10.2.1 Gas‐Phase Ion Generation of Biomolecules

To attain quantitative and qualitative data with extreme precision and accuracy by MS, efficient ionisation techniques are first required to generate analytes suitable for transmission into the mass analyser. Unless they are already in the gas phase, analytes need to be first vaporised, which can occur by a variety of methods including exposure to heat, high electric fields, laser irradiation and/or bombardment with atoms or ions. Analyte ionisation may occur before, during and after transfer to the gas phase, and can give rise to ions with either a positive or negative charge that can be selected by controlling the polarity of the applied electric field at the ion source. Selection of an appropriate ionisation method is critical and can often be the most significant determinant of a successful MS analysis.

Since the vapour pressure of biopolymers is negligible and biomacromolecules are often susceptible to degradation at high temperature, ion desorption techniques are principally required for the ionisation of analytes in biomolecular MS. Such ionisation approaches include ESI (and its variants such as nanoelectrospray and desorption electrospray ionisation, DESI) and MALDI. The type of analyte under investigation or the question to be answered will dictate which ionisation type is the most applicable and practical. While an incredible number of alternative ionisation approaches have been reported, for practicality here we focus only on those that are commonly applied in contemporary biomolecular analysis.

10.2.1.3 Electrospray Ionisation

In ESI, gas‐phase ions are generated from molecules in solution by passing the sample through a capillary at a low flow rate, typically in the nl/min to μl/min range. The tip of the capillary is held at a high voltage (typically 1.5–4 kV) [5] with respect to a counterelectrode and positive or negative ions are selected by controlling the polarity of this applied field. Charge accumulation at the capillary terminus causes the liquid surface to adopt a conical shape known as a ‘Taylor cone’, the tip of which is drawn into an elongated filament that becomes unstable as the charge density increases [15,16]. At a given point, this instability results in a spray of finely divided and electrically charged droplets. As the charged droplets move across both an electric field gradient and a pressure gradient towards the counterelectrode and high vacuum components of the mass spectrometer, their size decreases as the solvent evaporates. Typically, desolvation is assisted by either a weak counterflow of hot nitrogen gas or passage of the ions through a heated capillary. With evaporation of the solvent, charge concentration in the droplets increases to the point where Coulomb repulsion overcomes surface tension (the Rayleigh limit), ultimately resulting in fission of the droplets and a repeated size decrease and desolvation (Figure 10.2a). Two models are widely proposed for the formation of isolated gaseous ions from ESI, namely the ion evaporation model (IEM) or the charged residue model (CRM) [15]. The IEM proposes that ions are directly emitted from the small droplets due to electrostatic repulsion as the droplet radius decreases. In contrast, the CRM suggests repeated droplet fission continues to the point where no further evaporation can occur. In all likelihood both mechanisms are probable, with low molecular weight analytes following the IEM and the CRM being applicable to large globular species such as proteins [17]. A chain ejection model (CEM) has also been proposed for disordered polymers [18].

ESI is a very ‘soft’ ionisation method whereby little energy is provided to the analyte in the process of transfer to the gas phase. It is largely for this reason that electrospray ionisation mass spectrometry (ESI‐MS) is an essential technique in the study of biomolecules, which are often labile and otherwise susceptible to fragmentation. Furthermore, the non‐covalent interactions often important in the formation of biomolecular assemblies can be representatively transferred into the gas phase, allowing detection of binding interactions and complex stoichiometries [19,20]. ESI‐MS is also a sensitive method; low concentration of analyte (micromolar to femtomolar) is adequate. Finally, another useful feature of the ESI process is the formation of multiple charges on macromolecular ions, where these charges are statistically distributed across the ionisable sites of the analyte, giving rise to multiple peaks in the mass spectrum at differing m/z ratios (Figure 10.3a). Since the mass of large biopolymers typically falls outside the range of many mass analysers, increasing the charge state decreases the measured m/z ratio, effectively extending the mass range of the analyser to accommodate species up to the megaDalton range. The molecular weight of an ion can be determined from the ESI mass spectrum by analysis of any two adjacent charge state signals along the m/z axes, by solving simultaneous equations given the peaks have the same mass and of related charge state (i.e. z and z + 1).

Solvent volatility and acid/base properties are important considerations for ESI and, typically, polar solvents such as methanol, acetonitrile or acetonitrile:formic acid:water mixtures are used to facilitate the ESI process. The analyte can be introduced to the source in solution either from a syringe pump or commonly as the eluent flow from liquid chromatography (LC), allowing direct coupling of ESI‐MS to online separation methods. In the case of protein analysis, these specific solvent conditions induce protein unfolding/denaturation, where the unfolding allows the ionisation of all basic residues (in positive mode), which aids in accurate mass determination (Figure 10.3). To retain physiologically relevant structural information, ESI can also be performed using other non‐denaturing volatile buffers such as ammonium acetate or ammonium bicarbonate [21]. Most classes of biological polymers are amenable to ESI ionisation, though proteins are usually analysed in the positive ion mode while oligonucleotides, for example, tend to be preferably ionised in the negative ion mode given the abundance of anionic phosphate groups.

A variant of ESI using lower flowrates is nanoESI, introduced by Wilm and Mann in 1994 [16]. In this mode of operation, the sample is typically sprayed from a borosilicate glass capillary that has been pulled to a fine tip, and a conductive metal coating (such as gold or platinum) on the outside of the glass capillary or a thin platinum wire introduced from the large end of the capillary is used to supply the electric potential. These experiments require much less analyte solution and, unlike the syringe‐forced flow in normal ESI, the pull exercised by the applied electric field causes, for non‐viscous solutions, a self‐flow controlled by the capillary tip diameter. Due to the very small capillary tip orifices (1–5 μm in diameter) and the absence of external pumping, the primary droplet sizes are much smaller, improving desolvation processes and decreasing sample consumption. Furthermore, the tolerance to salt contamination from buffers or additives common in samples from biological preparations is increased in nanoESI [22].

Developments in biomolecule ionisation have also seen moves towards ambient ionisation approaches, which allow the ionisation of untreated samples in an open environment. In 2004 DESI, the first of now almost 30 ambient ionisation methods for MS, was first described [23]. The DESI experiment directs an electrospray plume at an acute angle (approximately 45°) towards analyte deposited on an insulating surface a few millimetres away. The splashed droplets then carry desorbed, ionised analytes into the atmospheric pressure interface for sampling by the mass spectrometer. Therefore, DESI represents a combination of ESI and desorption mechanisms. Recent studies have shown that data obtained from DESI experiments are comparable to those obtained in nanoESI experiments [24], though with the key advantage of the relatively short time it takes to analyse samples, with minimal sample preparation. The DESI approach also allows biomolecular interactions to be observed ‘on the fly’ as various analytes such as lipids, ligands and cofactors may be applied to the surface and allowed to react on the millisecond scale with other molecules, such as proteins, from the spray source. Finally, DESI can be used to investigate samples in situ, specifically to observe both spatial and temporal distributions [25]. However, some disadvantages to DESI, including low spatial resolution (200 μm), lower mass acquisition range (up to 2000 m/z compared to MALDI reaching 50 000 m/z) and lower ionisation efficiency have limited its more widespread use.

10.2.2 Mass Analysers

Once ions have been generated and transferred to vacuum regions of the mass spectrometer, they can be selectively and specifically manipulated using an array of MS approaches. In all cases the underlying principles of measurement are the same, which relate to the ability of magnetic and/or electric fields to influence the motion of charged atoms and molecules. As described by Newton's second law of motion, the acceleration of an object as a result of a net force is directly proportional to (and in the direction of) the magnitude of the force and inversely proportional to the mass of the object. Consequently, the trajectory of two identically moving and equally charged particles will be altered to different extents under the influence of an electric and/or magnetic field if they have a different mass. Similarly, the particle's charge is important as, according to the Lorentz force law, it relates directly to the extent of the force developed by the applied field. Therefore, by controlling the applied forces to manipulate particle trajectories, packets of ions with the same m/z ratio are separated in space or time from other ions and selectively detected. It is possible to deconvolve these ion motions to infer the mass‐to‐charge ratio (m/z) of distinct particles, forming the basis of the mass measurement. Results are usually presented in the form of a mass spectrum, with the m/z ratio plotted on the x‐axis and the signal intensity plotted on the y‐axis.

After selecting an appropriate ionisation method, the next major consideration in an MS experiment is the choice of mass analyser. There is a wide range of mass analysers available, differing in their compatibility with various ion sources. For example, continuous mass analysers that maintain a continuous flow of ions are compatible with ESI, while pulsed mass analysers are compatible with MALDI or otherwise require accumulation of ions for pulsed injection to be compatible with continuous ionisation sources. Analysers also vary in their ability to investigate ions of certain types, as well as in differing analytical considerations, such as sensitivity, accuracy, mass range and duty cycle, amongst others (Table 10.1). Sector, ToF, quadrupole, ion trap and ion cyclotron resonance (ICR) mass analysers evolved in parallel and are now the most commonly used spectrometers for the study of biomolecules [27]. The operating principles of these instruments are described in more detail here.

10.2.2.1 Sector Analysers

The first commercial mass spectrometers available in the 1940s employed magnetic sector mass analysers based largely on the prototypes developed by Arthur Dempster [28] and remain widely utilised in the MS to date. In these instruments, ions leaving the source are focused and accelerated to a high velocity before passing through a magnetic sector. Here a magnetic field is applied perpendicular to the ion motion, causing the ions to be directed on a curved trajectory (Figure 10.4). If all ions are accelerated to the same kinetic energy prior to introduction to the magnetic field, then ions of a specific m/z will have a unique radius of curvature (r) (for a constant velocity v and magnetic field strength B), as given by Eq. (10.1), where e is the unit of elementary charge:

10.1

Since ions of higher m/z ratio are deflected to a lesser extent than those of lower m/z, scanning the magnetic field enables ions of different m/z to be focused on to an exit slit for selective transmission of a particular ion or, alternatively, a detector array can be used for simultaneous recording of multiple m/z ions with varying spatial location.

Later developments in magnetic sector analysers led to significant improvements in resolution and other performance characteristics. Perhaps the most significant of these was the production of a double‐focusing instrument [28], which adds a second sector with an electrostatic field (‘E’ sector), either before or after the magnetic sector (‘E’ sector), known as EB or BE geometries, respectively. A mass spectrum is usually obtained by scanning the magnetic sector over a desired m/z range, while the electrostatic field of the E sector is kept constant to allow passage of ions whose kinetic energy‐to‐charge ratio is equal to eV (where V is the acceleration voltage). Since mass resolution in sector instruments is dependent on both the spatial dispersion of the incoming ion beam as well as the ion kinetic energy spread, the magnetic sector filters ions with differing momentum and the electrostatic mass analysers are efficient ion kinetic energy filters. Hybrid instruments combining these two analysers therefore enable double focusing, and hence offer extremely high resolution.

Sector analysers are well suited to monitoring selected ions at a single m/z with high resolution, particularly useful in quantitative applications. Additionally, the presence of a second analyser allows MS/MS experiments to be performed. In this case, ion fragmentation experiments occur in a high collisional energy regime, allowing access to fragments that are often not otherwise detected.

10.2.2.2 Time‐of‐Flight Analysers

ToF mass analysers were conceptualised more than 50 years ago, with the first report of a ‘pulsed mass spectrometer with time dispersion’ in 1946 [29]. They are based on very simple physical principles, whereby in the ToF analyser an accelerating potential (V) will give an ion of charge (z) an energy of zV, which can be equated to kinetic energy (E _kin ) according to

10.2

where m and v are the mass and velocity of the ion, respectively. Since velocity can be related to distance (d) and time (t), the above equation can be rewritten as shown below:

10.3

If the ions travel a fixed distance to a detector through a field free region, those of larger m/z will have a longer ToF. Therefore, the differences in the time taken for ions to arrive at the detector from the pusher are determined and converted to a measure of m/z in the mass spectrum (Figure 10.5). With no theoretical upper mass limit, ToF mass analysers are ideally suited to the study of biomacromolecules, particularly coupled with MALDI.

In what is termed the ‘linear mode’, ions extracted from the source are unidirectionally accelerated by application of an electrostatic field in short pulses, moving into a drift space containing no field (Figure 10.5a). A limitation of this configuration is that not all ions have the exact same initial position and velocity, and therefore a spread in arrival time results, leading to the formation of broad, low amplitude signals and hence limiting resolution and sensitivity. One approach to improve resolution in ToF mass analysers involves orthogonal acceleration of ions. Here ions are typically sampled from an ion beam from a continuous ion source. The applied electric field is designed to generate a pulsed force that is exclusively at right angles to the initial axis of the ion beam. As the beam is nearly parallel, the ions in it have zero average velocity in the direction of this force prior to its application, therefore reducing the complication of initial velocity dispersion [30]. Correction of the initial energy distribution can also be achieved using a reflectron device consisting of a series of electric fields that repulse the ions back along the flight tube, usually at a slightly displaced angle (Figure 10.5b). Here, for two ions with identical m/z but different velocities, the faster ion will penetrate deeper into the decelerating region of the reflectron and hence have a longer flight path, compensating for its greater velocity. Firstly, the resultant increase in ion path lengths leads to greater separation between packets of ions and, secondly, the ions are thus refocused, thereby greatly increasing the resolution of the measurement.

The advantages offered by ToF instruments including a very high duty cycle, high transmission efficiency, extended m/z range, fast repetition rates and compatibility with pulsed ionisation sources make them very popular analysers suitable for a wide variety of applications in the biological sciences.

10.2.2.3 Quadrupole Mass Filters

‘Mass filter’ devices achieve ion separation due to their ability to selectively maintain stable trajectories for ions of certain m/z ratios, while others become unstable. One such example, the quadrupole mass analyser, therefore can be considered as a ‘tunable’ mass filter that transmits ions within a narrow m/z range (typically a 1 m/z transmission window). The quadrupole analyser is comprised of four precisely parallel rods with a hyperbolic cross‐section, to which a direct current (DC) and alternating RF potentials are applied to produce oscillating electric fields [31]. Ions are introduced in a continuous beam by means of a low accelerating potential along the central axis. The ions are sequentially repelled and attracted by the pairs of rods due to the oscillating fields, and hence they oscillate in the yz‐ and xz‐planes as they travel through the quadrupole filter. Only ions of a particular m/z are able to traverse the quadrupole region and all other ions are radially ejected or hit the rods (Figure 10.6a).

The ion path transforms to a complex spiral‐like propagation, according to the Mathieu equation [32], regardless of the ion's initial velocity or initial position. Stable solutions of Mathieu's equation are usually simplified by defining the a and q terms, where a is proportional to the DC and q is proportional to the RF. These solutions are commonly presented as ‘stability diagrams’ (Figure 10.6b), highlighting the limited number of combinations of a and q that lead to stable trajectories for ions of a defined m/z to traverse the quadrupole analyser. The quadrupole is operated with a fixed ratio of DC and RF voltages, which determines the resolution of the device. Varying the voltages while keeping this ‘working’ ratio fixed gives a scan line that passes consecutively through respective regions of stability for ions of differing m/z. In this way, a mass spectrum can be obtained for ions with m/z ranging from small to large. Another important consideration from the stability diagram is that the quadrupole will allow passage of all ions if the DC component is set to zero. This is commonly termed the ‘rf only mode’, whereby the quadrupole simply operates as an ion guide rather than an ion filter.

The m/z range of a typical quadrupole mass spectrometer is limited to m/z 2000, though it is possible to increase this by variation in the applied electric fields, albeit at a cost in resolution. The scan rate of a typical quadrupole mass spectrometer is high enough to allow direct coupling to chromatographic separation and this type of mass analyser is extensively used in tandem MS experiments (described later).

10.2.2.4 Quadrupole Ion Traps

The notion of utilising a quadrupolar field to trap and store ions arose as a natural extension of quadrupole mass filter development and introduction of the 3D ion trap represents an important development in quadrupole technology. A quadrupole ion trap device confines ions by the formation of a potential well, when appropriate potentials are applied to three electrodes (two end‐caps and one ring electrode) of hyperbolic cross‐sections. The internal volume of a typical 3D ion trap is approximately 1 cm³.

Initially, ions are pulsed into the analyser under the influence of a field that maintains them in a stable oscillation (produced by an appropriate low RF potential applied to the central ring electrode), and they are therefore ‘trapped’ (Figure 10.7). The amplitude and frequency of the applied RF fields determine the mass range of trapped ions. A helium bath gas is used to stabilise the ion trajectories in the trap by acting as an energy sink, keeping the ions in tight orbits in the centre of the trap. Filling the ion trap in practice leads to enhanced full‐spectrum sensitivity compared to the linear quadrupole mass analyser, but too many ions adversely affects mass resolution and accuracy due to space‐charge effects. To acquire a mass spectrum, the fundamental RF voltage applied to the ring electrode is ramped and resonant absorption of energy progressively increases the amplitude of oscillations of the ions, destabilising the ion trajectories. Ions of increasing m/z are therefore sequentially ejected from the trapping volume to the detector, and based on the frequency being used at the time of detection, the m/z of the ion can be calculated.

A quadrupole ion trap instrument has a limited mass range and lower resolution compared with a ToF analyser, but transmission can be enhanced when proper ion populations are provided by pre‐accumulation. Importantly, in a similar fashion to the quadrupole filter, superimposing RF and DC potentials on the ring electrode allows for storage of ions of a particular m/z. Once the ion of interest has been isolated, they can be induced to undergo energetic collisions with the bath gas and the increase in ion internal energy can give rise to fragmentation. Scanning the fragment ions gives rise to an MS/MS spectrum, or alternatively a specific fragment can be selected for a further round of fragmentation and structure interrogation, in what is known as multistage tandem MS or simply MS ⁿ experiments. Typically, MS⁵ can be achieved based on the ion capacity of the trap, as the ion population is depleted with each subsequent MS stage.

More recently, 2D or linear versions of the ion traps have been introduced, which creates a cylindrical space for the ion cloud by a radially confining 2D RF field. Such analysers provide much greater storage capacity and avoid the limitations imposed by space‐charge distortion.

10.2.2.5 Ion Cyclotron Resonance

Ion confinement to a limited volume can also be achieved by using a combination of electrostatic and magnetic fields, and gives rise to instruments that currently provide highest mass accuracy in the form of a Fourier transform–ion cyclotron resonance (FT‐ICR) MS. In an FT‐ICR instrument, a strong magnet surrounds an ICR cell consisting of three pairs of electrodes (trapping plates, excitation plates and receiver plates). In the magnetic field, ions of a given m/z describe cyclotron motions with a radius r perpendicular to the magnetic field lines (Figure 10.8a). The principles of mass measurement in ion cyclotron resonance mass spectrometry (ICR‐MS) relate to the ion cyclotron resonance frequency (f) of each ion as it rotates in a magnetic field (B). A spectrum is obtained by scanning the magnetic field of an electromagnet to bring ions of different m/z to resonate, based on

10.4

For a population of ions whose orbiting motions are in phase, an image current will be induced on detector plates with a characteristic angular frequency. Conversion from the time domain to the frequency domain through Fourier transformation allows the cyclotron frequency of each population to be determined and corresponding m/z values measured.

Since resolution is linearly increased with increasing magnetic field strength, large magnets up to 25 T are employed. Furthermore, the method is non‐destructive, so the ions can be monitored for longer periods to increase resolution. Finally, frequency is a physical parameter that can be measured very accurately and, consequently, FT‐ICR‐MS offers the advantage of being able to detect ions with exceedingly high resolution, typically exceeding 10⁵ (defined as m/Δm _x ) in commercial instruments and far outperforming other mass analysers excluding the orbitrap. While image current detection is generally less sensitive that other ion counting methods used in other instruments, data acquisition can be carried out with the same ion population over an extended period of time, thereby enhancing the signal‐to‐noise ratio to the point that FT‐ICR has been used to trap and detect individual macromolecular ions [33].

Fragmentation of the ions isolated in the ICR cell can be induced by a variety of means, including collision‐induced dissociation, photoactivation and electron‐based methods. Since FT‐ICR MS is a trapping‐based method, multiple stages of fragmentation can be performed in sequence, often utilising a combination of activation techniques to improve structure interrogation.

10.2.3 Ion Detection

The final stage of the MS experiment is to produce a mass spectrum describing the relative abundance of ions for any given m/z. For this, the role of the detector is to convert a measure of the incoming ions to a signal that can be registered electronically and transferred to the computational system of the instrumentation for analysis. Ion detection may be accomplished in a variety of ways and is dependent on the instrument being used. For example, for instruments that produce and transmit a continuous ion beam, such as discussed for FT‐ICR and orbitrap instruments, ions arriving at the detector represent an electrical current (and hence the term MS is used rather that mass spectroscopy). Faraday cup collectors are predominantly used for detection in isotope ratio measurements and some inorganic MS. Otherwise, the most commonly employed detectors are the electron multiplier and the microchannel plate [36]. In both cases, ions striking a metal plate give rise to a cascade of secondary electron emissions that result in amplification of the signal, which can then be recorded as a function of m/z or at selected m/z values.

10.2.4 Hybrid Instrumentation

The combination of upstream separation methods with mass spectrometric detection is a well‐developed field and has enabled analysis and identification of molecules from increasingly complex mixtures and biological matrices. For example, gas chromatography provides a direct means for sample separation and introduction to the mass spectrometer for volatile, low molecular weight compounds [37]. Most commercial mass spectrometers now allow for integration of LC coupled to ESI sources, and particularly in the field of proteomics, reverse phase columns are commonly utilised for pre‐fractionation of analytes. Capillary electrophoresis has also been successfully integrated with ESI in biological applications, although this is technically more difficult due to the need for high voltages in electrophoresis separation.

A further ion separation method that has attracted significant attention in recent years for biomolecule analysis relies on the differing mobility of ions of varied three‐dimensional structure through a buffer gas in the presence of a low electric field (E). In these low field conditions where diffusion processes dominate, the velocity (v) of an ion is inversely proportional to its collisional cross‐section (CCS) according to

10.5

where z is the ion charge, μ is the reduced mass of the ion–gas pair (μ = mM/(m + M), where m and M are the ion and gas particle masses), k _B is Boltzmann's constant and N is the number density of the buffer gas. Therefore, measuring the drift time of an ion through a gas cell of intermediate pressure and defined distance yields the CCS (typically measured in Ų) of a molecule of interest. It also provides an orthogonal dimension of separation to m/z that can resolve structurally different ion populations such as configurational or conformational isomers.

The combination of multiple analysers in a single MS instrument has been utilised with great success. For example, typical triple quadrupole instruments consist of three identical quadrupoles placed in series, utilising the central quadrupole as a collision cell and ion guide [38]. In this geometry, various types of tandem MS experiments are possible that are particularly useful for studies requiring quantitation [39]. Furthermore, an increasing number of hybrid mass spectrometers are emerging that combine more than one type of analyser, often capitalising on the advantages of each component. For example, the combination of a quadrupole mass filter, ion mobility cell and a ToF mass analyser has been particularly popular (Figure 10.9) and offers tandem MS capabilities, extended mass range, high resolution and fast analysis.

10.3.1 Tandem Mass Spectrometry

While molecular weight information is a useful descriptor of a compound, it is not sufficient in most analyses to provide unambiguous identification, even for relatively small analytes at high mass accuracy. Furthermore, even if the elemental composition can be deduced, the molecular weight gives no indication of the structure of the molecule and therefore additional methods are required to this end. Tandem MS (also known as MS/MS or MS ⁿ ) is commonly used to further probe the structure of an analyte based on the manner in which that ion fragments with increasing activation energy. Due to the unambiguous nature of MS/MS in terms of ion peak assignment, it is a vital tool for the characterisation of biomolecules that cannot be achieved using MS on its own. In an MS/MS experiment, ions of interest at a selected m/z (precursor ions) from an initial mass spectral acquisition are isolated and fragmented, and the m/z of the resulting product ions are then measured. The sites of fragmentation, and hence the fragment ions observed, are often predictably and characteristically dependent on the structure of the molecule and therefore can be interpreted to provide details of molecular structure. This approach is widely applied in both organic and biomolecular MS and is particularly well suited for the sequencing of biopolymers [40].

The primary sequence determination of peptides and proteins by MS represents an excellent example of the power of tandem MS [40]. Since most proteins and peptides are linear polymers, cleavage of a single covalent bond along the backbone generates a fragment ion (or two complementary fragment ions) classified as shown in Figure 10.10 [41], depending on the position of the bond that is cleaved and whether the charge is retained on the N‐ or C‐terminal portion of the peptide. As a result of the heterogeneous nature of fragmentation, an array of peptide bonds are cleaved to give a series of fragment ions with distinctive mass differences, allowing for the identification of sequential amino acid residues. Cyclic and disulphide‐linked polypeptides are a special case, whereby a single bond cleavage does not necessarily produce distinct (physically separated) fragments.

Tandem MS can be performed in an ion trap‐type instrument, whereby all ions are first ejected except those at the m/z of interest. The ions are then excited to induce fragmentation and the fragments are sequentially ejected for mass measurement. Using such an instrument, multiple cycles of selection and fragmentation are possible to reveal greater structural detail, giving rise to MS ⁿ experiments, which is primarily limited by ion abundances. Alternatively, MS/MS analysis can be achieved through the coupling of two distinct mass analysers, separated by some activation regime. For example, in the hybrid Q‐ToF instrument shown in Figure 10.9, the quadrupole is used for ion selection, which is then separated from the ToF mass analyser by a pressurised collision cell to induce fragmentation. Other instrument configurations such as triple quadrupole and ToF/ToF instruments are also common to enable tandem analyses.

10.3.2 Gas‐Phase Ion Activation

There are many advantages to manipulating the energy of ions in the gas phase, including the opportunity to induce fragmentation in a highly informative manner for structure determination, as described for the tandem MS analysis above. In addition, slightly increasing the ion internal energy can help improve spectral resolution by a process that has been termed ‘collisional cleaning’. This process essentially removes excess buffer, salts or other contaminants that may be non‐specifically bound to the analyte, increasing the signal‐to‐noise ratio and enhancing peak shape, without inducing dissociation or unfolding of the biomolecules being analysed, thereby indirectly improving the accuracy of the molecular weight measurement [42].

10.3.3 Solution Phase Chemistry

Various forms of solution‐phase chemistry are used to introduce covalent modifications in biomolecules that can be localised by MS analysis to derive information regarding structure and molecular dynamics. Here we limit discussion to three key areas, particularly directed at protein structure investigation, namely covalent labelling, chemical cross‐linking mass spectrometry (CXL‐MS) and hydrogen–deuterium exchange mass spectrometry (HDX‐MS). Each provide a key structural constraint that can be combined in an integrated MS‐based approach to develop models of protein structure, particularly for cases not amenable to traditional high resolution structural biology methods such as nuclear magnetic resonance spectroscopy and X‐ray crystallography.

10.3.3.1 Covalent Labelling

Covalent labelling involves the introduction of irreversible modifications at reactive sites in a biomolecule to obtain information about solvent exposed regions. This provides conceptually similar information to hydrogen–deuterium exchange (HDX) methods, although the irreversibility of the chemistry involved means sample handling and processing are more similar to standard proteomics methods. A range of different labelling strategies is possible, depending on the type of chemistry involved, and can be limited to a few amino acids with specific labelling reagents or broadly distributed to improve spatial resolution. The simultaneous modification of many residue types is possible by oxidative labelling, typically with hydroxyl radicals, which is also frequently described as ‘footprinting’ (Figure 10.11) [56,57]. Proteins that have been covalently modified are most commonly analysed by a proteomics workflow, regardless of the actual labelling chemistry used, where enzymatic digestion gives rise to peptide mixtures for liquid chromatography mass spectrometry (LC‐MS) analysis and sequencing by MS/MS. Modified amino acids are identified by their specific mass shifts compared to unmodified residues with the help of database search software and can be incorporated into structural models as a measure of surface accessibility [56].

10.3.3.2 Chemical Cross‐Linking

Chemical cross‐linking (CXL) of biomolecules is an extremely useful analytical approach capable of probing inter‐ and intramolecular distances within and across biomolecules, both in vitro and in vivo. The CXL method for obtaining structural information on proteins is based on the formation of a covalent bond between two spatially proximate residues. This bond formation can be within a single or between two polypeptide chains and provides an upper distance constraint for incorporation into structural models [58–60].

Conventional CXL reagents typically contain two reactive sites connected through a spacer or linker region, generally an alkyl chain. More sophisticated designs include features to address analytical challenges, such as incorporation of additional reactive groups or isotopic labels, MS cleavable sites and affinity purification tags. Most commonly, the reactive groups of the CXL reagents target primary amino groups of lysine residues and the N‐terminus using N‐hydroxysuccinimidyl or sulfosuccinimidyl esters. However, despite these esters exhibiting fast conjugation, they are also susceptible to rapid hydrolysis in aqueous solutions with half‐lives on a scale of tens of minutes under physiological conditions (pH 7; 25–37 °C). More targeted CXL reactions can be carried out by linking cysteine residues using maleimide functional groups, as the relative abundance of cysteine (<2%) in proteins/peptides provides higher specificity.

Following CXL, proteins of interest are typically enzymatically digested with trypsin and peptides are separated and identified by LC‐MS/MS. Cross‐linked peptides represent just a small fraction of the total peptide components, as the resulting mixture consists of a majority of unmodified or modified, but not cross‐linked, peptides (termed ‘dead‐end’ or ‘mono‐links’). Based on their precursor mass and tandem MS fragmentation patterns, putative cross‐linked peptides and their site‐specific residue linkages can be assigned. Consequently, unambiguous charge state information for both precursor and fragment ions and accurate mass measurements are important for interpretation of CXL‐MS data. Dedicated analysis software for interpretation of CXL‐MS data has also been developed to assist with cross‐link identification. This approach has been extremely useful in identifying conformational changes in macromolecular assemblies, for example upon post‐translational modification (Figure 10.12).

10.3.3.3 Hydrogen–Deuterium Exchange

HDX reactions occurring in solution and monitored by ESI‐MS form the basis of investigation of structure and conformational dynamics for biomolecules, in particular proteins. This methodology takes advantage of the fact that acidic hydrogens in the protein can readily exchange with deuterium from D₂O to produce a mass shift (approximately 1.006 Da per D atom) that can be detected by MS. The exchange of backbone amide hydrogens occurs on a timescale well suited to MS analysis, and are largely dependent on the higher‐order structure, or specifically the solvation and H‐bonding patterns of the analytes. Consequently, increased or decreased rates of isotopic exchange can correlate with structural differences and thus HDX‐MS is a versatile tool for monitoring dynamic conformational changes, for example in the case of protein folding/misfolding [61,62] or protein– ligand [63,64] and protein–protein interactions [65].

While HDX is facile in solution, the exchange process is reversible. Therefore, it is vital that the deuterated form of the protein at a selected time point is preserved/stabilised, which is usually achieved by quenching the solution to a much lower temperature (typically 2–4 °C) and pH (approximately 2.5). In most applications of HDX, labelled proteins are then enzymatically digested into peptides using pepsin, which is active under acidic conditions, and the amount of deuterium incorporated at the peptide level can be determined by the mass shift from the unmodified peptide, often assisted by dedicated software packages. Due to the lack of proteolytic specificity of pepsin, peptide identification is achieved using LC‐MS/MS. To increase the spatial resolution from the peptide level to the individual amino acid level, data from overlapping peptides are combined. Differential rates of HDX in peptide regions are often mapped on to high resolution protein structures where available to highlight regions of conformational diversity [63].

An example of the HDX data can be seen in Figure 10.13 for prolyl oligopeptidase (PREP), a highly conserved proteolytic enzyme involved in numerous processes including brain function (memory and learning) and neuropathology. In this work, HDX‐MS provided the first near‐residue resolution analysis of global dynamics in the presence or absence of inhibitor bound in the active site, providing a clear structural basis for the activation mechanism [63].

10.3.4 Experimental Considerations for Biomolecular Mass Spectrometry

The analyses of biomolecules (e.g. drugs, lipids, proteins, etc.) for accurate mass measurement is enabled by high quality sample preparation, particularly when isolating analytes from complex mixtures such as crude extracts and cell lysates [66]. This section focuses on sample isolation and preparation approaches in the context of proteins, whereby these principles can also be applied to the characterisation of DNA/RNA, carbohydrates and other biomolecules. The majority of sample preparation techniques applied for the MS analyses of proteins involve the use of protein precipitation, using ammonium sulphate, to separate this class of compounds from cellular debris. Further separation and purification is achieved using fast protein liquid chromatography (FPLC), where proteins of interest can be separated from complex mixtures by properties such as binding affinity, hydrophobicity, size and charge. It is important to consider the extraction and purification process carefully in order to maximise the yield of purified product and minimise the possibilities of misfolding or aggregation that can hamper downstream characterisation by MS.

Samples for MS analyses need to be prepared in suitable buffer systems depending on the type of experiments being performed. For accurate mass determination of biomolecules, denaturing solvents such as methanol (20% v/v) or formic acid and acetonitrile (1 : 40% v/v) are used, which maximise sensitivity and mass resolution. In the case where the native structures and interactions of biomolecules are of interest, analytes must be exchanged into volatile buffers such as ammonium acetate and ammonium bicarbonate. It is important to note that solutions containing sodium and chloride ions are not suitable for MS analyses due to their deleterious influence on the spectra due to ionisation suppression and/or adduct formation (by Na⁺ ions). Typically, protein sample concentrations needed for native MS analyses are on the order of 0.1–50 μM, whereas lower concentrations are suitable for non‐native/denatured samples, though this is of course somewhat dependent on the MS instrumentation used.

The instrument parameters employed during an MS experiment will determine what is observed in a spectrum, which can be manipulated in a variety of ways by subtle adjustments at various stages of the acquisition. Overall, optimisation of instrument parameters is an extensive and paramount process for attaining accurate and meaningful MS data. For example, in the case of native MS where it is important to preserve the solution state of the biomolecules, parameters associated with desolvation, ionisation, ion transmission and activation must be adjusted to prevent protein unfolding and/or dissociation of the non‐covalent interactions responsible for maintaining structure. This can be dependent on the size and stability of the biomolecular assembly, whereby harsher conditions are required to aid in the desolvation and transmission of larger complexes compared to those that are smaller. Typically, the conditions that aid in high mass resolution and sensitivity come at the expense of unwanted structural changes and therefore a balance needs to be determined and due caution exercised when interpreting MS data.

Determining the mass of a macromolecular ion is now relatively straightforward and software development has advanced such that mass analysis and deconvolution have become automated and robust with increasing accuracy. Deconvolution of complicated spectra where multiple charge state series are present with a considerable degree of overlap (e.g. polydisperse proteins) has traditionally been performed manually and is extremely time consuming and can be subject to significant error and misinterpretation. In the light of this, research groups have developed custom software that is freely accessible and shown to be quite effective in accurate deconvolution [67].

10.4.1 Elemental Analysis

Accurate determination of elements in various sample types is essential for many fields, including health, medicine and environmental science, and from research to industry. ICP‐MS has undergone significant development over the last decade and while early adopters of the technology were largely from the geochemical field due to the superior ability of this technology to detect rare earth elements, it has been increasingly applied in other areas including the life sciences.

One key feature of ICP‐MS is the ability to measure nearly 70% of the elements in the periodic table, offering a unique insight into the metal components of biological samples in particular. As a recent example, ICP‐MS was utilised in a high‐throughput fashion to reveal assimilated metals and metalloproteins from the biomass of a prototypical microbe (Pyrococcus furiosus) [68]. Metal ion cofactors afford proteins with unique reactivity and have significant effects on protein stability and, consequently, metalloproteins are critical to most biological processes. However, it remains difficult to predict in an organism from the genome sequence alone as metal coordination sites are diverse and poorly recognised. In this example, shifting from a protein‐based purification to a metal‐based identification allowed the determination of all metals assimilated by the organism from its environment and identification of its metalloproteins on a genome‐wide scale. This study enabled the detection of metals known to be utilised by the organism (cobalt, iron, nickel, tungsten and zinc) and identified others that were originally thought not to be assimilated (lead, manganese, molybdenum, uranium and vanadium). Purification of eight of the 158 unexpected metal containing components yielded four novel nickel‐ and molybdenum‐containing proteins, while another four purified proteins contained substoichiometric amounts of misincorporated lead and uranium. This application demonstrates that metalloproteomes are much more extensive and diverse than previously recognised and promise to provide key insights in cell biology.

In addition to detection of particular elements, the interrogation of specific isotopes of an element can also give useful biological insight. Because the isotope ratios of elements such as carbon, hydrogen, oxygen, sulphur and nitrogen can become locally enriched or depleted through a variety of kinetic and thermodynamic factors, measurement of the isotope ratios can be used to differentiate between samples that otherwise share identical chemical compositions. Isotope ratio determinations are usually achieved using magnetic sector instruments and are used in a variety of applications, including geological dating, and have become an analytical standard in a wide range of fields from forensics to food science. For example, since technology was first reported in 1994 to distinguish between endogenous and exogenous (synthetic) testosterone and its metabolites [69], doping control laboratories have utilised analysis of the carbon isotope ratios of endogenous steroids by gas chromatography mass spectrometry (GC‐MS) to distinguish between naturally elevated steroid profiles and their illicit administration. One such high profile case relates to the 2006 Tour de France winner, Floyd Landis. Soon after the race conclusion it was reported that the urine specimen obtained from Landis was found to be positive for synthetic testosterone, where the final confirmation of this exogenous substance was determined by measuring the ¹³C/¹²C stable isotope ratios in four metabolites of testosterone via isotope ratio MS. Although Landis denied the doping, the Court of Arbitration for Sport announced a unanimous finding against him. Interestingly, some reservations have since been bought to light about the quality of the MS analysis [70], highlighting the importance of rigorous interpretation of MS data.

10.4.2 Mass Spectrometry of Organic Compounds

MS is also the tool of choice for small‐molecule analysis in areas such as natural products discovery and particularly metabolomics, which aims to provide a comprehensive assessment of a wide range of endogenous metabolites in a given biological sample. Most simply, based on the measured m/z, characteristic fragment ions and their peak intensities, the formula and chemical structure of organic molecules can be determined manually and/or by comparison with reference spectral libraries. However, the molecular diversity in small organic compounds is incredibly expansive; for compounds comprised of only carbon, hydrogen, oxygen, nitrogen, sulphur and phosphorus, with a molecular weight less than 2000 Da and that are subject to the constraints of the ‘seven golden rules of metabolomics’, more than 2 billion compounds are possible and ∼600 million of those are highly probable [71]. The Human Metabolome Database contains records for more than 42 000 metabolites, from peptides to sugars to enzyme cofactors, although the total is likely to be significantly higher. To probe the complexities of biological systems, huge numbers of molecules must therefore be inventoried, offering an extreme challenge in development of MS‐based approaches to address the needs in sensitivity, selectivity, accuracy, dynamic range and resolution.

The number of MS‐based metabolomics studies have increased exponentially over the last decade. To date such approaches have been applied to track metabolic pathways and measure dynamic flux in the metabolome, for example in response to drugs, toxins and various disease states, amongst others. Even individual cells within the same population may differ dramatically, with cell‐to‐cell heterogeneity stemming from differences in cell lifecycle, environmental influences and stochastic factors, and can provide new insight into differing phenotypes. Detecting metabolic changes at such low levels requires sensitive analysis, ideally suited to MS approaches. For example, using state‐of‐the‐art MS‐based metabolomics approaches and microarray technology, it is possible to profile individual cells. One recent ground‐breaking study utilised such a platform to present examples of biological insight at the single‐cell level, including metabolite–metabolite correlations, and visualisation of coexisting subpopulations within a genetically identical sample – one characterised by low levels of the metabolite fructose 1,6‐bisphosphate and another with high levels [72].

Advances in ambient ionisation methods for imaging, such as DESI and MALDI, have opened new avenues for real‐time detection of small organic molecules for the characterisation of biological specimens such as tissue samples, and shows immense utility for clinical applications [73]. One example is in surgical intervention for cancer therapy, where the main goal is to maximise tumour resection while preserving healthy tissue. However, existing techniques do not afford the molecular information needed to define tumour boundaries. While histopathology has long been held as the gold standard in imaging of tumours, diagnostic information is only accessible on the timeframe of hours and is limited to a few samples. Widespread studies have demonstrated the diagnostic potential of imaging disease tissues with examples including bladder, kidney, prostate and brain cancers. In the latter case, pioneering work to rapidly analyse and classify brain tumours based on lipid profiles acquired by desorption electrospray ionisation mass spectrometry (DESI‐MS) has been shown to discriminate gliomas and meningiomas from surgical specimens who underwent brain tumour resection (Figure 10.14). The samples analysed included tumours of different histological grades and tumour cell concentrations. The molecular diagnosis derived from DESI‐MS imaging correlated exceedingly well to histopathology, demonstrating the ability of this approach to providing rapid diagnosis and tumour margin assessment in near‐real time [74]. Overall, MALDI and, to a lesser extent DESI, imaging approaches have been able to provide significant insights and potential in both research and diagnostic settings.

10.4.3 Mass Spectrometry for Macromolecules

All processes that underpin biological activity in living organisms are ultimately reliant on macromolecules such as oligonucleotides, polysaccharides and proteins. Consequently, elucidating the structures, interactions and dynamics of these macromolecules is an essential pursuit in the understanding of health, disease and biological activity. For these reasons, the development of ionisation methods that enabled the gas‐phase generation of large ions revolutionised the discipline of biological MS, with the field of proteomics arguably the quintessential application of this technology in the modern era.

The term ‘proteomics’ was first used to describe the large‐scale characterisation of the entire protein complement of a cell, tissue or organism [76], and has more recently come to be directly associated with the MS identification of proteins in a biological sample. In a generic proteomics experiment, the proteins are first isolated and often fractionated to define the ‘subproteome’ for analysis. Since MS detection of proteins is less sensitive than that of peptides, and mass measurement and fragmentation of intact proteins are often insufficient for unambiguous identification, the proteins are typically analysed by a ‘bottom‐up’ proteomics approach (Figure 10.15), whereby the protein is enzymatically digested, often by trypsin. Following separation of the peptides, usually by online LC coupled to ESI, mass spectra of the eluting peptides are recorded along with a series of MS/MS experiments for abundant precursor ions. The MS and MS/MS data are commonly searched against a protein sequence database to allow the identity of the constituent peptides and therefore the initial proteins (or partial gene products) to be determined.

Determination of post‐translational modifications of proteins, such as the carbohydrate portion of glycoproteins or occupancy of phosphorylation sites provides another analytical challenge to researchers interested in the proteomic description of the cell, especially when only minimal amounts of sample are available. Reasons for this include decreases in ionisation and fragmentation propensities of modified peptides and, in particular, the isomeric structural heterogeneity and their very low abundance. Characterisation of oligosaccharides is more challenging than that of proteins and oligonucleotides due to the isomeric nature of the sugar subunit and its ability to form heterogeneous, branched structures. However, it is possible to determine the structures of oligosaccharides using tandem MS in an analogous fashion to peptides. For a complete proteomics analysis, it is not only necessary to define the identity of the protein components, but also the relative abundances of the proteins as a function of time, environmental stimulus or biological state. Consequently, it is increasingly desired to add a quantitative dimension to proteomics experiments, for example, quantifying the abundance of particular peptides under various conditions (e.g. healthy and diseased states). This is commonly achieved utilising stable‐isotope dilution, in which pairs of chemically identical analytes of different isotopic composition are differentiated in the mass spectrometer by their mass difference and the signal intensities for such analyte pairs is used to give an accurate representation of the relative abundance. Stable isotope tags can be introduced to proteins via metabolic labelling, enzymatic transfer or chemical bioconjugation using isotope coded mass tags [77].

Illustrating the power and importance of proteomics, the genome sequencing project of the malaria parasite Plasmodium falciparum has recently been complemented by large‐scale proteomics efforts. A large number of proteins were identified in the sexual and non‐sexual human stages of the parasite, and relative quantitation was achieved between stages. From this work, a set of more than 200 proteins has been identified as possible site‐specific drug or vaccine targets for future research [78].

The analysis of protein complexes is another area where MS‐based methods have had a significant impact, particularly since such massive, dynamically interacting ensembles are often not amenable to traditional structural biology techniques such as X‐ray crystallography and nuclear magnetic resonance spectroscopy. It has been known for more than 20 years that information derived from the mass spectra of these macromolecules, particularly for ESI‐MS analysis of proteins, can be correlated with their solution structural properties. For example, the extent of multiple charging on proteins is found to differ substantially using ESI from non‐denaturing or denaturing conditions, and is attributed to conformational changes taking place in solution that are reflected in the number of accessible ionisable sites on the protein [79]. Consequently, the ESI charge state distribution can be used as a measure of a three‐dimensional macromolecular structure. The soft nature of ESI has also been shown to maintain the structural integrity of protein–protein and protein–ligand interactions throughout the ionisation process, enabling the study of higher order structures and binding associations through ‘native’ MS. The overwhelming majority of structural MS studies have focused on proteins, although similar principles are also applicable to other macromolecular complexes.

The combination of ion mobility (IM) with MS has been used extensively to determine the structures of compounds from small molecules such as drugs and explosives to subunit architecture of large macromolecular assemblies such as molecular chaperones [80,81] and ATPases [82]. IM‐MS is also capable of discerning between structural isomers such as L‐ and D‐isoforms of peptides [83], heterogeneous glycans of identical molecular weight [84], differing unsaturation sites in lipids [85] or conformationally different protein states amongst many other examples, offering unrivalled separation of conformers and unique capabilities for the analysis of a 3D structure (Figure 10.16). IM‐MS is commonly used in conjunction with gas‐phase dissociation methods to extensively probe the structural dynamics of proteins [86]. More recently, it has been utilised to provide modelling constraint data, in conjunction with complementary biophysical techniques, to determining the structure and conformation of macromolecular complexes such as integral membrane proteins [87] and viruses [88]. Overall, IM‐MS is a key component of the MS toolbox for elucidating the structure and dynamics of biomolecules as a means of providing a structural rationale for their function.

One of the most straightforward applications of native MS is the determination of subunit stoichiometry in a biological complex, which, with few exceptions, shows excellent consensus between that derived by other methods. This has led to successful analysis of an extensive range of non‐covalent biomolecular complexes, including impressive examples such as the intact ribosome [89]. With the introduction of commercially available IM‐MS instrumentation, this technology has achieved a rapid rise in popularity to study the overall structure of biomolecular assemblies, monitor changing protein conformations or reduce sample complexity by complementing the mass measurement with another gas‐phase separation dimension. Combined with gas‐phase activation methods, the stoichiometry, conformation, binding interactions and dynamics of a structural assembly can be potentially determined from a single MS analysis (Figure 10.16). This can certainly be further complemented by utilising an integrated approach including other structural constraints from MS‐based methods such as HDX, CXL and chemical labelling [90], as well as other biophysical techniques. The benefits of an increasingly integrated structural biology approach are effectively illustrated by the Mediator complex, a multiprotein assembly that functions as a transcriptional coactivator in all eukaryotes, but is refractory to traditional structural biology methods due to its conformational flexibility and diverse composition. The middle module of the Mediator complex was first investigated by numerous MS‐based experiments (native MS, tandem‐MS and IM‐MS), which, in combination with light scattering, small‐angle X‐ray scattering and pull‐down assays, allowed for determination of its overall subunit topology [91]. This putative model of the complex was further refined using CXL‐MS [92]. Similarly, a structure of the Mediator head module was determined by X‐ray crystallography and CXL‐MS [93] and, most recently, a model of the full Mediator‐Pol II pre‐initiation complex comprising 52 protein subunits has been proposed largely from MS‐based analyses [94].

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Further Reading

1. Gross, J.H. (2017). Mass Spectrometry – A Textbook, 3e. Heidelberg: Springer‐Verlag.
2. Hillenkamp, F. and Peter‐Katalinic, J. (eds.) (2007). MALDI‐MS: A Practical Guide to Instrumentation, Methods and Applications. Weinheim: Wiley‐VCH.
3. de Hoffmann, E. and Stroobant, V. (2007). Mass Spectrometry – Principles and Applications, 3e. Wiley.
4. Kaltashov, I.A. and Eyles, S.J. (2005). Mass Spectrometry in Biophysics: Conformation and Dynamics of Biomolecules. Wiley.
5. Kinter, M. and Sherman, N.E. (2000). Protein Sequencing and Identification Using Tandem Mass Spectrometry. New York: Wiley‐Interscience.
6. Kool, J. and Niessen, W.M.A. (2015). Analyzing Biomolecular Interactions by Mass Spectrometry. Wiley.
7. Lehmann, W.D. (2010). Protein Phosphorylation Analysis by Electrospray Mass Spectrometry: A Guide to Concepts and Practice. Cambridge: The Royal Society of Chemistry.
8. Pottiez, G. (2015). Mass Spectrometry: Developmental Approaches to Answer Biological Questions. Springer.
9. Schalley, C.A. and Springer, A. (2009). Mass Spectrometry and Gas‐Phase Chemistry of Non‐covalent Complexes. Hoboken: Wiley Interscience.
10. Watson, J.T. and Sparkman, O.D. (2007). Introduction to Mass Spectrometry – Instrumentation, Applications and Strategies for Data Interpretation, 4e. Chichster: Wiley.