Desorption ion source with dopant-gas assisted ionization

Abstract

Disclosed is a device to generate ions from a deposited sample, comprising: A chamber which is arranged and designed to keep the deposited sample in a conditioned environment comprising a dopant gas, A desorption device which is arranged and designed to desorb the deposited sample in the chamber using an energy burst, An ionization device which, for the purpose of ionization, is arranged and designed to irradiate the desorbed sample in the chamber using coherent electromagnetic waves or expose it to an electric discharge, a plasma, or light of an arc discharge lamp with broadband emission spectrum, which are chosen such that the dopant gas is receptive to them, and An extraction device which is arranged and designed to extract ions from the desorbed sample and transfer them into an analyzer. Disclosed is also a method which is preferably conducted on such a device.

Claims

1. A device to generate ions from a deposited sample, comprising: a chamber which is arranged and designed to keep the deposited sample in a conditioned environment, where the conditioned environment comprises a dopant gas, a desorption device which is arranged and designed to desorb the deposited sample in the chamber using an energy burst, an ionization device which, for the purpose of ionization, is arranged and designed to irradiate the desorbed sample in the chamber using coherent electromagnetic waves which are chosen such that the dopant gas is receptive to them, and an extraction device which is arranged and designed to extract ions from the desorbed sample and transfer them into an analyzer.

2. The device according to claim 1, wherein the dopant gas is selected from the groups: (i) polar aprotic solvents such as acetone, anisole, and chlorobenzene, (ii) polar protic solvents such as isopropanol, and/or (iii) non-polar solvents such as toluene.

3. The device according to claim 1, wherein the chamber has a feed-in device which is arranged and designed to feed in a gas with low reactivity as buffer gas for the conditioned environment.

4. The device according to claim 3, wherein the feed-in device is arranged and designed to admix the dopant gas to the buffer gas.

5. The device according to claim 1, wherein the chamber is connected to a vacuum source to evacuate the environment of the deposited sample.

6. The device according to claim 5, wherein the vacuum source is arranged and designed to maintain a pressure which is substantially higher than a high vacuum (>10.sup.−3 hectopascal) and lower than around 10.sup.2 hectopascal.

7. The device according to claim 1, wherein the desorption device is arranged and designed to direct an energetic beam onto the deposited sample to trigger the energy burst.

8. The device according to claim 7, wherein the energetic beam is a pulsed laser beam to ablate the deposited sample.

9. The device according to claim 7, wherein the energetic beam and the coherent electromagnetic waves are not aligned in parallel, but have directions of propagation which are at an angle to each other.

10. The device according to claim 1, wherein the coherent electromagnetic waves have a wavelength longer than around 140 nanometers.

11. The device according to claim 1, wherein the ionization device is arranged and designed to irradiate the desorbed sample with a pulse of coherent electromagnetic waves tempo-rally coordinated with the energy burst.

12. A device to generate ions from a deposited sample, comprising: a chamber which is arranged and designed to keep the deposited sample in a conditioned environment, where the conditioned environment comprises a dopant gas, a desorption device which is arranged and designed to desorb the deposited sample in the chamber using an energy burst, an ionization device which, for the purpose of ionization, is arranged and designed to expose the desorbed sample in the chamber to an electric discharge, a plasma, or light of an arc discharge lamp with broadband emission spectrum, which are chosen such that the dopant gas is receptive to them, and an extraction device which is arranged and designed to extract ions from the desorbed sample and transfer them into an analyzer.

13. A method to generate ions from a deposited sample, comprising: keeping the deposited sample in a conditioned environment, which comprises a dopant gas, desorbing the deposited sample using an energy burst, ionizing particles in the desorbed sample by exciting molecules of the dopant gas and providing a charge carrier transfer between the dopant gas molecules and the particles in the desorbed sample, and extracting ions from the desorbed sample and transferring the ions into an analyzer.

14. The method according to claim 13, wherein the dopant gas molecules are excited using coherent electromagnetic waves.

15. The method according to claim 13, wherein the dopant gas molecules are excited by an electric discharge, a plasma or light of an arc discharge lamp with broadband emission spectrum.

16. The method according to claim 13 wherein said charge carrier transfer is a transfer from the dopant molecules to the particles in the desorbed sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention can be better understood by referring to the following illustrations. The elements in the illustrations are not necessarily to scale, but are primarily intended to illustrate the principles of the invention (mostly schematically). The same reference numbers designate the same elements in the various diagrams.

(2) FIG. 1A shows schematically a first example embodiment of a device to generate ions from a deposited sample, comprising a chamber, desorption device, ionization device, and an extraction device.

(3) FIG. 1B illustrates measurement results of the improved detection sensitivity, which were obtained with a Spectroglyph-Orbitrap®-MS; Q Exactive Plus Orbitrap®, Thermo Fisher Scientific (Bremen, Germany).

(4) FIG. 1C further illustrates measurement results of the improved detection sensitivity, which were obtained with a Spectroglyph-Orbitrap®-MS; Q Exactive Plus Orbitrap®, Thermo Fisher Scientific (Bremen, Germany).

(5) FIG. 1D yet further illustrates measurement results of the improved detection sensitivity, which were obtained with a Spectroglyph-Orbitrap®-MS; Q Exactive Plus Orbitrap®, Thermo Fisher Scientific (Bremen, Germany).

(6) FIG. 2 shows schematically a second example embodiment of a device to generate ions from a deposited sample, comprising a modified chamber, desorption device, ionization device, and modified extraction device.

(7) FIG. 3 shows schematically a third example embodiment of a device to generate ions from a deposited sample, comprising a chamber, modified desorption device, ionization device, and an extraction device.

DETAILED DESCRIPTION

(8) While the invention has been illustrated and explained with reference to a number of embodiments thereof, those skilled in the art will recognize that various changes in form and detail can be made without departing from the scope of the technical teaching, as defined in the attached claims.

(9) The invention increases the yields of molecular ions in mass spectrometry (especially in laser mass spectrometry such as MALDI mass spectrometry; MALDI stands for matrix assisted laser desorption/ionization), mobility mass spectrometry, or combined mobility-mass spectrometry, which are low for many biomolecules—yield means the ratio of molecules that are ionized and can thus be detected by means of a mass spectrometer, and the total molecules ablated from a deposited sample.

(10) Furthermore, matrix effects such as ion suppression effects, i.e., the suppression of specific classes of molecule by other, very easily ionized classes of molecules in the sample, are reduced.

(11) The invention has already been successfully tested in experiments by appropriate modification of two commercially available mass spectrometers with fundamentally different operating principles.

Example 1 (Spectroglyph-Orbitrap® MS; Q Exactive Plus Orbitrap®, Thermo Fisher Scientific)

(12) FIG. 1A is a schematic diagram of a vacuum chamber (10), in which ions can be generated. By sealing the vacuum chamber (10) as tightly as possible, there are only two ways for gas to escape from the chamber (10): firstly via an interface to a mass analyzer (MS), via which ions and also small numbers of gas particles are fed out, and secondly via a port with a pump connected (not shown), which maintains the general pressure level in the chamber (10). In the design shown, the gas is introduced into the chamber (10) via only a single gas inlet (12), through which both molecular nitrogen as a buffer gas, which displaces residual quantities of oxygen-containing air and thus contributes to the low reactivity of the vacuum environment, as well as admixed dopant gas, such as acetone, are fed into the chamber (10). A pressure gauge (14) displays the prevailing pressure level so that the gas flows into and out of the chamber (10) can be adjusted in the event of a deviation from the desired value. Parameter range settings may include: total pressure: 2 to 20 hectopascal; partial pressure (acetone) up to 2 hectopascal.

(13) At the interface to the mass analyzer (MS), which requires pressure levels below medium vacuum, there is an ion guide operated by an RF voltage (16), said guide consisting of a plurality of ring or apertured-plate electrodes arranged in series, some of which have a constant inside diameter and thus form a short tunnel section (16a), whereas the inside diameters of others taper towards the analyzer and thus form a funnel section (16b), which constricts ions guided through it into a quite narrow spatial region about the axis of the ion guide (16) so as to transfer them in a more compact form, and thus more efficiently, to downstream components. A DC voltage gradient from the entrance of the tunnel section (16a) to the exit of the funnel section (16b), which can be pulsed in temporal coordination with the ion generation in the chamber (10), if necessary, drives the ions forward to feed them out of the chamber (10).

(14) A translation device (18), on which there is a mass spectrometric specimen slide (20), is located in the lower section of FIG. 1A. An ablation laser (22) is positioned in the upper section of FIG. 1A and aligned such that its pulsed beam (24) can be guided through separate apertures (not shown) in the ring electrodes in the funnel section (16b) of the ion guide and impinge on the specimen slide (20) at a predetermined location. The translation device (18), e.g., a vacuum-compatible x/y translation stage with low mechanical abrasion, can move the specimen slide (20) that sits on it in two spatial directions in the plane that is almost perpendicular to the ablation laser beam (24), and can thus bring different points of the surface into the focus of the ablation laser (22) each time, as is systematically done for example when scanning a tissue section or a regular arrangement of individual preparations, e.g., on AnchorChip™ plates.

(15) A transparent window (26) is built into the side wall of the chamber (10), through which a post-ionization laser pulse (28) can be beamed laterally into the chamber (10) and focused at a position directly above the specimen slide (20) so as to interact with the desorbed neutral molecules (30) created by the desorption of the deposited sample and with the dopant gas, which is omnipresent in the background. A beam dump (not shown) can be mounted on the opposite side wall of the chamber (10) to prevent damage to the chamber wall and undesired scattered photons.

(16) The dopant gas, e.g., acetone, is removed from the headspace in a container (32) over the surface of the liquid (“headspace method”), and introduced into the vacuum chamber (10) at around 3 to 15 hectopascal via a separate feed-in. The dopant gas feed and the total source pressure with molecular nitrogen N.sub.2 as buffer gas can be manually controlled by a needle valve (34). The sample can be a porcine brain homogenate, for example, coated with the MALDI matrix substance 2,5-dihydroxyacetophenone (DHAP) according to standard protocol and ablated with the standard Nd:YLF laser (wavelength 349 nanometers). The post-ionization can be achieved by means of a frequency-quadrupled Nd:YAG laser (wavelength 266 nanometers, Ekspla, at 28 picosecond pulse duration). In both the positive and the negative ion mode, significant signal increases can be demonstrated for a large number of biochemically relevant analytes, for example various glycerophospholipids.

(17) FIGS. 1B, 1C, and 1D illustrate spectra of a thin section of the same porcine brain homogenate preparation, which was coated with the matrix substance DHAP. The measurement was conducted with a Spectroglyph-Orbitrap® coupling, as outlined in FIG. 1A, under optimized conditions in each case in the positive ion mode (MALDI-2) using the dopant gas acetone (upper diagram in each case) and without dopant gas for comparison (lower diagram in each case). As can be seen from the various mass range sections m/z in the spectra, the intensity of the mass signals increases by around one order of magnitude (factor×10), whereas the signature or profile of the mass signals remains more or less constant. The increase in sensitivity is therefore also achieved across a broad mass range.

Example 2 (Quadrupole Time-Of-Flight MS; Synapt G2-S, Waters Corporation)

(18) FIG. 2 is a schematic diagram of a two-part vacuum chamber (10a, 10b), in whose lower part (10a) ions can be generated. By sealing the vacuum chamber (10a, 10b) as tightly as possible, there are only two ways for gas to escape from the chamber (10a, 10b): firstly via an interface to a time-of-flight analyzer (not shown), via which ions and also gas particles are fed out, and secondly via a port with a pump connected (not shown), which maintains the general pressure level in the chamber (10a, 10b). In the design shown, the gas is introduced into the chamber (10a, 10b) via only a single gas inlet (12), through which both molecular nitrogen as a buffer gas, which displaces residual quantities of oxygen-containing air and thus contributes to the low reactivity of the vacuum environment, as well as admixed dopant gas, such as acetone, are fed into the lower part of the chamber (10a). A pressure gauge (14) monitors the pressure level set by the operator and communicates with a gas feed device (36) so that the buffer gas flow into and out of the lower part of the chamber (10a) can be automatically adjusted in the event of a deviation from the desired value. Parameter range settings may include: total pressure: 0.2 to 4 hectopascal; partial pressure (acetone) up to 2 hectopascal.

(19) An arrangement of voltage-controlled extraction electrodes (38) is located at the interface to the vacuum region of the time-of-flight analyzer, which requires a pressure level lower than medium vacuum (the boundary between the lower and the upper part of the chamber). An extraction electrode (38a) with a conical opening extends into both parts of the chamber (10a, 10b) and its truncated end is located opposite a sample desorption region in the lower part of the chamber (10a). Further annular extraction electrodes (38b) in the upper part of the chamber (10b) lead to an RF multipole ion guide (40) in a hexapole design, which can guide ions into further connected components of the time-of-flight analyzer. A DC voltage gradient from the truncated conical electrode (38a) to the hexapole (40), which can be pulsed in temporal coordination with the ion generation in the lower part of the chamber (10a), if necessary, drives the ions forward in order to feed them out of the lower part of the chamber (10a).

(20) A translation device (18), on which there is a mass spectrometric specimen slide (20), is located in the lower part of the chamber (10a). An ablation laser is positioned in the top left section of FIG. 2 outside the upper part of the vacuum chamber (10b) and aligned such that its pulsed beam (24) is directed through a transparent window (42) of the vacuum system and through separate apertures (not shown) in the arrangement of annular extraction electrodes (38b), and impinges on the specimen slide (20) at a predetermined location at a distinct angle to the surface normal. The translation device (18), e.g., a vacuum compatible x/y translation stage with low mechanical abrasion, can move the specimen slide (20) that sits on it in two spatial directions in the plane that is perpendicular to the alignment of the arrangement of extraction electrodes (38) and the hexapole (40), and can thus bring different points of the surface into the focus of the ablation laser each time, as is systematically done for example when scanning a tissue section or an array of individual preparations, e.g., on AnchorChip™ plates.

(21) A transparent window (26) is built into the side wall of the lower part of the chamber (10a), through which a post-ionization laser pulse (28) can be beamed laterally into the lower part of the chamber (10a), and focused at a position directly above the specimen slide (20) so as to interact with the desorbed neutral molecules created by the desorption of the deposited sample and with the dopant gas, which is omnipresent in the background. A beam dump (not shown) can be mounted on the opposite side wall of the lower part of the chamber (10) to prevent damage to the chamber wall and undesired scattered photons.

(22) The dopant gas, such as acetone, is introduced into the lower part of the vacuum chamber (10a) via the central buffer gas inlet (12) at around 0.5 to 4 hectopascal, using the headspace method. The dopant gas feed is manually controlled via a needle valve (34); the total source pressure is automated via pneumatic valves (44) and controlled in communication with the pressure gauge (14). As an example of a sample, a porcine brain homogenate can be coated with 2,5-DHAP matrix, according to standard protocol, and ablated with the standard Nd:YLF laser (wavelength 349 nanometers). The post-ionization is achieved by a frequency-quadrupled Nd:YAG laser (wavelength 266 nanometers, Ekspla, at 28 picosecond pulse duration). In both the positive and the negative ion mode, significant signal increases can be demonstrated for a large number of biochemically relevant analytes, for example various glycerophospholipids.

(23) FIG. 3 is a schematic diagram of a chamber (10) in which ions can be generated. A conditioned environment is maintained by ensuring the chamber (10) is sealed as tightly as possible. There are fundamentally only two ways for gas to escape from the chamber (10): firstly—following a pressure gradient—via an interface to a mass analyzer (MS), a mobility analyzer, or a combined mobility-mass analyzer, through which ions and also gas particles are fed out, and secondly via a port with a connected pump (46), which maintains the general pressure level in the chamber (10) in coordination with the gas supply for the conditioned environment. In the design shown, gas is fed into the chamber (10) via only a single gas inlet (12), through which both a low-reactivity buffer gas, which displaces residual quantities of oxygen-containing air and thus contributes to the low reactivity of the conditioned environment, as well as admixed dopant gas are fed into the chamber (10). A pressure gauge (not shown) monitors the pressure level set by the operator so that the gas flows into and out of the chamber (10) can be adjusted in the event of a deviation from the desired value, possibly automatically in direct communication with valves of the gas inlet (12). The chamber (10) can essentially be operated at or close to atmospheric pressure, or in a medium vacuum, depending on the equilibrium of the gas inflows and outflows into and out of the chamber (10).

(24) A voltage-assisted extraction device (48) comprising several electrodes (not shown) is located at the interface to the analyzer, which usually requires pressure levels below medium vacuum, e.g., a high vacuum (>10.sup.−3 hectopascal). A DC voltage gradient across the extraction device (48), which can be pulsed in temporal coordination with the ion generation in the chamber (10), if necessary, drives the ions forward in order to feed them out of the chamber (10).

(25) A translation device (18) with a recess into which a mass spectrometric specimen slide (20) is placed, is located in the lower section of FIG. 3. With an appropriately transparent design of the chamber floor and the specimen slide (20), a pulsed ablation laser beam (24) can act in transmission at a predetermined location on the deposited sample through the specimen slide (20), and desorb the sample, e.g., by means of transmission MALDI (t-MALDI). The translation device (18), e.g., a vacuum compatible x/y translation stage with low mechanical abrasion, where appropriate, can move the specimen slide (20) located in the recess in two spatial directions along the floor of the chamber in the plane that is almost perpendicular to the ablation laser beam (24), and can thus bring different points of the surface into the focus of the ablation laser each time, as is systematically done for example when scanning a tissue section or an arrangement of individual preparations. A UV-transmitting glass plate, as used in microscopy, can be used as the specimen slide (20), for example. The surface of the glass plate which bears the sample can be designed so as to be conductive, e.g., by means of a coating.

(26) A transparent window (26) is built into the side wall of the chamber (10), through which a post-ionization laser pulse (28) can be beamed laterally into the chamber (10) and focused at a position directly above the specimen slide (20) so as to interact with the desorbed neutral molecules (30) created by the desorption of the deposited sample and with the dopant gas, which is omnipresent in the background. In contrast to the designs explained previously, where the energy burst is effected by beams (24) which impinge at an angle on the sample side, the neutral molecules are desorbed to a large extent along the normal to the surface on the sample side because the impact is frontal from the rear surface. A beam dump (50) is mounted on the opposite side wall of the chamber (10) to prevent damage to the chamber wall and undesired scattered photons.

(27) The dopant gas, which is fed into the chamber as a continuous flow or in pulses, and is thus omnipresent, e.g., a polar aprotic solvent such as acetone, anisole, and chlorobenzene, a polar protic solvent such as isopropanol, a nonpolar solvent such as toluene, or a mixture of the aforementioned, assists in the post-ionization of the sample desorbed by bombardment with the pulsed transmission laser beam (24) directly above the specimen slide (20) by increasing the number of locally available charge carriers, in particular by photochemical excitation of the neutral dopant gas molecules and subsequent transfer of charge carriers, e.g., protons, to desorbed neutral sample molecules.

(28) A common feature of all the embodiments explained above is that material is desorbed spatially resolved from a sample (prepared with matrix, if necessary) with the aid of an energy burst from a primary energy source, e.g., an ablation laser (for example by LDI/MALDI). A secondary post-ionization laser, which emits coherent electromagnetic waves, is focused into the desorption cloud produced, which drastically increases the ion yield (e.g., by MALDI-2).

(29) In addition, a dopant, which is volatile under the chosen pressure conditions and which is receptive to the coherent electromagnetic waves (e.g., acetone, toluene, anisole, chlorobenzene, isopropanol), is now fed permanently (or in pulses) from a reservoir into the gaseous phase. The feed-in can be effected by pressure gradients from the headspace over the surface of the liquid of a bottle (or other container) filled with the liquid dopant, or by means of injection, directly into the conditioned environment of the ion source. Furthermore, the volatile dopant can be fed into the ion source through a separate gas pipe (Orbitrape®) or by a gas mixer with a mass flow control via the gas pipe of the N.sub.2 buffer gas (Synapt).

(30) The gaseous phase portion of the dopant gas can be regulated by means of a fine adjustment valve (manually with a needle valve or by means of electrically controlled valves) and the pressure controlled via pressure gauges. By regulating the buffer gas (in the case of the Synapt, example 2: automated), the optimal dopant gas partial pressure and total source pressure can be set and kept sufficiently constant over several hours. A secondary energy source (e.g., in the form of a plasma, an electric discharge, an arc discharge lamp with broadband emission spectrum or, in the examples shown, a post-ionization laser) excites the omnipresent dopant gas in the gaseous phase, which enables effective post-ionization of the desorbed material of the sample.

(31) Further embodiments of the invention are conceivable in addition to the embodiments described by way of example. With knowledge of this disclosure, those skilled in the art can easily design further advantageous embodiments, which are to be covered by the scope of protection of the appended claims, including any equivalents as the case may be.