unimolecular event activated by laser energy, in which a highly energetic (metastable) ion spontaneously fragments [196]. PSD also typically causes backbone fragmentation. ECD and ETD rely on a single impact of an electron with a peptide precursor either by capture of a thermal electron or transfer of electrons from radical anions to the protonated peptide/protein. This high- speed impact immediately imparts sufficient energy to fragment the precursor. Like CID and PSD, ETD and ECD also typically produce backbone fragmentation, however ECD is only workable in FT-ICR mass spectrometers, whereas ETD is used in traps.