MALDI target plate

Abstract

A MALDI ion source is disclosed comprising: a target plate (2) having a front surface (4), a rear surface (6), and at least one sample receiving well (9) for receiving a liquid sample or at least one sample receiving channel (8) extending from an opening (12) in the rear surface (6) to an opening (14) in the front surface (4) for receiving a liquid sample (10), wherein each well (9) or channel (8) has a volume of ≥1 μL. The ion source also comprise a laser (16) for ionising a liquid sample (10) on or in the target plate (2), wherein the laser (16) is a pulsed laser set up and configured to have a pulsed repetition rate of ≥20 Hz, or is a continuous laser.

Claims

1. A liquid MALDI ion source comprising: a target plate having a front surface, a rear surface, and at least one sample receiving well for receiving a liquid sample or at least one sample receiving channel extending from an opening in the rear surf ace to an opening in the front surface for receiving a liquid sample, wherein each well or channel has a volume of ≥50 μL; and a laser for ionising a liquid sample on or in the target plate, wherein the laser is a pulsed laser set up and configured to have a pulsed repetition rate of ≥30 Hz, or is a continuous laser.

2. The ion source of claim 1, wherein for any given channel the opening on the rear side of the target plate has a larger area than the opening on the front side of the target plate.

3. The ion source of claim 1, comprising at least one sample supply capillary connected to the opening in the rear side of target plate of the at least one channel.

4. The ion source of claim 3, comprising a pump connected to the capillary for pumping the sample or another liquid to the channel through the capillary; and/or comprising a liquid chromatography column connected to the opening in the rear side of target plate of the at least one channel.

5. The ion source of claim 1, comprising a pump for creating a pressure differential between the rear and front openings of the at least one channel so as to urge sample towards the front opening.

6. The ion source of claim 1, wherein said at least one channel is configured such that desorption of the sample at the channel opening in the front side of the target plate draws the remainder of the sample through the channel under capillary action to the opening in the front side.

7. The ion source of claim 1, wherein the cross-sectional area of any given channel continuously tapers or is stepped from a first area arranged towards the rear side of the target plate to a second smaller area arranged towards a front side of the target plate.

8. The ion source of claim 1, wherein the laser is a pulsed laser having a laser pulse rate of ≥40 Hz, ≥50 Hz, ≥60 Hz, ≥80 Hz, ≥100 Hz, ≥200 Hz, ≥300 Hz, ≥400 Hz, ≥500 Hz, ≥600 Hz, ≥700 Hz, ≥800 Hz, ≥900 Hz, ≥1 kHz, >2 kHz, ≥3 kHz, ≥4 kHz, ≥5 kHz, ≥10 kHz, or ≥50 kHz.

9. The ion source of claim 1, wherein each of said at least one channel or well has a volume of: ≥60 μL, ≥70 μL, ≥80 μL, ≥90 μL, ≥100 μL, ≥200 μL, ≥300 μL, ≥400 μL, ≥500 μL, ≥600 μL, ≥700 μL, ≥800 μL, ≥900 μL, ≥1 mL, ≥2 mL, ≥3 mL, ≥4 mL, or ≥5 mL.

10. The ion source of claim 1, wherein the ion source is an atmospheric pressure ion source.

11. The ion source of claim 1, wherein the target plate comprises a 1D or 2D array of said channels or wells spaced in the plane orthogonal to the direction between the front and rear surfaces of the target plate.

12. The ion source of claim 1, comprising a laser controller for moving a laser beam from the laser between different ones of said channels or wells at different times; and/or comprising a target plate carrier configured for moving the target plate so that the laser beam is incident on different ones of said channels or wells at different times; and comprising a position control system having one or more detector for sensing the laser beam and/or target plate position and a controller for controlling this position so as to direct the laser beam onto an opening of the channel or well.

13. The ion source of claim 12, wherein the one or more detector comprises a photodetector arranged on the opposite side of the target pate to the laser, wherein the control system is configured to control the position of the laser beam and/or target plate so that the laser beam passes through the channel to be incident on the photodetector.

14. The ion source of claim 1, comprising at least one voltage source arranged and configured for charging the liquid sample and to provide an electric field for urging the liquid sample through the channel or well towards the front side of the target plate.

15. A mass spectrometer comprising the ion source of claim 1 and a mass analyser and/or ion mobility analyser for analysing ions from the ion source.

16. A method of ionising a sample comprising: providing an ion source as claimed in claim 1; providing a liquid sample to said target plate; and ionising said sample.

17. The method of claim 16, wherein the sample is a liquid sample and said step of ionising the sample is performed by directing the laser onto the liquid sample.

18. The method of claim 17, comprising driving the liquid sample through the target plate whilst ionising said liquid sample on or in the target plate; or wherein ionisation of the liquid sample on or in the target plate draws the sample through the at least one sample receiving well or channel.

19. A liquid MALDI ion source comprising: a target plate having at least one sample well extending only partially through the thickness of the target plate for receiving a liquid sample, wherein each well has a volume of ≥50 μL; and a laser for directing a laser beam onto the at least one well for ionising a sample in said well, wherein the laser is a pulsed laser set up and configured to have a pulsed repetition rate of ≥100 Hz, or is a continuous laser.

20. A liquid MALDI ion source comprising: a target plate having a front surface, a rear surface, and at least one sample receiving channel extending from an opening in the rear surface to an opening in the front surface for receiving a liquid sample, wherein for any given channel the opening on the rear side of the target plate has a larger area than the opening on the front side of the target plate; and a laser for ionising a liquid sample at the opening of the at least one channel in the front surface of the target plate, wherein the laser is a pulsed laser set up and configured to have a pulsed repetition rate of ≥30 Hz, or is a continuous laser, wherein said at least one channel is configured such that desorption of the liquid sample at the opening in the front surf ace of the target plate by the laser draws the remainder of the liquid sample through the channel under capillary action to the opening in the front side.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments will now be described, by way of example only, and with reference to the accompanying drawings in which:

(2) FIG. 1 shows a target plate according to an embodiment of the present invention having sample receiving channels;

(3) FIG. 2A shows a view of the front side of the target plate, and FIG. 2B shows a view of the rear side of target plate;

(4) FIG. 3 the ion signal obtained as a function of laser pulse rate for an embodiment of the invention; and

(5) FIG. 4 shows an embodiment of the invention having sample receiving wells.

DETAILED DESCRIPTION

(6) Embodiments of the present invention relate to sample analysis using pulsed lasers operating at a relatively high rate. The operation of such faster lasers (e.g. kHz lasers) has not been demonstrated previously. However, it has been realised that for pulsed laser rates up to 1 kHz, e.g. in an AP-MALDI ion source, the ion current generated may be linearly proportional to the laser firing rate.

(7) Operating the pulsed laser at such high repetition rates enables the experiment time to be reduced, since the sample may ionised and hence analysed more rapidly. In conventional MALDI techniques the sample is deposited on the target plate as laterally spaced droplets and the laser is moved between the droplets so as to ionise the material therein. Each of the sample droplets is held in place on the target plate by its surface tension, which limits the volume that the droplet can have. Typically, sample droplets exceeding around 1-2 μL in volume will “burst” and spill onto adjacent sample droplets. As the sample size of any given droplet in conventional MALDI techniques is so low, sample depletion would be a problem if operating the pulsed laser at higher rates.

(8) The inventor has realised the above problems and recognised that they may be overcome or mitigated by use of the embodiments described herein.

(9) FIG. 1 shows a schematic of a target plate 2 according to an embodiment of the present invention. The target plate 2 comprises a front side 4 for being arranged towards the inlet or inlet tube 5 of a mass spectrometer and an opposing rear side 6. The target plate 2 comprises a plurality of channels 8 extending through it from the rear side 6 to the front side 4, and for receiving sample 10 to be analysed. The target plate 2 may comprise a 1D or 2D array of such channels 8 through the target plate 2 (or even only a single such channel). The opening 12 of each channel 8 on the rear side 6 of the target plate 2 may be relatively large, whereas the opening 14 of each channel 8 on the front side 4 of the target plate 2 may be smaller than its opening 12 on the rear side 6. For example, each opening 14 in the front side 4 may be circular and have a diameter of 0.1 to 0.2 mm. The narrowing of each channel 8 from the rear side to the front side enables each channel 8 to have a relatively large volume for holding a relatively large sample 10 (e.g. 5-100 μL or higher), whilst providing a relatively small sample area at the front side 4 of the target plate 2 so that a relatively small laser spot can be efficiently used to illuminate and desorb the sample at the front side 4. As each channel 8 is able to hold a relatively large sample 10, a laser 16 may be used that is operated at a relatively high pulse rate without depleting the sample 10 in each channel 8 too quickly. For example, the laser 16 may be operated at a repetition rate exceeding 20 Hz.

(10) In the embodiment shown in FIG. 1, each channel 8 has a first length of constant cross-sectional size extending from the rear side 6 of the target plate 2 into the plate, connected to a second length of smaller constant cross-sectional size extending from the front side 4 of the target plate 2. However, other channel configurations are contemplated. For example, the channels 8 may taper down in other manners towards the front side of the target plate, such as by tapering continuously or in a conical fashion. Alternatively, the channel 8 may have the same size opening in the front and rear sides of the target plate (or may even have a smaller opening in the rear side than the front side), but may have a length between the front and rear openings of a cross-sectional area that is larger than that of the front and/or rear openings. Alternatively, it is contemplated that the entire channel 8 may have a constant cross-sectional area throughout, i.e. which is the same as that of the openings in the front and rear sides. This still allows a large sample volume loading per channel, since the sample volume may be defined by the thickness of the target plate (rather than the surface tension of the sample, as in conventional techniques where the samples are deposited on top of the front surface of the target plate). It is contemplated that the channel 8 may even have a cross-sectional area between the front and rear openings that is smaller than that of the rear and/or front openings.

(11) In operation, one or more sample 10 to be analysed is loaded into the channels 8 in the target plate 2 through their openings 12,14 in the rear and/or front sides 4,6. This may be achieved by loading the sample(s) 10 into the openings 12 in the rear sides 6. This avoids having to interfere with any instrument components adjacent the front side 4 of the target plate 2. This also enables one or more sample source to remain connected to the channel 8, even when the laser 16 is being fired at the front side 4 of the target plate 2. For example, the channel openings 12 may be connected to one or more capillary 15 for delivering liquid into the channel 8, e.g. for using an infusion pump 17 to replenish a channel or for delivering liquid into the channel directly from a liquid chromatography column 19 in an on-line LC-MALDI technique. Also, if the channel openings 12 in the rear side 6 of the target plate 2 are larger than those in the front side 4 of the target plate 2, then this more easily facilitates injection of the sample into the rear side of the target plate.

(12) Once the sample 10 is loaded into the channels 8 of the target plate 2, the target plate is arranged proximate the inlet of a mass spectrometer. Alternatively, the sample may be loaded into the target plate whilst the target plate is proximate the inlet to the mass spectrometer. In the example shown in FIG. 1, the mass spectrometer has an inlet tube 5 for receiving the analyte and arranged in front of the inlet of a vacuum chamber of the mass spectrometer. A laser 16 is then directed onto the front side 4 of the target plate 2, at the opening 14 of one of the channels 8. The laser beam 16 causes the liquid sample 10 at the front opening 14 of the channel 8 to be desorbed and ionised. The analyte ions 18 then pass into the inlet tube 5, which may be heated so as to assist in the desorption and/or ionisation of the analyte. The analyte ions 18 then pass into the inlet of the vacuum chamber of the mass spectrometer. The analyte ions may be drawn into the inlet by a gas flow, e.g. due to the target plate being in a higher pressure region (e.g. atmospheric pressure) than the vacuum chamber.

(13) As the liquid sample 10 is ionised and leaves the channel opening 14 in the front side of the target plate, the liquid 10 in the channel 8 moves towards the front opening 14 and may subsequently be ionised by the laser 16. The channels 8 may have a cross-sectional size and may be configured such that this motion of the liquid 10 is performed under capillary action. Alternately, or additionally, the liquid motion to the front side may be driven by applying a pressure differential across the target plate 2. For example, for any given channel 8, the opening 12 in the rear side of the target plate may be maintained at a higher pressure than the opening 14 in the front side of the target plate. This may be achieved by arranging the target plate as the interface between different pressure regions. Alternatively, a pump may be connected to the rear side of the channels 8 and used to apply pressure to the opening 12 in the rear side of the target plate.

(14) It is also contemplated that the liquid sample 10 may be charged and a potential difference, such as an electrostatic field, may be applied between the target plate 2 and an electrode in front of the target plate (e.g. the inlet tube 5 or the vacuum chamber inlet) so as to urge the charged liquid towards the electrode, i.e. to force the liquid through the channels 8 to the front surface 4 of the target plate. For example, a 3 kV potential difference may be applied between the target plate 2 and the inlet tube 5. The liquid 10 may be electrically charged by applying a voltage directly to the liquid or by using an electrically conductive target plate 2 and applying a voltage to the target plate.

(15) The pulsed laser 16 may be directed on one channel until it is desired to ionise the sample in another channel, at which point the laser beam 16 may be redirected so as to be incident on the next channel. The laser can be stepped between the various channels in this way. Alternatively, rather than redirecting the laser to ionise the sample in another channel, the sample plate 2 may be moved so that the laser 16 is incident on said another channel. The movement of the sample plate may be stepped so that the laser beam is incident on the different channels. It is also contemplated that multiple lasers or multiple laser beams may be used to illuminate multiple different channels.

(16) One or more detector 20 may be provided for sensing the laser beam 16 and/or target plate position and controlling this position to optimise the direction of the laser onto any given channel. The one or more detector may form part of a control system for controlling the position of the laser beam and/or target plate. For example, a photodetector may be used to detect light from the laser beam to ensure the laser is in the correct position relative to a channel. The photodetector may be arranged on the opposite side of the target pate to the laser and may be used to determine when the laser beam is in the correct position, e.g. when light from the laser is passing through the channel onto the detector (e.g. with maximum intensity).

(17) The above-described target plate structure ensures that the surface area of the sample at each opening 14 in the front side of the target plate is relatively small, allowing a high sample density per unit area. Also, this small surface area of the sample in each channel helps to define the electric field more precisely than the electrostatically undefined liquid spots normally used.

(18) In order to illustrate the effectiveness of the embodiments of the invention, a specific example will now be described. An AP-MALDI source assembly was fitted to a Synapt G2 Si instrument. The standard ESI source housing of the instrument was removed. A heated ion transfer/desolvation inlet tube was fitted and a target plate loaded with sample was positioned in front of the ion transfer tube on an X-Y target plate carrier, i.e. as shown in FIG. 1. Each channel was configured as in FIG. 1 and had a volume of 10 μL. A 5 μL bradykinin peptide solution (10 pm per μL) had been spotted onto the channel from the rear of the sample plate together with 5 μL of the liquid matrix (50 mg of 2,5-DHB dissolved in 100 μL of 50:50 water/acetonitrile solution followed by the addition of 60% glycerol by volume). A potential difference of 4 kV was applied between the MALDI target plate and the ion transfer tube. The samples were then irradiated by a pulsed DPSS Nd:YLF laser (349 nm; ˜8 ns).

(19) FIG. 2A shows a view of the front side 4 of the target plate 2, including a view of the tip of heated inlet tube 5 and the laser spot 7 from a laser (firing at 1 kHz).

(20) FIG. 2B shows a view of the rear side 6 of target plate 2, showing the relatively large opening 12 into the channel 8 and the laser fluorescence of the liquid AP-MALDI sample/matrix solution 10.

(21) FIG. 3 shows the ion signal obtained as a function of laser pulse rate using the above described embodiment for 2+ bradykinin (5 pm per μL) with DHB and glycerol. More specifically, the graph illustrates the signal (summation of 10×1 second scans) as a function of laser repetition rate.

(22) The target plate structure according to the embodiments of the invention allows high rate laser repetition acquisition from significantly larger sample volumes than conventionally used. Experimental speeds, for a given sample volume, can be significantly increased, e.g. by at least two orders of magnitude.

(23) Although a reflection mode MALDI technique has been described, wherein the laser beam illuminates the side of the target plate from which the analyte ions are emitted, it is also contemplated that a transmission mode MALDI technique may be used. In such a transmission mode technique, the laser may be directed from the rear side of the target plate, through the target plate and onto the sample in the channel and such that analyte ions are emitted from the front side of the target plate.

(24) FIG. 4 shows a less preferred embodiment of the invention that is substantially the same as that shown and described in relation to FIG. 1, except that high volume wells 9 (e.g. ˜1 mL each) are provided in the target plate 2 rather than providing channels through the target plate. The target plate may comprise a 1D or 2D array of such wells (or even only a single such well). For example, the target plate may comprise 96 wells in a 2D array.

(25) Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.

(26) For example, additional chromatographic material may be added to the sample, optionally for desalting.

(27) Salts or other additives may be added to the sample, e.g. to enhance the liquid dipole features and hence the electrostatic drive through the target plate.

(28) The target plate may be a microfabricated plate, e.g. with additional functional structures or interconnected channels for mixing of different solutions.

(29) Although the various channels and well configurations are described herein, it is contemplated that a target plate may comprise a plurality of these different configurations of channels or a plurality of these different configurations of wells. It is also contemplated that a target plate may comprise both one or more of the channels and one or more of the wells.

(30) Although embodiments have been described in which the sample is ionised by a MALDI technique, it is contemplated that the target plate may be used in other ionisation techniques, such as Laser Desorption Ionisation (“LDI”), Solvent Assisted Inlet Ionisation (“SAII”), Desorption Electrospray Ionisation (“DESI”), Rapid Evaporative Ionization Mass Spectrometry (REIMS), Laserspray Ionisation (“LSI”), Ultrasonic Desorption, Atmospheric Sample Analysis Probe (ASAP) ionisation or other ambient ionization techniques.