WO2005057610A1

WO2005057610A1 - Devices and methods for the separation, detection and/or capture of particles

Info

Publication number: WO2005057610A1
Application number: PCT/GB2004/005150
Authority: WO
Inventors: Shiv Sharma; Terek Schwarz
Original assignee: Ge Heathcare Uk Limited
Priority date: 2003-12-11
Filing date: 2004-12-07
Publication date: 2005-06-23
Also published as: GB0328697D0

Abstract

Mass spectrometer for separating and detecting and/or capturing molecules and/or particles independent of their charge in which molecules and/or particles are accelerated along a flight tube by radiation pressure. When used to detect the mass of molecules and/or particles their times of arrival at a detector used to determine the mass of each detected molecules and/or particle. When used to capture molecules or particles, the mass spectrometer is provided with a capture surface.

Description

Devices and methods for the separation, detection and/or capture of particles

Field of the Invention

The present invention relates to separating, detecting and or capturing devices for detecting and/or capturing single molecules, groups of similar molecules, trains of differing molecules and particles, methods for separating, detecting and/or capturing these using said detecting and/or capturing devices, and the use of such devices and methods to separate, detect and/or capture such molecules and particles.

Prior Art

In prior art devices and methods such as matrix assisted laser ablation time of flight mass spectrometers (MALDI -TOF MS), for measuring the time of flight (TOF) of molecules and/or particles (where a particle is defined as being a body of finite mass and internal structure but negligible dimensions), the molecules and/or particles are ablated from a matrix by a laser pulse and charged molecules and/or particles are accelerated towards a timing detector by an electric field along the length of a vacuum flight tube. The timing detector is usually a micro channel plate detector (MCP), which is an electron multiplier and needs a certain number of detectable particles (i.e. particles having kinetic energy above a threshold detector value) to hit it before a count is registered. The timing detector measures the time from the ablating laser pulse to a detectable number of particles (having substantially the same mass/charge ratio and sufficient in number to be registered) hitting the timing detector, i.e. the time that the particle(s) take to travel down the flight tube and hit the detector is determined. A problem with these devices is that the limitations in sensitivity of the microchannel plate detectors means that they are not suitable for detecting single particles. Another difficulty is that higher mass particles (i.e. those with masses greater than about 10000 Daltons), which are often important in biological measurements, are accelerated relatively slowly by the electrical field, resulting in low particle velocities which produce lower signals at the detector. These low signals may be too low to be reliably detected and hence TOF MS is not suitable for the detection of higher mass particles. Additionally, only charged particles can be accelerated by an electrical field and therefore the ablated particles must be ionised during ablation. Present methods of ionisation are inefficient and it is estimated that about only 1 in 10⁴ to 1 in 10⁷ analyte molecules in a sample are ionised. The ions of many analyte molecules are often unstable. Some ionised analyte molecules are so unstable that they disintegrate before they reach the detector. Other analyte molecular ions can rapidly decay in the acceleration region of the mass spectrometer and during their passage through the time of flight tube. The decay of ions is a major cause of peak broadening and loss of resolution. Furthermore, the resolution of prior art MADLI-TOF MS is limited by the angular spread of analyte ions. This is seen as ions of a particular mass hitting the detector at different times and increasing the width of the detected peak. The angular spread is caused by some or all of the following reasons: a) The ions may be formed in different locations. Ions formed close to the surface of the sample plate, will pass through a larger electrical potential and will be accelerated to a higher kinetic energy than the ion formed at the front of the gas plume. b) Two ions of the same mass may be formed at the same location but with different kinetic energies. The ions will travel at different velocities through the time of flight tube and consequently may be detected at different times. c) Two ions of the same mass and the same initial kinetic energy may start moving in opposite directions. The ion moving away from the time-of-flight tube will travel against the electrical field, stop, turn around and be accelerated to the same energy as the other ion that was initially moving toward the time-of-flight tube. These two ions will be accelerated to the same velocity and will therefore maintain a constant difference in arrival times.

It is often difficult to identify all the components in a mixture of analytes. The phenomenon is known as analyte-analyte suppression (Knochenmuss et al 2000, J. Mass. Spectrom 35: 1237- 1245, Knochenmuss & Zenobi 2003, Chem. Rev. 103: 441-452). For example less than half the peptides generated by a tryptic digest are usually observed in a MALDI spectrum (Kratzer et al 1998, Electrophoresis 19: 1910-1919).

Summary of the Invention

In devices and methods in accordance with the present invention the separation, and subsequent detection, of the molecules of a volatilised analyte can be achieved independently of the charged state of the molecules. This is achieved by the volatilised analyte molecules being accelerated by radiation pressure, preferably photon pressure, towards detecting means where the times of arrival of the analyte molecules are detected.

In the present invention analyte molecules can be used in the pure state or mixed with a matrix. The matrix may be a liquid, for example glycerol or water, or a solid. The matrix may be chosen solely on its ability to ablate the analyte molecules at a specified ablation wavelength and light intensity.

In method and devices in accordance with the present invention, the analyte and matrix (if necessary) are provided inside a chamber in a mass spectrometer. The analyte and any matrix may be placed on a sample plate (possibly transparent to the radiation, e.g. photon, source) or a reflecting or metal surface in a vacuum inside an evacuable mass spectrometer chamber. Alternatively, analyte molecules may be injected into the mass spectrometer, for example as an analyte molecule-laden gas or liquid stream that is inputted to the evacuable chamber of the mass spectrometer though an input device, such as a capillary, communicating with the chamber. The mass spectrometer chamber preferably has a proximal input end containing the sample plate and/or input device, a detecting means for detecting particles positioned at or near a distal end and a flight chamber connecting the proximal and distal ends. The analyte molecules are put into motion inside the mass spectrometer and then directed and, preferably, driven towards the mass spectrometer detecting means using photon pressure. The photon pressure can be provided by at least one intense pulse of laser light that drives the analyte molecules towards the detecting means. Preferably the wavelength of the light is chosen to maximise the separation and minimise the degradation of the analyte molecules. The laser beam may hit the sample from any suitable angle, e.g. underneath or from behind, such that the laser beam pushes the analyte molecules towards the end of the time of flight chamber and the detector. When molecules are driven by photon pressure, the molecules with the largest volumes (i.e. the heavier molecules) will be accelerated more than molecules with smaller volumes (i.e. the lighter molecules). Photon pressure increases the momentum in direct proportion to M (where M = mass of the analyte molecule).

Optionally, apertures may be placed between the sample and the detecting means so that ablated analyte molecules which are not moving substantially directly towards the detecting means are removed in order to prevent them reaching the detector means. The operating principle of the present invention is as follows:

The amount of energy imparted to a particle such as molecule in a beam of photons is dependent on the number of photons absorbed by the particle and this is dependent on the particle's mass.

This can be shown as follows:

The energy of a photon = hv Where h = Planck's Constant = 6.628 x 10^"34 Joules.sec (m². kg. s^"1) v= frequency of light = 6 x 10¹⁴ m.sec^'1 for visible light.

The kinetic energy of a particle = Vτ Mu² Where u = particle velocity and M = mass

Equating the two energies we get:

or u² = 2hvM

For a given photon - λ

Velocity u per absorbed photon = V2hv/M

Number of photons absorbed by a particle is determined by its absorption cross-section which is proportional to the square of the mass of the particle (M²).

Therefore Velocity of a particle = U = M². 2hv/M

So the final velocity of a particle, U, is proportional to:

U = M^3/2.V(2hv) Example:

Calculation to predict the velocity at which a 100 kilo-Daltons (JDa) molecule will move when it is hit by a single photon: M =1.67 x 10^"19 g (for a 100,000 Daltons particle) = 1.67 x l0^"22 kg

{6 x 10²³ molecules (each weighing 100,000 Daltons) weigh 100,000g Therefore 1 molecule = 100,000/6 x 10²³ g = 1.67 x l0^"19 g.} u² = 2hv/M

= 2 x 6.628 x l0^" 3^J4 _.m .kg. s -^"1 , x- 6 x 1l ΛO+⁺1¹4V -17 IΛl. _6-7-τ „x ιl ΛO-^~22 ^■kg

2 6.628 l0²/1.67 m². s^"2

= 794 m². s^"2

or u = 28 m/s

Hence the velocity of the molecule of mass of 100,000 Daltons (100 kDA) after it absorbs a photon is 28 m/s in the direction of the photon.

Performing the same calculation for a 10 kDA molecule, shows that when hit by one photon a 10 Da molecule will have a velocity of u = 89 m/s.

In a beam of photons, each particle will be hit by many photons and this leads to that larger molecules will move at a higher velocity than smaller molecules when placed in the path of a beam of photons. This can be shown as follows:

The number of photons that a small particle adsorbs is determined by the absorption cross section of that particle. The absorption cross section of a small particle is proportional to the square of the particle's mass, i.e., the number of photons that a particle absorbs α M².

Thus take two particles of masses 10,000 Daltons (10 kDA) and 100,000 Daltons (lOOkDA). The velocity changes per photon absorbed are 89 m/s and 28 m/s respectively as shown above.

The masses of the two particles differ by a factor of 10. The larger particle would absorb 10² = 100 times more photons than the smaller one.

If the smaller particle is placed in beam of photons long enough for it to absorb one photon, its velocity change would be 89 m/s whereas the larger particle would, when placed in the same beam of photons for the same length of time, absorb 100 photons resulting in its velocity change being 2800 m/s. This enables particles of different masses to be easily separated.

Furthermore, in traditional, ion-based time of flight mass spectrometers, the time of flight of an ion is proportional to the square root of the mass of the ion - thus the difference in the time of flight for two different ions is proportional to the square root of the difference in the masses of the two ions. Consequently it is difficult to resolve the difference in the flight time of two similarly charged ions which differ in mass by only a few Daltons. Conversely, in mass spectrometers in accordance with the present invention, the time of flight of a particle is proportional to the M^{3 2} (where M = the mass of the analyte molecule/particle) - thus the difference in the time of flight for two different particles is proportional to M^{3 2} of the difference in the masses of the two particles - making it easier to resolve the difference in the time of flight of two particles which differ by the same few Daltons. The separation of analytes using the present invention is M (i.e. M divided by M ) times greater than by prior art Time of Flight methods

Further advantages of the present invention are that: a) Photon pressure can impart high velocities in the direction of the detecting means to the analyte molecules. This high velocity can be much higher than undesirable lateral velocities imparted to the analyte molecules during volatilisation and this high velocity towards the detecting means leads to most of the volatilised molecules reaching the detecting means before their lateral motion has moved them outside of the detecting area of the detecting means. This is a very efficient method for separating most of the molecules that have been ablated from a sample. b) Molecules are simultaneously drawn into the centre of a continuous photon beam as the same time as they are accelerated in the direction of the light beam as observed by Bjorkholm JE et all 1978, "Observation of focusing of neutral atoms by the dipole forces of resonance-radiation pressure", Physical Review Letters 41:1361-1364. If the accelerating light beam is directed towards the detecting means then this effect helps ensure that the maximum number of particles reaches the detecting means. c) Charged molecules are inefficiently made when a laser ablates the analyte and matrix molecules. Most of the analyte molecules in the gas phase after laser desorption are not charged. Estimates of the ratio of ions to neutral analyte molecules in the gas phase indicate that only one in 10⁴ to one in 10 ⁷ analyte molecules are ions depending on the analyte molecule (Puretzky et al 1999, Phys. Rev. Lett. 83: 444-447, Mowry et al 1993, Rap. Comm. Mass. Spectrom. 7: 569-575, Quist et al 1994 Rap. Comm. Mass. Spectrom 8: 149-154 , and Ens et al 1991, Rap. Comm. Mass. Spectrom. 5: 117-123). In the present invention it is possible to detect substantially all the analyte molecules in the gas phase leading to an increase of sensitivity by 10⁴ to 10 ⁷ compared to MALDI-TOF. d) Additionally, it is difficult to optimise sample preparation and ablation conditions for the detection of each analyte ion, The fraction of molecules in a sample that are detected varies wildly in MALDI-TOF. It depends on the analyte molecules, the nature of the other ions formed after desorbtion, the solvents present, the pH, the laser frequency, the concentration ratio of analyte to matrix and the matrix used. If the wrong matrix is used, or if no suitable matrix is available, it can be very difficult to ionise some analytes as the efficiency of ionisation of an analyte is dependent on the choice of matrix used. These problems do not affect the present invention as it does not need particles to be charged to detect them. e) It will not be necessary to modify analytes so that analyte ions are stable in the gas phase (i.e. for DNA analysis - Tang et al 1997, Anal. Chem. 69: 302-312).

1) It will not be necessary to add charge tags to molecules that are difficult to ionise (i.e. for DNA analysis - Gut et al 1997 Rapid Comm. Mass Spectrom. 11 : 43-50, for peptide analysis - Liao & Allison 1995, J. Mass Spectrom. 30: 408). f) It is usually not possible to obtain accurate quantitative data of the amount of the different analytes in a mixture partly because there are often dramatically non-linear concentration effects of analyte concentration and MALDI ion signal. (Knochenmuss et al 1998, Rapid Comm. Mass Spectrom. 10: 871, Knochenmuss et al 1998, Rapid Comm. Mass Spectrom. 12: 529). g) A major advantage of the present invention is that the resolution of separation of analytes increases with the mass of the analyte. Thus, in devices in accordance with the present invention for the separation and detections of molecules and/or particles using radiation dispersion, the resolution of the device increases as the mass of the particles or molecules being detected increases. This is the opposite of prior art mass spectrometers in which the resolution decreases as the masses of the molecules or particles being detected increase. This will be particularly useful for sequencing DNA and identifying peptide and protein modifications. When DNA is sequenced, it is necessary to distinguish between a fragment ending in A from the same fragment ending in a T. This is a difference in MW of 9. Similarly, when characterising proteins and peptides, a modified peptide or protein will have a small increase in mass. Some examples are; N-terminal palmitoylation -» +238 Da N-terminal myristylation - +210 Da Phosphorylation & Sulphation -» +80 Da N-terminal acetylation —» +42 Da Carboxylation - +30 Da N-terminal formylation -» +28 Da Hydroxylation ~» +16 Da N-methylation & O-methylesterification -> +14 Da

The present invention will also be useful for the analysis of other biological molecules. The binding of drugs and ligands to their targets (protein, nucleic acid or lipid) will be detectable without the use of radioactive or dye labels as will the detection of enzyme-substrate complexes. The present invention will also facilitate the identification and analysis biological complexes (i.e. protein-protein and protein-lipid interactions).

Thus, in the present invention, at least some of the problems with the prior art are solved by means of devices having the features present in the characterising portions of claim 1 and claim 2, and by methods having the features mentioned in the characterising portion of claim 6. h particular, devices in accordance with the present invention can detect particles independently of any charge on the particle. Furthermore the present invention gives a high sensitivity for larger mass particles, which are difficult to detect in prior art mass spectrometers as their accelerating electrical fields impart low velocities to heavy ions, but which are relatively easy to detect using devices in accordance with the present invention in which the heavier a particle is, the more photons it absorbs and the higher its velocity becomes.

Brief Description of the Figures

Figure la) shows schematically a lateral view of a first embodiment of a device in accordance with the present invention;

Figure lb) shows schematically an enlarged section through line I-I of the device of figure la); and

Figure 2 shows a schematically a second embodiment of a device in accordance with the present invention.

Detailed Description of Embodiments Illustrating the Invention

Figures la and lb show schematically, and not to scale, a first embodiment of a mass spectrometer 1 in accordance with the present invention. Mass spectrometer 1 has at its proximal end 2 a sample chamber 3 in which a sample 5 to be analysed can be ablated, by ablating means such as an ablation laser 7. Sample 5 can be any ablatable substance, for example, biological material such as a piece of tissue, bio-molecules such as nucleic acids, proteins, protein complexes, viruses, etc. as well as non-biological materials. An example of a suitable ablation laser is a nitrogen gas laser with a pulse length of the order of 1 nanosecond. Other lasers are also conceivable and may depend on the nature of the sample being ablated. The wavelength of the light emitted by the laser is preferably chosen to be sufficiently long that the amount of sample ionised is minimised. The sample may be any substance of interest, for example a biological sample in the form of a piece of tissue or a sample of fluid or a smear or blot or the like, or a sample comprising one or more chemical compounds that need to be identified or a substance, the composition of which is being investigated, etc. Sample chamber 3 has an orifice 9 which leads into an elongated flight chamber 11. The choice of the length of flight chamber depends on the accuracy and resolution required from the mass spectrometer. In general longer flight chambers give better accuracy and resolution as the longer the distance that the particle travel, the larger the time differential is between fast and slow moving particle. Preferably the flight chamber is at least 10 cm long and less than 2 m long. When the mass spectrometer 1 is being used, air is evacuated from flight chamber 11 so that it contains a near vacuum. Ablation laser 7 is positioned outside the sample chamber. Sample chamber 3 is provided with a first window 13 between ablation laser 7 and the sample 5 to be analysed. Ablation laser 7 is positioned, and optical devices such as lenses, prisms and/or mirrors (not shown) are optionally provided as necessary, so that an ablating laser beam 15 emitted from ablation laser 7 to the sample 5 travels along a path 17 that is perpendicular to the longitudinal axis L of the flight chamber 11. The purpose of the ablating laser 7 is merely to volatilise the analyte and not to impart a high velocity to the volatised analyte molecules. The position of the ablation laser 7 and the angle at which the ablation laser beam 15 strikes the sample are not critical to the performance of methods and devices in accordance with the present invention and other ablation laser positions and laser beam paths, such as parallel to the longitudinal axis L or at an angle to the longitudinal axis L are also conceivable. The distal end 19 of flight chamber 11 is provided with detecting means 21, such as a multichannel plate detector (MCP) which detects the impact of particles on its detector surface 23, or a photomultiplier tube, or a scattered light detector (as described in PCT patent application WO2002/086945) or equivalent, and/or a capture means such as a capture surface 21' upon which the particles/molecules can stick. Preferably such a capture means is removable to allow further analysis of the captured molecules/particles. Sample chamber 3 is provided with a second window 25 on the sample chamber wall opposite orifice 9. Particle accelerating means, for example a particle accelerating laser 27, is positioned outside sample chamber 3 and arranged such that an accelerating laser beam 29 emitted from it travels substantially parallel to the longitudinal axis of the flight chamber and towards the distal end 19 of the flight chamber. The accelerating laser is preferably a pulsed laser with sufficiently high energy in the pulses to drive the particles in the direction of the detector at the far end of the tube. The velocity imparted to the particles by the accelerating laser is preferably sufficiently high that most or all particles which had been given a sideways velocity vector by the ablating means move so rapidly to the detecting means 21 that they are detected before their sideways vector moves them outside the detector surface 23. Sample 5 is supported on a sample support 31 which is preferably movable so as to be able to position the sample 5 in the path of ablating laser beam 15 so that the sample can be ablated, and a small distance, e.g. 1- 10 mm away from the path of accelerating laser beam 29, so that the accelerating laser beam 29 does not hit the sample 5.

Ablating laser 7, particle accelerating laser 27 and detecting means 21 are controllable by control means 29. Control means 29 may be a computer, microprocessor or the like which is able to cause the lasers 7, 27 to emit laser beams, detecting means 21 to detect the impact of particles on its detector surface and, optionally, the position of sample support 31 and any pumps (not shown) for evacuating the sample chamber and flight chamber. Control means 29 maybe provided with operator command inputting means such as a keyboard, switches, a pointing device e.g. a computer mouse, tracker ball or touch-sensitive screen, etc. for inputting operator commands. Computing means, optionally integrated in control means 29, are preferably provided for calculating the mass of particles detected by the detecting means 21. Display means, e.g. a computer monitor 33, for displaying operator input menus, operating conditions, system information, the results of the analysis of a sample and other information, and storage means, e.g. a hard disc 35, for storing the results of the analysis of a sample may also be provided.

An example of a method for detecting the mass of particles volatilised from a solid sample comprises the following steps: the sample 5 to be examined is applied to sample support 31 in sample chamber 3, optionally a matrix which improves volatilisation of the sample is applied to the sample; air is evacuated as necessary from sample chamber 3 and flight chamber 11 ; sample 5 is positioned in the path of the ablation laser beam 15 from the ablation laser 7; ablation laser 7 is activated to produce a pulse of laser light which, in the form of ablation laser beam 15, hits sample 5. This volatilises the portion of the sample 5 that it hits and causes volatised, sample particles to be ejected from the sample - while most of said volatised sample particles will be uncharged, it is possible that some will be charged; within a short predetermined period of time, accelerating laser 27 fires an accelerating laser beam 29 towards the distal end 19 of the flight chamber 11. Any particles, uncharged or charged, in the path of accelerating laser beam 29 that are hit by photons from the laser beam 29 are accelerated in the direction of the laser beam. The timing of the acceleration laser beam can be adjusted according to the initial ejection velocity of the particles and/or the distance that the particles have to travel from the sample support 31 to the path of the accelerating laser beam 29 and/or which particle masses the operator is interested in analysing - for example, heavy particles may be ejected with a lower velocity than lighter particles from the sample and increasing the period of time between firing the ablation laser pulse and starting the accelerating laser will allow faster moving particle to pass out of the path of the accelerating laser while allowing slower moving particle to enter the path of the accelerating laser. Particles that are in the path of the accelerating laser beam 29 and are hit by the photons in the accelerating laser beam 29 are accelerated along the flight chamber 11 towards the detecting means. The arrival times of the particles at the detector surface 23 of detecting means 21 can be recorded, their times of flight calculated and their masses calculated.

Figure 2 shows an embodiment of a device 201 in accordance with the present invention which is suitable for detecting the mass of particles volatilised from a fluid sample.

Components of this device which are similar to those of the device shown in figures la) and lb) have the same reference numbers as used in those figures and will not be described again. Mass spectrometer 201 comprises a fluid inlet means 251 which projects into sample chamber 3. Fluid inlet means 251 may be in the form of a capillary 253 leading from a fluid sample reservoir 255, e.g. a beaker, or ajar, or the outlet from an instrument such as a chromatography column or the like, etc. A pump 257 is optionally provided to assist the flow of sample from reservoir 255 to capillary 253. A valve 259 is optionally provided to control the flow of sample from reservoir 255 to capillary 253. In this device the sample fluid flows through capillary 253 into the evacuated sample chamber 3. Sample fluid may be made to flow continuously or as pulses of fluid. As sample fluid enters the chamber the low pressure in the chamber causes the fluid to vaporise and form a cloud of sample particles which continue to move in the same general direction that the fluid was travelling in as it left the capillary. The capillary can be aligned so that these sample particles pass through the path of the acceleration laser beam 29. The acceleration laser 27 can be pulsed on and off and the particles in the path of the acceleration laser beam 29 which are hit by photons in the beam are accelerated towards the detecting means 21 where they may be detected. In order to minimise the number of particles arriving at the same time at the detecting means 21, the accelerating pulses can be made of short duration and/or the flow rate of fluid from the capillary can be minimised by reducing the pressure that it is pumped at by pump 257 and or throttling the flow with valve 259.

A method for detecting the mass of particles volatilised from a fluid sample comprises the following steps: air is evacuated from sample chamber 3 and flight chamber 11; the sample supplied to the sample chamber 3 via a fluid inlet means 251 pointing towards the path of the acceleration laser beam 29 from the accelerating laser 27. As the sample enters the evacuated chamber the low pressure causes the sample to vaporised forming sample particles. The vaporised sample particles continue to move towards the path of the accelerating laser 27; accelerating laser 27 fires an accelerating laser beam 29 towards the distal end 19 of the flight chamber 11. Any vaporised sample particles, uncharged or charged, in the path of accelerating laser beam 29 that are hit by photons from the laser beam 29 are accelerated in the direction of the laser beam along the flight chamber 11 towards the detecting means. The times of arrival of particles at the detecting means can be recorded, the time of flight of the particles calculated and the masses of the particles can subsequently be calculated.

The acceleration laser beam can be in the form of one or more pulses of laser light and preferably, the duration, power and, when appropriate, the repetition rate of the acceleration laser beam pulses are controllable.

Optionally, the analysis of a sample is achieved by performing a series of ablation/acceleration/detection steps. In order to investigate the full range of particles ejected from a sample, the timing difference between the ablation and acceleration lasers may be adjusted during the series to optimise the yield of the particles ejected at different velocities (and hence arriving at different times in the path of the accelerating beam) from the sample.

In order to achieve the highest possible sensitivities, if a photomultiplier tube is used to detect particles, it is possible to cool the photomultiplier tube in order to reduce its background noise, referred to as background counts.

While in the present invention the acceleration of particles towards the detecting means present invention is independent of the charge state of the particles, it is possible that ionised particles could react with other particles and reduce the accuracy or resolution of a mass spectrometer in accordance with the present invention. In order to prevent this it is possible to provide a device in accordance with the present invention with electrical field producing means arranged to prevent ionised particles travelling towards the detecting means. This electrical field producing means could be in the form of charged plates in the vicinity of the sample chamber or its orifice which attract or repulse ionised particles in order to prevent them reaching the detecting means. i the case that a device in accordance with the present invention has capture means, e.g. a surface, for capturing the molecules/particles after they have been separated as well as a detector surface, it may be provided with means for arranging for the molecules/particles to be directed to either the detector means or the capture means. It is also conceivable to provide means for post-separation fragmentation of the molecules/particles in order to permit further analysis of them.

The present invention may be used to take a mass spectrum image of a sample. This can be achieved by, for example, ablating the sample in a grid pattern, detecting the particles ablated from each grid point thereby obtaining a mass spectrum point by point in a raster manner to obtain a mass spectrum image of an area of a sample and displaying the results of each ablation as pixels in an image.

The present invention may be used for the analysis of biological molecules. Examples are: DNA sequencing (using either Sanger's method or Maxam-Gilbert), detection of protein modifications (i.e. detection of phosphorylation), detection of the biological molecule (peptide, protein, nucleic acid, lipid) to which a Hgand or drug binds to without the use of labels, detection of enzyme-substrate complexes, identification and analysis of biological complexes (i.e. protein-protein and protein-lipid interactions).

It is also conceivable to provide devices in accordance with the present invention with movable particle capture targets. The movable capture targets can be moved transverse the direction of movement of the particles from a sample so that the particles, for example proteins, are captured as one or more lines of particles on the capture target. This could be used to produce arrays of proteins which could then be used to determine protein-drug interactions, protein-protein interactions for the analysis of protein complexes, etc.

The above mentioned embodiments are intended to illustrate the present invention and are not intended to limit the scope of protection claimed by the following claims.

Claims

1. Mass spectrometer for separating and detecting and/or collecting neutral charged molecules and/or particles by radiation dispersion which comprises: an evacuable sample chamber connectable to molecule and/or particle detector means, and/or molecule and or particle capture means, wherein said sample chamber comprises a sample holder and ablating means for ablating a solid sample on said sample holder, and/or fluid sample inputting means, in order to provide one or more molecules and/or particles of sample inside said sample chamber; molecule and particle accelerating means for accelerating at least one of said one or more molecules and/or particles of sample towards said detector means; means for determining the length of time, for a detected molecules and/or particle of sample, that elapsed between said molecule and/or particle starting to be accelerated towards said detector means and said molecule and/or particle of sample arriving at said detector means and/or means for directing said particle and/or molecules to said capture means .

2. Mass spectrometer in accordance with claim 1 characterised in that said molecule and particle accelerating means comprises an accelerating beam of radiation such as photons.

3. Mass spectrometer in accordance with claim 2 characterised in that said beam of radiation is a beam of photons.

4. Mass spectrometer in accordance with any of the previous claims wherein said ablating means comprises an ablating laser.

5. Mass spectrometer in accordance with claim 1 wherein said fluid sample inputting means is a capillary.

6. Method for detecting and/or capturing molecules and or particles comprising the steps of: providing one or more molecules and/or particles of sample inside an evacuated sample chamber; accelerating one or more of said molecules and/or particles towards a detecting means and/or a capturing means: and detecting the arrival at said detecting means of one or more of said molecules and/or particles, and/or capturing said molecules and/or particles on said capturing means.

7. Method in accordance with claim 6 wherein the step of accelerating one or more of said molecules and/or particles includes the step of firing an accelerating beam of radiation at one or more of said molecules and/or particles.

8. Method in accordance with claim 6 or 7 wherein the step of providing one or more molecules and/or particles of sample inside an evacuated sample chamber comprises the step of ablating a solid sample.

9. Method in accordance with claim 6 or 7 wherein the step of providing one or more molecules and/or particles of sample inside an evacuated sample chamber comprises the step of providing a flow of fluid into said sample chamber.

10. The use of a mass spectrometer according to any of claims 1-5 to analysis biological molecules.

11. The use of a mass spectrometer according to any of claims 1 -5 to take a mass spectrum image of a sample.

12. The use of a mass spectrometer in accordance with any of claims 1-5 to produce an array of particles from a sample.