WO2009109534A1

WO2009109534A1 - Improvements relating to mass spectrometry

Info

Publication number: WO2009109534A1
Application number: PCT/EP2009/052415
Authority: WO
Inventors: Jason Blair Greenwood; Ian David Williams
Original assignee: The Queen's University Of Belfast
Priority date: 2008-03-01
Filing date: 2009-02-27
Publication date: 2009-09-11
Also published as: GB0803885D0

Abstract

A mass spectrometer (1) for analysing the mass of molecules of an analyte is provided, comprising an ion trap (3) comprising a sample holder (5) which is adapted to receive a sample comprising the analyte, a first laser (7) which emits laser radiation onto the sample which detaches molecules of the analyte to form a plume of analyte molecules, a second laser (9) which emits pulses of laser radiation having a full width half maximum bandwidth of at least 20nm into the plume of analyte molecules which ionizes at least some of the analyte molecules and produces analyte ions which are trapped in the ion trap, and a detector (11) which detects a characteristic of the analyte ions, which characteristic is used to produce a measure of the mass of the analyte molecules. The first laser is used to achieve detachment of the analyte molecules and the second laser is used to achieve ionization of the analyte molecules. There is therefore a clear separation of the detachment and ionization processes.

Description

Improvements relating to Mass Spectrometry

This invention relates to improvements in mass spectrometry.

Mass spectrometry is a widely-used technique, for the identification or structural study of a large range of substances, by measuring the mass of particles or sub-components of particles of the substance. Conventionally, the mass of ionized particles of a substance, or analyte, is determined using a mass spectrometer. The ionized particles may then be fragmented into sub-components thereof, and the mass of the subcomponents determined using the mass spectrometer.

Mass spectrometry may be used to measure the mass of particles of many kinds of substances. For example, the determination of the mass of particles of biomolecular substances, such as proteins, is a commonly- used analysis technique for such substances.

Mass spectrometers vary greatly in the features that they include. For example, there are many techniques for the production of ionized particles, similarly there are many techniques for the fragmentation of the ionized particles into sub-components, and for the determination of the mass of the ionized particles and of the sub-components thereof. Each of the techniques has advantages and disadvantages.

According to a first aspect of the invention there is provided a mass spectrometer for analysing the mass of molecules of an analyte comprising an ion trap comprising a sample holder which is adapted to receive a sample comprising the analyte, a first laser which emits laser radiation onto the sample which detaches molecules of the analyte to form a plume of analyte molecules, a second laser which emits pulses of laser radiation having a full width half maximum bandwidth of at least 20nm into the plume of analyte molecules which ionizes at least some of the analyte molecules and produces analyte ions which are trapped in the ion trap, and a detector which detects a characteristic of the analyte ions, which characteristic is used to produce a measure of the mass of the analyte molecules.

The first laser is used to achieve detachment of the analyte molecules and the second laser is used to achieve ionization of the analyte molecules. There is therefore a clear separation of the detachment and ionization processes.

The second laser may emit pulses of laser radiation having a full width half maximum (FWHM) bandwidth in the range of approximately 20nm to approximately 200nm, for example approximately 50nm to approximately 200nm. The bandwidth of the pulses of laser radiation are inversely related to the duration of the pulses. Thus, as the bandwidth of the pulses increases, the duration of the pulses will decrease. A pulse having a FWHM bandwidth of approximately 50nm will have a duration of approximately 20fs. The second laser may emit pulses of laser radiation wherein each pulse has a FWHM bandwidth giving rise to a pulse duration which is less than a shortest vibrational period of the molecules of the analyte. As motion of the atoms in the molecules of an analyte generally occurs in timescales longer than 20fs, the molecules are frozen since they do not have time to adjust to the pulses of laser radiation. This means that many of the possible dissociation pathways of the molecules may be suppressed in favour of ionization. The second laser may emit pulses of laser radiation having an energy of approximately 0.5mJ. The second laser may emit pulses of laser radiation having peak intensities greater than 1 x10¹² WcnrT². The second laser may emit pulses of laser radiation having peak intensities depending on the analyte being analysed. For example the second laser may emit pulses of laser radiation having peak intensities between 1x10¹³ and 1x10¹⁴ Won^"2 when the analyte comprises a rare gas, and the second laser may emit pulses of laser radiation having peak intensities of approximately 1 x10¹³ Wcm^"2 when the analyte comprises biomolecular ions. At these intensities, substantially all molecules of the analyte will be ionized, thus providing an efficient ionization source. Ionization can proceed without the excitation of a resonant transition in the molecules, and so does not require the presence of a strongly absorbing chromophore in the analyte. The second laser may emit pulses of laser radiation having intensities whose maximum is limited to promote single ionization of the molecules of the analyte. This reduces the occurrence of multiple ionization followed by Coulomb explosion and thus fragmentation of the molecules.

The second laser may emit pulses of laser radiation which are elliptically polarised. Fragmentation of analyte ions may occur through collision of the analyte ions with an ionized electron. This is suppressed by elliptical polarisation of the laser pulses, which induces the ionized electrons to spiral away from the analyte ions, to a sufficient distance that collision between the electrons and the ions is reduced.

The second laser may comprise a titanium:sapphire lasing medium.

The mass spectrometer may comprise a focussing apparatus for focussing the pulses of laser radiation emitted by the second laser to provide an ionization volume in which the pulses of laser radiation interact with analyte ions. The focussing apparatus may focus the pulses of laser radiation such that the intensity of the pulses in the ionization volume is greater than 2x10¹³ Won^"2 . The focussing apparatus may focus the pulses of laser radiation such that the volume of the ionization volume is approximately 1x10^"5 cm³. The focussing apparatus may comprise a focussing element which is oriented to shape the ionization volume along a longitudinal axis of the ion trap. The focussing apparatus may comprise a focussing element which is oriented to shape the ionization volume along a transverse axis of the ion trap. The focussing apparatus may comprise a focussing element comprising a spherical lens which provides an ionization volume which is substantially cylindrical in shape. The focussing apparatus may comprise a focussing element comprising a cylindrical lens which provides an ionization volume which is substantially pancake-shaped.

The shape and orientation of the ionization volume will influence the number of analyte ions which are created. To maximise the number of analyte ions which are created, the ionization volume should be maximised. The shape and orientation of the ionization volume will influence the number of analyte ions which are subsequently trapped in the ion trap, and the time dispersion of oscillations of the trapped analyte ions in the ion trap. If the shape and orientation of the ionization volume results in an elongate ionization volume along the transverse axis of the ion trap, ions will be created at a plurality of positions along the transverse axis. As the position of creation increases along the transverse axis away from the longitudinal axis, the probability that an ion will be stably trapped decreases. Further, the dispersion of the positions of creation of the ions along the transverse axis results in a dispersion of oscillation times of the ions which are stably trapped. This limits the mass resolution of the mass spectrometer. If the shape and orientation of the ionization volume results in an elongate ionization volume along the longitudinal axis of the ion trap, ions will be created at a plurality of positions along the longitudinal axis. Although the probability of trapping of all these ions is high, the dispersion of the positions of creation of the ions along the longitudinal axis results in a dispersion of energies of the ions, and this, in turn, results in a dispersion of oscillation times of the ions. This again limits the mass resolution of the mass spectrometer. The shape and orientation of the ionization volume must therefore be chosen to achieve a satisfactory trade-off between the number of analyte ions which are created, and the trapping efficiency and the time dispersion (and hence mass resolution) of the ions.

The analyte ions may be generated in the ion trap. This removes the need for an external injection method, which avoids loss of ions between formation and injection into stable trajectories in the ion trap.

The ion trap may comprise a linear electrostatic ion trap. The ion trap may comprise a kilovolt electrostatic ion reflection analyser (KEIRA) ion trap. The ion trap may operate with a partial vacuum. The ion trap may comprise electric field generation apparatus. The electric field generation apparatus may generate a static electric field or a time-varying electric field. The electric field generation apparatus may comprise a first electrostatic mirror and a second electrostatic mirror. The first electrostatic mirror and the second electrostatic mirror may comprise planar electrostatic mirrors.

The first electrostatic mirror may comprise a plurality of mirror plates. The second electrostatic mirror may comprise a plurality of mirror plates. For example, the first and second electrostatic mirrors may each comprise seven mirror plates. The mirror plates may each comprise an apertured plate, or a grid-like plate, or a plane surface plate. The mirror plates may have an aperture diameter of approximately 16mm. The mirror plates may have a thickness of approximately 3mm. At least some of the mirror plates may have a separation of approximately 7mm. A first and second mirror plate of each electrostatic mirror may have a separation of approximately 14mm. The mirror plates may each comprise an electrode. The mirror plate electrodes may each have a voltage applied thereto. For each electrostatic mirror, a series of decreasing voltages or a series of increasing voltages may be applied to the mirror plate electrodes. The mirror plate electrodes may then provide a voltage gradient. For example, for the first and second electrostatic mirror, a series of decreasing voltages may be applied to the mirror plate electrodes comprising 4800V, 3900V, 3200V, 2400V, 1600V, 800V, and OV. For each electrostatic mirror, a first mirror plate electrode may have a voltage applied thereto which is greater than a maximum energy of the analyte ions, to reflect the analyte ions. For each electrostatic mirror, the mirror plate electrodes may provide a high voltage gradient, thus providing a hard electrostatic mirror. For each electrostatic mirror, the mirror plate electrodes may provide a low voltage gradient, thus providing a soft electrostatic mirror. The soft electrostatic mirror may be achieved using a large number of mirror plate electrodes having gradually decreasing or increasing voltages applied thereto.

The first electrostatic mirror may comprise a cylinder provided with a electrically-resistive coating. The second electrostatic mirror may comprise a cylinder provided with a electrically-resistive coating. The cylinders may be made of glass. The cylinders may be hollow or may be provided with apertures therethrough. Each cylinder may have a voltage applied between the ends thereof, which passes through the electrically- resistive coating which provides a linear voltage gradient along the cylinder length. The first electrostatic mirror may be positioned at a first end of a longitudinal axis of the ion trap. The second electrostatic mirror may be positioned at a second end of a longitudinal axis of the ion trap. The first and second electrostatic mirrors may constrain the analyte ions to move along at least one longitudinal axis of the ion trap. The ion trap may comprise an electric field-free region provided between the first electrostatic mirror and the second electrostatic mirror. The first and second electrostatic mirrors may constrain the analyte ions to oscillate between the first and second electrostatic mirrors, passing through the electric field-free region. The period of oscillation of the analyte ions will be dependent on the mass of the ions. Thus the electrostatic mirrors act to trap the analyte ions in oscillatory trajectories in the ion trap.

For each electrostatic mirror, a voltage gradient may be provided along the longitudinal axis of the ion trap. For each electrostatic mirror, a voltage gradient may be provided along the longitudinal axis which decreases towards the electric field-free region of the ion trap. Each voltage gradient may accelerate the analyte ions into the electric field-free region. The analyte may gain an energy in the electric field-free region equal to a potential energy with which they were created. As the analyte ions exit an electrostatic mirror, their potential energy is converted to kinetic energy, resulting in velocity differences for ions with different mass to charge ratios.

The ion trap may comprise electric field generation apparatus which may comprise a first electrostatic lens. The ion trap may comprise electric field generation apparatus which may comprise a second electrostatic lens. Each electrostatic lens may comprise a saddle potential electrostatic lens. Each electrostatic lens may comprise a first electrode situated between second and third electrodes. The first electrode may have a first voltage applied thereto, the second electrode may have a second voltage applied thereto and the third electrode may have a third voltage applied thereto. The second and third voltages may be lower or higher than the first voltage. For example, for the electrostatic mirror voltages detailed above, the first voltage may be approximately -4800V or approximately +2500V, the second voltage may be approximately OV, and the third voltage may be approximately OV. A negative voltage is preferred for the first voltage. Each electrode may comprise a planar electrode. Each planar electrode may have a thickness of approximately 3mm. Each planar electrode may have a diameter of approximately 16mm. Each planar electrode may have a separation of approximately 7mm. The first electrostatic lens may be provided adjacent the first electrostatic mirror. The second electrostatic lens may be provided adjacent the second electrostatic mirror.

The electrostatic lenses may each act as focussing lenses. The electrostatic lenses may focus the analyte ions by exerting a transverse force on the analyte ions, with respect to a longitudinal axis of the ion trap. The force exerted on the ions may increase as the position of an ion away from the longitudinal axis increases. Thus analyte ions may be directed towards a focal point on the longitudinal axis, the location of which along this axis depends on the voltages applied to the lenses.

As discussed above, ions with a plurality of initial positions along a longitudinal axis of the ion trap result in a plurality of ion energies and therefore a plurality of ion oscillatory trajectories with different periods, i.e. time dispersion of oscillations of the trapped ions. The time dispersion of the oscillations of the ions results in a finite analysis time for the ions, as eventually the ions spread out and become distributed throughout the length of the spectrometer and the characteristic of the ions can no longer be detected and their mass deduced. This limits the achieveable mass resolution of the spectrometer. The plurality of ion energies and therefore the time dispersion of oscillations of the trapped ions, results from the presence of the voltage gradients of the electrostatic mirrors and the electrostatic lenses. The time dispersion of the oscillations of the ions may be reduced by adjusting the potential gradient of the first and second electrostatic mirrors. The potential gradient of the first and second electrostatic mirrors may be adjusted to increase travel time of higher energy ions within the electrostatic mirrors. In this way, the higher energy ions penetrate deeper into the mirror regions, but attain approximately the same longitudinal position in the field-free region as ions with lower energies.

As further discussed above, the probability of an analyte ion being trapped in an oscillatory trajectory will depend on the position along a transverse axis of the ion trap at which the ion is created. Ions created further along the transverse axis have less chance of being trapped. In addition, ions with a plurality of initial positions along a transverse axis of the ion trap result in a plurality of ion oscillatory trajectories with different periods, i.e. time dispersion of oscillations of the trapped ions. As already mentioned, specific arrangements of the focussing of the pulses from the second laser can be used to influence the trapping probability and the time dispersion of the ion oscillations, by reducing the number of ions created at larger distances along the transverse axis. The electrostatic lenses can also be used to influence the trapping probability and the time dispersion of the ion oscillations, by focussing the ions along the transverse axis towards the longitudinal axis of the ion trap. The mass spectrometer may further comprise one or more limiting apertures. The limiting apertures can also be used to influence the trapping probability and the time dispersion of the ion oscillations. The or each limiting aperture may be positioned in the ion trap to impede the oscillation of ions which are created at positions along the transverse axis of the ion trap which are a pre-determined distance from the longitudinal axis of the ion trap. The pre-determined distance may be, for example, approximately 3mm. The or each limiting aperture may be positioned in the field-free region of the ion trap. The or each limiting aperture may be provided by grounded aperture electrodes, with a diameter of twice the pre-determined distance.

In the mass spectrometer, there will exist a volume of space within the ion trap, from which newly-formed analyte ions will subsequently be trapped into oscillatory trajectories. Ion generation and ion trap loading is very efficient, and confinement of the ions in the trajectories is very efficient, resulting in a high sensitivity spectrometer.

The detector may comprise a charge detector. The charge detector may detect a characteristic of the analyte ions comprising image charge of the analyte ions. The charge detector may comprise at least one pick-up electrode. The charge detector may comprise a series of pick-up electrodes. The or each pick-up electrode may be in the form of a pick-up ring. The charge detector may be positioned in the mass spectrometer such that the analyte ions pass the or each pick-up electrode. When the or each pick-up electrode comprises a pick-up ring , the charge detector may be positioned in the mass spectrometer such that the analyte ions pass through the or each pick-up ring. The charge detector may be positioned in the electric field-free region of the ion trap provided between the electrostatic mirrors. The or each pick-up electrode may measure image charge of the analyte ions as they pass the pick-up electrode. The or each pick-up electrode may measure image charge of the analyte ions over many passes of the ions. The measurements of the image charge may be used to determine the mass of the analyte ions. A Fourier transform of a charge measurement may be used to give a frequency spectrum in which the frequency is proportional to the square root of charge/mass of the analyte ions.

The charge detector provides a non-destructive system for measuring a characteristic of the analyte ions. This system depends on the magnitude of the charge of the analyte ions, and does not therefore suffer loss of sensitivity for higher mass ions. However, pick-up electrodes can be noisy, which reduces the sensitivity of the detector. The sensitivity is limited by noise due to, for example, environmental electromagnetic fields, microphonic vibrations, and the sensitivity of a pre-amplifier when used. The noise can be reduced if the charge detector is made mechanically stable, is well shielded and is cooled. For example, the charge detector may comprise shielding around the or each pick-up electrode, except for a region thereof over which the ions pass.

The detector may comprise a particle detector. The particle detector may detect a characteristic of the analyte ions which comprises products of collisions of the analyte ions with a background gas in the ion trap. The particle detector may be located outside the ion trap, and may detect products which are ejected from the ion trap. The background gas may comprise a neutral species gas. The measurements of the collision products may be used to determine the mass of the analyte ions. A Fourier transform of the measurements will give a frequency spectrum in which the frequency is inversely proportional to the square root of the mass of the analyte ions.

The sample holder may be provided by a mirror plate of one of the electrostatic mirrors, for example a first mirror plate of the first electrostatic mirror. The sample may be deposited on a surface of the mirror plate. The first laser may emit laser radiation having a frequency which is readily absorbed by the sample holder. The laser radiation from the first laser may lift the analyte off the sample holder by desorption, to form the plume of analyte molecules. The desorption of the analyte may be due to evaporation of the analyte from the sample holder, caused by a heating effect provided by the radiation from the first laser. The laser radiation from the first laser may be directed onto the sample from a range of angles relative to the sample holder, e.g. 30 degrees. The laser radiation from the first laser is not resonantly absorbed by the analyte of the sample, so the analyte generally remains intact.

The sample may comprise the analyte and a matrix, for example the analyte mixed in solution with the matrix. The first laser may emit laser radiation having a frequency which is readily absorbed by the matrix. The laser radiation from the first laser may ablate the matrix from the sample holder, lifting the analyte off the sample holder by desorption, and forming the plume of analyte molecules. The plume of analyte molecules is therefore produced by a matrix assisted laser desorption technique. The laser radiation from the first laser may be focussed onto the sample. The laser radiation from the first laser is again not resonantly absorbed by the analyte of the sample, so the analyte generally remains intact.

The sample may be placed on a surface comprising silicon or graphite. The sample may be placed on a nano-patterned surface. The first laser may emit laser radiation having a frequency which is readily absorbed by the surface. The laser radiation from the first laser may ablate the analyte off the sample holder by desorption, to form the plume of analyte molecules. The plume of analyte molecules is therefore produced by a surface assisted laser desorption technique. For samples comprising a matrix, the creation of analyte ions by the second laser will also cause creation of matrix ions. Generally, for such samples, the matrix ions will be lighter than the analyte ions. The matrix ions will therefore travel through the ion trap with a higher velocity than that of the analyte ions. The matrix ions may interfere with the measurement of the mass of the analyte ions, this can be suppressed by ejecting the matrix ions from the ion trap. This can be achieved by controlling the voltage gradients provided by the electrostatic mirrors, for example by carefully timed switching of the voltages applied to the mirrors. For instance, if the analyte and matrix ions are created in the vicinity of the first electrostatic mirror, the voltage applied to the second mirror may initially be lower than that applied to the first mirror. The analyte and matrix ions will therefore be constrained to move towards the second mirror. The matrix ions will travel faster, and will reach the second mirror first, and can travel through the second mirror and exit the ion trap through an aperture provided therein in the proximity of the second mirror. Once the matrix ions have been ejected from the ion trap, the voltage applied to the second mirror can be raised, to trap the analyte ions in the trap.

Analyte and matrix ions can also be created by the laser radiation from the first laser, as well as by the laser radiation from the second laser. These analyte and matrix ions will be created just above the surface of the sample holder, and some of these ions may become trapped and oscillate in the ion trap. These oscillatory trajectories may interfere with the measurement of the mass of the analyte ions created by the second laser. This can be suppressed by ejecting the analyte and matrix ions created by the first laser, by pulsing the voltage on the first electrostatic mirror. If the analyte ions are created in the vicinity of the first electrostatic mirror, the voltage applied to the second mirror may initially be lower than that applied to the sample holder. The analyte ions will therefore be constrained to move towards the second mirror, and can travel through the second mirror and exit the ion trap through an aperture provided therein in the proximity of the second mirror. Following application of the first laser, the voltage applied to the first mirror can be lowered, before the second laser is deployed. Therefore, the second laser generates ions at a time when voltages on the first and second electrostatic mirrors are equal and thus are trapped. If, instead, it is wished to analyse the mass of the analyte ions created by the first laser, the second laser is not deployed, allowing analysis of the analyte ions created by the first laser without interference from analyte ions subsequently created by the second laser.

After each oscillation, the analyte ions will return close to the position where they were created, with very low kinetic energy. For conditions of low time dispersion within the analyte ion trajectories, all the ions will return to this position at approximately the same time. The analyte ions will therefore lie within the focus of the second laser, and can be irradiated again. The second laser may emit pulses of laser radiation into the analyte ions which fragments at least some of the analyte ions, to produce sub-components of the analyte ions. These pulses of radiation from the second laser are emitted subsequent to the pulses which created the analyte ions.

One or more parameters of the pulses of laser radiation from the second laser may be controlled to induce a chosen fragmentation of the analyte ions. One or more parameters of the pulses of laser radiation from the second laser may be controlled to induce breakage of specific bonds in the analyte ions. Any of the duration, wavelength, amplitude and polarisation of the pulses of laser radiation from the second laser may be controlled. For example, phases of spectral components of these pulses of laser radiation from the second laser may be controlled to give rise to pulse durations which are longer than those used to induce ionization of the analyte molecules. The control of one or more parameters of the pulses of laser radiation from the second laser may be carried out dynamically within a single laser pulse. The control of one or more parameters of the pulses of laser radiation from the second laser may be carried out using a feedback loop with, for example, a genetic algorithm. Multiple pulses of laser radiation from the second laser may be used in a pump-control-probe arrangement, where an initial pulse induces vibrational or electronic excitation in an analyte ion and subsequent pulses can be used to control the distribution of this vibrational or electronic energy.

For pulses of laser radiation from the second laser which have longer duration than the pulses used for ionization, or for a series of shorter pulses separated in time by less than one ns, fragmentation of analyte ions may proceed as there is time for vibrational energy to be redistributed throughout the ions. This allows the structure of the ions to change between successive laser pulses or evolve in the presence of a longer pulse laser field. The parameters of the pulses of laser radiation from the second laser can therefore be adjusted to induce breakage of chosen, specific bonds in the analyte ions.

The sub-components of the analyte ions are created in the same spatial volume as the analyte ions, and are also trapped in oscillatory trajectories within the ion trap. The period of the trajectories of the analyte ion subcomponents will be shorter than those of the analyte ions, as the subcomponents have lighter masses. The detector may detect a characteristic of the analyte ion sub-components, and use this to produce a measure of the mass of the analyte ion sub-components. The second laser may emit further pulses of laser radiation which fragment the analyte ion sub-components to produce further sub-components. The mass of the further sub-components can be analysed. The mass spectrometry cycle can therefore be repeated many times, with the parameters of the pulses of laser radiation adjusted to optimise particular fragmentation outcomes. The invention allows for true tandem mass spectrometry of the analyte, as the ionization stage can be clearly separated from subsequent fragmentation steps which could be applied N times.

The mass spectrometer may be used to analyse the mass of biomolecules. The mass spectrometer may be used to analyse the mass of polymeric biomolecular ions. The mass spectrometer may be used to determine the primary structure of biomolecular ions from analysis of the mass of components of the biomolecular ions, e.g. protein biomolecular ions which comprise peptides, or peptide biomolecular ions which comprise amino acids. The mass spectrometer may be used to analyse the mass of nucleotides to, for example, obtain structural information and sequencing of DNA bases.

According to a second aspect of the invention there is provided a method of analysing the mass of molecules of an analyte using a mass spectrometer of the first aspect of the invention, comprising placing a sample comprising the analyte in a sample holder of an ion trap of the spectrometer, deploying a first laser to emit laser radiation onto the sample to detach molecules of the analyte to form a plume of analyte molecules, deploying a second laser to emit pulses of laser radiation having a full width half maximum bandwidth of at least 50nm into the plume of analyte molecules to ionize at least some of the analyte molecules and produce analyte ions which are trapped in the ion trap, and operating a detector to detect a characteristic of the analyte ions, and using the characteristic to produce a measure of the mass of the analyte molecules.

Embodiments of the present invention will now be described by way of example only, with reference to the accompanying drawings, in which:

Figure 1 is a schematic representation of a first embodiment of a mass spectrometer according to the first aspect of the invention;

Figure 2 is a schematic representation of a second embodiment of a mass spectrometer according to the first aspect of the invention, and

Figure 3 is a schematic representation of part of an alternative sample holder of the mass spectrometers of Figures 1 and 2.

Referring to Figure 1 , the first embodiment of the mass spectrometer 1 comprises an ion trap 3, a sample holder 5, a first laser 7, a second laser 9 and a detector 11.

The ion trap 3 is a linear electrostatic ion trap, comprising a kilovolt electrostatic ion reflection analyser (KEIRA) ion trap. The ion trap operates with a partial vacuum, and comprises electric field generation apparatus which generates a static electric field. The electric field generation apparatus comprises a first planar electrostatic mirror 13 and a second planar electrostatic mirror 15, each having seven mirror plates, in the form of apertured plates. The mirror plates have a thickness of approximately 3mm, and a diameter of approximately 16mm. First and second mirror plates have a separation of approximately 14mm, as shown, and the remainder of the mirror plates have a separation of approximately 7mm. The mirror plates each comprise an electrode, and have a voltage applied thereto. The mirror plates of the first electrostatic mirror 13 are positioned at a first end of the ion trap 3, and the mirror plates of the second electrostatic mirror 15 are positioned at a second end of the ion trap, as shown. A series of decreasing voltages are applied to the mirror plates of each mirror, from a mirror plate electrode adjacent to an end of the ion trap 3. The mirror plates of each electrostatic mirror 13, 15 therefore provide a voltage gradient along a longitudinal axis of the ion trap. For example, for the first and second electrostatic mirrors, a series of decreasing voltages may be applied to the mirror plates comprising 4800V, 3900V, 3200V, 2400V, 1600V, 800V, and OV. The electric field generation apparatus further comprises a first electrostatic lens 17, and a second electrostatic lens 19, each comprising a saddle potential electrostatic lens. The electrostatic lenses 17, 19 comprise a first electrode situated between second and third electrodes. The first electrode has a voltage, for example approximately -4800V, applied thereto which is greater than a voltage, for example approximately OV, applied to the second electrode and the third electrode. Each electrode is a planar electrode, having a thickness of approximately 3mm, a diameter of approximately 16mm, and a separation of approximately 7mm. The first electrostatic lens 17 is positioned adjacent the first electrostatic mirror 13, and the second electrostatic lens 19 is positioned adjacent the second electrostatic mirror 15.

The sample holder 5 is provided by a mirror plate of the first electrostatic mirror 15, which mirror plate is adjacent a first end of the ion trap 3. The sample is deposited on a surface of the mirror plate, and comprises the analyte mixed in solution with a matrix. The first laser 7 emits laser radiation having a frequency which is readily absorption by the matrix of the sample, for example a frequency in the ultra violet frequency range. The laser radiation from the first laser 7 is directed onto the sample on the mirror plate 5 from a range of angles relative to the sample e.g. 30 degrees, and may be focussed onto the sample. The laser radiation from the first laser 7 is not directly inputted into the analyte, so this generally remains intact.

The second laser 9 comprises a titanium:sapphire lasing medium, and emits pulses of laser radiation having a FWHM bandwidth of at least

20nm. Thus such pulses have a duration of approximately 50fs or lower, and preferably less than a shortest vibrational period of the molecules of the analyte. The energy of the pulses is approximately 0.5mJ, and the intensity greater than 1x10¹³ WcnrT². At these intensities, substantially all molecules of the analyte will be ionized, thus providing an efficient ionization source. The mass spectrometer comprises a focussing apparatus (not shown), which focuses the pulses of laser radiation emitted by the second laser 9. The focussing apparatus comprises a focussing element, which focuses the pulses of laser radiation to provide a substantially cylindrical ionization volume, the axis of the volume lying along a longitudinal axis of the ion trap, and the diameter of the volume lying along a transverse axis of the ion trap. The diameter of the cylindrical ionization volume is less than the length of the volume. The second laser 9 emits pulses of laser radiation which are elliptically polarised.

The detector 11 comprises a charge detector, and is positioned in the ion trap 3 in an electric field-free region of the ion trap provided between the electrostatic mirror 13 and the lens 17 and the electrostatic mirror 15 and the lens 19. The charge detector 11 comprises at least one pick-up electrode, in the form of a pick-up ring. Shielding is provided around the or each pick-up ring. A shielded connection connects the pick-up ring to a pre-amplifier (not shown), which is connected to an oscilloscope (not shown), outside the vacuum of the ion trap 3. The charge detector 11 is made mechanically stable, is well shielded and can be cooled. The charge detector 11 is positioned in the ion trap 3 such that analyte ions pass through the or each pick-up ring, and the detector detects a characteristic of the analyte ions comprising image charge of the analyte ions.

To analyse the mass of molecules of an analyte, the analyte is mixed with a matrix, and a sample comprising the analyte and matrix deposited on a surface of the first mirror plate 5 of the first electrostatic mirror 13. The first laser 7 emits laser radiation into the ion trap 3 and onto the sample, and ablates the matrix from the sample holder 5, lifting the analyte off the sample holder by desorption, and forming a plume of analyte molecules. The second laser 9 emits laser radiation into the ion trap 3 and into the plume of analyte molecules, and ionizes at least some of the molecules to produce analyte ions.

In this invention, the analyte ions are generated in the ion trap 3. This removes the need for an external injection method, which avoids loss of ions between formation and injection into stable trajectories in the ion trap 3. The first laser 7 is used to achieve desorption of the analyte molecules and the second laser 9 is used to achieve ionization of the analyte molecules. There is therefore a clear separation of the desorption and ionization processes.

After their creation at the first end of the ion trap 3, the analyte ions are constrained to move along at least one longitudinal axis of the ion trap 3 by the first and second electrostatic mirrors 13, 15. The voltage gradient provided by the first electrostatic mirror 13 accelerates the analyte ions through the mirror plates of the first electrostatic mirror 13, and into the electric field-free region between the mirrors. The analyte ions gain an energy in the electric field-free region equal to a potential energy with which they were created. The analyte ions travel through the field-free region, and into the second electrostatic mirror 15. The voltage gradient provided by the second electrostatic mirror 15 decelerates the analyte ions as they pass through the mirror plates of this mirror, until they reach the second end of the ion trap 3. Here the analyte ions come to a stop, and are then accelerated by the voltage gradient of the second mirror 15 back into the field-free region. The first and second electrostatic mirrors 13, 15 therefore constrain the analyte ions to oscillate between them, passing through the electric field-free region provided between the mirrors. Thus the electrostatic mirrors act to trap the analyte ions in oscillatory trajectories in the ions trap. The period of oscillation of the analyte ions will be dependent on the mass of the ions. The analyte ions are focussed by the lens 17, 19 along the transverse axis of the ion trap 3.

As the analyte ions pass through the pick-up ring of the charge detector 11 , the image charge of the analyte ions is detected. The image charge signal is passed to the pre-amplifier which converts the signal to a voltage which can be displayed on the oscilloscope. The detector 11 measures the image charge of the analyte ions over many passes of the ions therethrough. The measurements of the image charge are then used to determine the mass of the analyte ions, using, for example, a Fourier transform of a charge measurement to give a frequency spectrum in which the frequency is proportional to the square root of charge/mass of the analyte ions. After each oscillation, the analyte ions will return close to the position where they were created, and will lie within the focus of the second laser 9, and can be irradiated again. The second laser 9 may emit pulses of laser radiation into the analyte ions which fragments at least some of the analyte ions, to produce sub-components of the analyte ions. The subcomponents of the analyte ions are also trapped in oscillatory trajectories within the ion trap 3. The period of the trajectories of the analyte ion subcomponents will be shorter than those of the analyte ions, as the subcomponents have lighter masses. The detector may detect the image charge of the analyte ion sub-components, and use this to produce a measure of the mass of the analyte ion sub-components.

Referring to Figure 2, a second embodiment of a mass spectrometer according to the invention is shown. Like reference numerals are used to denote like elements in the mass spectrometer in Figure 2, as in Figure 1. The mass spectrometer 1 comprises an ion trap 3, a sample holder 5, a first laser 7, a second laser 9 and a detector 11.

In this embodiment, the ion trap 3 comprises electric field generation apparatus in the form of a first planar electrostatic mirror 113 and a second planar electrostatic mirror 115, each comprising a glass cylinder provided with a electrically-resistive coating. The glass cylinders are provided with apertures therethrough, and each have a voltage applied between the ends thereof, which passes through the electrically-resistive coating to provides a linear voltage gradient along the cylinder length. The voltage gradient provided by the first electrostatic mirror 113 accelerates analyte ions through the cylinder of the first electrostatic mirror 113, and into the electric field-free region between the mirrors. The analyte ions travel through the field-free region, and into the second electrostatic mirror 115. The voltage gradient provided by the second electrostatic mirror 115 decelerates the analyte ions as they pass through the cylinder of this mirror, until they reach the second end of the ion trap 3. Here the analyte ions come to a stop, and are then accelerated by the voltage gradient of the second mirror 115 back into the field-free region. The first and second electrostatic mirrors 113, 115 therefore constrain the analyte ions to oscillate between them, passing through the electric field-free region provided between the mirrors, as before. The remaining elements of the mass spectrometer of this embodiment are the same as those of the embodiment of Figure 1 , and operate in the same manner.

It will be understood that modifications may be made to the embodiments described above. For example, the second laser may direct pulses of radiation along the longitudinal axis of the ion trap, instead of along the transverse axis of the ion trap.

The sample holder 5 of the embodiments of the mass spectrometer 1 of Figures 1 and 2 may be replaced by the sample holder 35 of Figure 3. The sample holder 35 comprises an approximately central aperture and a removable plug 37 placed in the aperture, as shown. The plug 37 defines an aperture 39 of, for example, approximately 1 mm diameter, which provides a passage through the sample holder 35.

In use, a gas is introduced into the ion trap 3 of the mass spectrometer 1 through the aperture 39 of the plug 37 of the sample holder 35. The second laser 9 emits laser radiation into the ion trap 3 and into the gas molecules, and ionizes at least some of the gas molecules to produce gas ions. After their creation at the first end of the ion trap 3, the gas ions are constrained to move along at least one longitudinal axis of the ion trap 3 by the first and second electrostatic mirrors 13, 15, as before. As the gas ions pass through the pick-up ring of the charge detector 11 , the image charge of the gas ions is detected, and measurements of the image charge are used to determine the mass of the gas ions. The gas may be a 'known' gas having a well-defined mass. Measurements of the mass of the gas ions can then be used to calibrate the mass spectrometer 1.

A test of the mass spectrometer 1 comprising the sample holder 35 has been carried out using a known gas comprising argon. The argon gas was injected into the ion trap 3 through the aperture 39 defined by the plug 37. The second laser 9 was operated to emit laser radiation into the ion trap 3 and into the gas molecules. The laser radiation comprised pulses of approximately 800nm width, 30fs duration and 1 mJ energy. The laser radiation was focussed using a focussing element comprising a spherical lens having a focal length of 25cm. This produced a cylindrical focal volume, elongated transverse to the longitudinal axis of the ion trap 3. The pressure in the ion trap was approximately 1x10^~6 mbar. At least some of the gas molecules were ionized to produce Ar⁺ and Ar²⁺ gas ions. Some of these gas ions were trapped and oscillated in the ion trap 3, for approximately 2ms. The gas ions were constrained to move along at least one longitudinal axis of the ion trap 3 by the first and second electrostatic mirrors 13, 15, as before. As the gas ions passed through the pick-up ring of the charge detector 11 , the image charge of the gas ions was detected. The measurements of the image charge were then analysed using a Fourier transform to give a frequency spectrum comprising a number of peaks at a fundamental frequency and approximately fifty harmonic frequencies of oscillation of each of the Ar⁺ and Ar²⁺ gas ions. For each peak, the resolution of the mass spectrometer may be defined as the frequency at a central point of the peak divided by twice the FWHM of the peak. For the Ar⁺ gas ions, the resolution was determined to be 500 for the fundamental frequency peak, and 4000 for the 35th harmonic frequency peak. The sample holder 35 may also be used to hold a sample comprising an analyte. The sample comprising the analyte (with or without a matrix) is placed on the sample holder 35, for example, on the plug 37 around the aperture 39. The first laser 7 is used to ablate the sample to produce a plume of analyte molecules, and the second laser 9 is used to ionize the analyte molecules, and the resultant analyte ions are analysed by the mass spectrometer 1 as before. Previous to this, a gas may be introduced into the ion trap 3 through the aperture 39 of the plug 37, and the mass spectrometer 1 calibrated. After waiting for a sufficient time for the gas ions to clear from the ion trap 3, the sample may then be ablated and analyte ions analysed. Alternatively, the gas may be introduced into the ion trap 3 and ionization of the analyte molecules (ablated by the first laser 7) and gas undertaken simultaneously by the second laser 9, and analysis of the gas ions and the analyte ions carried out together.

A test of the action of the first laser 7 of the mass spectrometer 1 comprising the sample holder 35, has been carried out. This comprised placing a sample comprising only a matrix, F20TPP, on the sample holder 35, i.e. the sample did not comprise an analyte. The first laser 7 was used to emit laser radiation into the ion trap 3 and onto the sample, and ablate the matrix from the sample holder 35, lifting the matrix off the sample holder by desorption. Matrix ions were created by the laser radiation from the first laser 7, just above the surface of the sample holder 35. The pressure in the ion trap was approximately 1x10^~7 mbar. Some of the matrix ions were trapped and oscillated in the ion trap 3, for approximately 10ms. The matrix ions were constrained to move along at least one longitudinal axis of the ion trap 3 by the first and second electrostatic mirrors 13, 15, as before. As the matrix ions passed through the pick-up ring of the charge detector 11 , the image charge of the matrix ions was detected. The measurements of the image charge were then analysed using a Fourier transform to give a frequency spectrum comprising a number of peaks at a fundamental frequency of oscillation of the ions and approximately fifty harmonic frequencies. As before for each peak, the resolution of the mass spectrometer may be defined as the frequency at a central point of the peak divided by twice the FWHM of the peak. The resolution was determined to be 400 for the fundamental frequency peak, and 4000 for the 24th harmonic frequency peak. The FWHM of the frequency peaks is inversely proportional to the lifetime of the ions in the ion trap 3. The ion lifetime is inversely proportional to the pressure within the ion trap 3. If the pressure is decreased, the ion lifetime will increase, the FWHM will decrease, and the resolution of the mass spectrometer will increase.

Claims

1. A mass spectrometer for analysing the mass of molecules of an analyte comprising an ion trap comprising a sample holder which is adapted to receive a sample comprising the analyte, a first laser which emits laser radiation onto the sample which detaches molecules of the analyte to form a plume of analyte molecules, a second laser which emits pulses of laser radiation having a full width half maximum bandwidth of at least 20nm into the plume of analyte molecules which ionizes at least some of the analyte molecules and produces analyte ions which are trapped in the ion trap, and a detector which detects a characteristic of the analyte ions, which characteristic is used to produce a measure of the mass of the analyte molecules.

2. A mass spectrometer according to claim 1 , in which the second laser emits pulses of laser radiation having a full width half maximum (FWHM) bandwidth in the range of approximately 20nm to approximately 200nm.

3. A mass spectrometer according to claim 2, in which the second laser emits pulses of laser radiation having a full width half maximum (FWHM) bandwidth in the range of approximately 50nm to approximately 200nm.

4. A mass spectrometer according to any preceding claim, in which the second laser emits pulses of laser radiation wherein each pulse has a FWHM bandwidth giving rise to a pulse duration which is less than a shortest vibrational period of the molecules of the analyte.

5. A mass spectrometer according to any preceding claim, in which the second laser emits pulses of laser radiation having intensities whose maximum is limited to promote single ionization of the molecules of the analyte.

6. A mass spectrometer according to any preceding claim, in which the second laser emits pulses of laser radiation which are elliptically polarised.

7. A mass spectrometer according to any preceding claim which comprises a focussing apparatus for focussing the pulses of laser radiation emitted by the second laser to provide an ionization volume in which the pulses of laser radiation interact with analyte ions, which ionization volume is substantially centred on a longitudinal axis of the ion trap, and is substantially cylindrical in shape, has a length greater than its diameter, and has a major axis oriented substantially along the longitudinal axis of the ion trap.

8. A mass spectrometer according to any preceding claim, in which the ion trap comprises electric field generation apparatus which comprises a first electrostatic mirror and a second electrostatic mirror.

9. A mass spectrometer according to claim 8, in which each electrostatic mirror comprises a plurality of mirror plates, the mirror plates each comprise an electrode and a series of decreasing voltages or a series of increasing voltages is applied to the mirror plate electrodes to provide a voltage gradient.

10. A mass spectrometer according to claim 8, in which each electrostatic mirror comprises a cylinder provided with a electrically- resistive coating.

11. A mass spectrometer according to claim 10, in which each cylinder has a voltage applied between the ends thereof, which passes through the electrically-resistive coating to provide a linear voltage gradient along the cylinder length.

12. A mass spectrometer according to any of claims 8 to 11 , in which the first electrostatic mirror is positioned at a first end of a longitudinal axis of the ion trap, the second electrostatic mirror is positioned at a second end of a longitudinal axis of the ion trap, and the first and second electrostatic mirrors constrain the analyte ions to move along at least one longitudinal axis of the ion trap.

13. A mass spectrometer according to any preceding claim, in which the ion trap comprises electric field generation apparatus which comprises a first electrostatic lens and a second electrostatic lens, which each act as focussing lenses to focus the analyte ions by exerting a transverse force on the analyte ions, with respect to a longitudinal axis of the ion trap.

14. A mass spectrometer according to any preceding claim, in which the detector comprise a charge detector, and detects a characteristic of the analyte ions comprising image charge of the analyte ions.

15. A mass spectrometer according to any preceding claim, in which the first laser emits laser radiation having a frequency which is readily absorbed by the sample holder, and the laser radiation from the first laser lifts the analyte off the sample holder by desorption, to form the plume of analyte molecules.

16. A mass spectrometer according to any preceding claim, in which the sample comprise the analyte and a matrix, the first laser emits laser radiation having a frequency which is readily absorbed by the matrix, and the laser radiation from the first laser ablates the matrix from the sample holder, lifting the analyte off the sample holder by desorption, and forming the plume of analyte molecules.

17. A mass spectrometer according to any preceding claim, in which the sample comprises the analyte, on a nano-patterned surface, the first laser emits laser radiation having a frequency which is readily absorbed by the surface, and the laser radiation from the first laser ablates the analyte off the sample holder by desorption, to form the plume of analyte molecules.

18. A mass spectrometer according to any preceding claim, in which the second laser emits pulses of laser radiation into the analyte ions which fragments at least some of the analyte ions, to produce sub-components of the analyte ions.

19. A mass spectrometer according to claim 18, in which one or more parameters of the pulses of laser radiation from the second laser are controlled to induce a chosen fragmentation of the analyte ions, and/or to induce breakage of specific bonds in the analyte ions.

20. A method of analysing the mass of molecules of an analyte using a mass spectrometer of the first aspect of the invention, comprising placing a sample comprising the analyte in a sample holder of an ion trap of the spectrometer, deploying a first laser to emit laser radiation onto the sample to detach molecules of the analyte to form a plume of analyte molecules, deploying a second laser to emit pulses of laser radiation having a full width half maximum bandwidth of at least 20nm into the plume of analyte molecules to ionize at least some of the analyte molecules and produce analyte ions which are trapped in the ion trap, and operating a detector to detect a characteristic of the analyte ions, and using the characteristic to produce a measure of the mass of the analyte molecules.