Two-dimensional separation and imaging technology for the rapid analysis of biological samples

Abstract

A method for ion mapping of a sample, which has the following features: Application of a liquid sample to a metallic target surface, Separation of components of the liquid sample on the metallic target surface according to one or more physicochemical properties in a first spatial dimension and in a second spatial dimension and Ionizing and mass-analyzing multiple separated areas of the separated sample to generate an ion map of at least a portion of the sample separated on the target surface, wherein the components of the sample are applied to the target surface and separated by mechanical, hydrodynamic and / or aerodynamic means, and wherein the one or more physicochemical properties include one or more from the group comprising surface binding, surface affinity, solubility, binding affinity and polarity.

Description

AREA OF INVENTION

[0001] The present invention relates to a method for ion mapping, a method for mass spectrometry, a device and a mass spectrometer. BACKGROUND

[0002] Attempts have been made to analyze complex biological samples (such as proteins) using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry. However, the complex biological sample must first undergo sample preparation, followed by chromatographic separation, such as thin-layer chromatography (TLC) or gel electrophoresis, prior to MALDI analysis. These chromatographic separation processes are relatively time-consuming, and the known approaches also suffer from the serious problem that the chromatographic separations are performed using a chromatographic substrate whose surface is not ideally suited for subsequent MALDI analysis.

[0003] US 6579719 B1 (Hutchens) discloses various methods for retentate chromatography. Analytes are separated in at least two non-spatial dimensions based on their ability to be adsorbed onto a stationary phase under at least two different selectivity conditions (i.e., by varying the adsorbent and the eluent). The analytes can then be analyzed by MALDI mass spectrometry. Several different adsorbents on a single substrate are disclosed, forming a multiple array with addressable sites. The substrate can also be in the form of a strip or a plate, wherein one or more binding properties can vary in a one- or two-dimensional gradient. To provide multidimensional analysis, each adsorbent site is washed with at least one first and one second different eluent. Fig. For example, 11 from US-6579719 B1 (Hutchens) shows a composite retention map of urine from premature infants exposed to defined selectivity conditions through six different adsorbents and three different eluents.

[0004] WO 2007 / 058893 A2 (Patton) discloses a method for separating a sample, wherein one sample is subjected to planar electrochromatographic separation in one selected direction and the other to thin-layer chromatographic separation in another direction. WO 2007 / 058893 A2 (Patton) relates to the separation of samples using a combination of electrically driven planar chromatography (PEC) and thin-layer chromatography (TLC), optionally followed by direct detection of analytes using mass spectrometry. A sample is loaded onto a planar stationary phase, and an electric field is applied to cause a first liquid mobile phase and / or the sample to migrate along the length of the stationary phase, thereby separating biomolecules.A second separation, based on chromatography, is then applied to cause a second liquid mobile phase to move along the length of the stationary phase in a second direction, thereby separating biomolecules. Typically, the second direction is perpendicular to the first.

[0005] One problem with the arrangements disclosed in both US-6579719 B1 (Hutchens) and WO 2007 / 058893 A2 (Patton) is that the chromatographic separation processes are relatively slow and that the surface of the chromatographic substrate is not ideally suited for subsequent MALDI analysis.

[0006] It is desirable to provide an improved method for mass spectrometry.

[0007] DE 10 2006 019 530 A1 describes a frozen tissue section which melts after being applied to a substrate and spreads on the substrate. US 2007 / 0187243 A1 deals with the two-dimensional separation of samples on silica gel plates for thin-layer chromatography (TLC) using a combination of electrically driven planar chromatography (PEC) and thin-layer chromatography (TLC). US 2009 / 0272890 A1 deals with the ionization of substances in a two-dimensional region of a sample using a two-dimensionally spread laser beam. SUMMARY

[0008] According to one aspect, a method for ion mapping or ion mapping is provided, which has the following features: Application of a liquid sample to a metallic target surface, Separation of components of the liquid sample on the metallic target surface according to one or more physicochemical properties in a first spatial dimension and in a second spatial dimension and Ionizing and mass-analyzing several separated areas of the separated sample to generate an ion map of at least a portion of the sample separated on the metallic target surface, wherein the components of the sample are applied to the target surface and separated by mechanical, hydrodynamic and / or aerodynamic means.

[0009] The approach according to various embodiments is particularly advantageous in that the method for applying the sample to the target and for separating the sample is quick and simple, and furthermore, the sample can be applied to a target that is particularly suitable for subsequent MALDI analysis. This contrasts with conventional arrangements.

[0010] One or more physicochemical properties can be the same or different.

[0011] According to various embodiments, a sample is applied to a target surface in two dimensions and separated by mechanical, hydrodynamic and / or aerodynamic means.

[0012] It is known to separate a sample in two dimensions on a surface using conventional chromatographic techniques. For example, WO 2007 / 058893 A2 (Patton) discloses a method for separating a sample wherein a sample is subjected to planar electrochromatographic separation in one selected direction and the sample is then subjected to thin-layer chromatographic separation in another direction. However, this approach does not use any mechanical, hydrodynamic, and / or aerodynamic means within the scope of the present invention to deposit the sample onto a target and then to effect a two-dimensional separation of the sample on the target surface.

[0013] The known approach is relatively time-consuming, and the separations are provided on a chromatography substrate surface that is not ideally suited for subsequent matrix-assisted laser desorption ionization (“MALDI”) mass spectrometry analysis.

[0014] In contrast, the present embodiments advantageously provide for a simple and rapid two-dimensional separation of components of a sample on a target surface using mechanical, hydrodynamic, and / or aerodynamic means. This enables the execution of a subsequent ion mapping procedure that is free of complex procedures. Furthermore, the subsequent ion mapping analysis can be performed on a surface that is essentially optimized for matrix-assisted laser desorption ionization (“MALDI”) or other similar forms of mass spectrometry analysis.

[0015] In the steps of applying the sample to the target and separating the sample, the sample can be applied to the target surface so that the sample is separated according to one or more physicochemical properties in a first dimension and in a second dimension.

[0016] One or more physicochemical properties can be the same or different.

[0017] Alternatively, in the steps of depositing and separating the sample, the sample can be applied to the target surface and then separated on the target surface according to one or more physicochemical properties in a first dimension and in a second dimension.

[0018] Alternatively, in the steps of applying and separating the sample, the sample can be applied to the target surface so that the sample is separated in a first dimension according to one or more physicochemical properties and separated in a second dimension.

[0019] The first dimension can be orthogonal to the second dimension.

[0020] The first dimension and the second dimension are spatial dimensions.

[0021] The sample can include a liquid sample.

[0022] The sample may be dissolved in a liquid solvent.

[0023] The method may also involve applying a liquid solvent to the sample on the target surface.

[0024] According to one embodiment, the target surface can comprise a substantially non-porous and / or substantially planar target surface. The sample can be applied to a non-porous target surface and separated in two dimensions.

[0025] It is known to separate a sample in two dimensions on a porous surface using conventional chromatographic techniques. For example, WO 2007 / 058893 A2 (Patton) discloses a method for separating a sample wherein one sample is subjected to planar electrochromatographic separation in one selected direction and the other sample is subjected to thin-layer chromatographic separation in another direction.

[0026] However, the known approach is performed on porous and uneven chromatography substrate surfaces, which leads to a broadening of mass spectrum peaks and a deterioration of mass accuracy when combined with axial time-of-flight mass analysis.

[0027] Furthermore, the known approach is time-consuming and provides separation on chromatography substrate surfaces that are not ideally suited for subsequent matrix-assisted laser desorption ionization (“MALDI”) mass spectrometry analysis.

[0028] In contrast, the present embodiments advantageously provide separations on a non-porous surface and include an ion mapping method that has improved mass accuracy, is free of complex procedures and can be performed on a surface optimized for matrix-assisted laser desorption ionization (“MALDI”) or other related mass spectrometry analysis.

[0029] The target surface can include a metallic target surface.

[0030] The target surface can include a polished metallic target surface.

[0031] The target surface can include a stainless steel target surface.

[0032] Other embodiments are also considered, in which the target may comprise a semiconductor target or an insulator target, but are not part of the present invention.

[0033] According to one embodiment, the target can have one or more elongated grooves for guiding a sample applied to the target surface.

[0034] The target may consist of a plate.

[0035] Alternatively, the target can have a staff.

[0036] Furthermore, in the steps for applying and separating the sample, the sample can be applied to the target surface using an atomizer.

[0037] Furthermore, in the steps for applying and separating the sample, the sample can be applied to the target surface using a sample syringe or a sample spotter.

[0038] Furthermore, in the steps for applying and separating the sample, a sample can be placed in a gas spray and the spray can be directed onto the target surface.

[0039] Furthermore, in the steps for applying and separating the sample, a sample applied to the target surface can be forced to move along the target surface under the influence of a gas and / or liquid flow.

[0040] The fluid flow can include a flow of a liquid solvent.

[0041] Furthermore, in the steps for applying and separating the sample, a sample applied to the target surface can be dispersed along the target surface under the influence of gravity.

[0042] In the steps for applying and separating the sample, a sample applied to the target surface can optionally be spread using a scraper or other device.

[0043] Furthermore, the sample and / or the target can be heated during the steps for applying and separating the sample.

[0044] During the steps for applying and separating the sample, the target can also be shifted, moved, rotated and / or vibrated.

[0045] The mechanical, hydrodynamic and / or aerodynamic means may include an atomizer for applying the sample to the target surface.

[0046] The mechanical, hydrodynamic and / or aerodynamic means may further include a sample syringe or sample spotter for applying the sample to the target surface.

[0047] The mechanical, hydrodynamic and / or aerodynamic means may further include a device that is set up and designed to introduce a sample into a gas spray and to allow the spray to strike the target surface.

[0048] The mechanical, hydrodynamic and / or aerodynamic means may further include a device that is set up and designed to force a sample applied to the target surface to move along the target surface under the influence of a gas and / or liquid flow.

[0049] The mechanical, hydrodynamic and / or aerodynamic means may further include a device that is set up and designed to disperse a sample applied to the target surface along the target surface under the influence of gravity.

[0050] The mechanical, hydrodynamic and / or aerodynamic means may further include a device that is set up and designed to spread a sample applied to the target surface, optionally using a spreader or other device.

[0051] The mechanical, hydrodynamic and / or aerodynamic means may further include a device that is set up and designed to heat the sample and / or the target.

[0052] The mechanical, hydrodynamic and / or aerodynamic means may further include a device that is set up and designed to displace, move, rotate and / or vibrate the target.

[0053] According to one embodiment, the target has a rod covered with one or more layers, wherein, after the steps of applying and separating the sample in the process, the one or more layers are further transferred to a second target.

[0054] The step of ionizing and mass-analyzing several separate areas of the sample to generate an ion map of at least a part of the sample applied to the target surface can be carried out after the one or more layers have been transferred to the second target.

[0055] According to one embodiment, cells in biological samples, either bound to the target or moving along the target surface, can undergo cell lysis under extreme conditions of turbulence, microvorticity, and ultrasound, etc. Accordingly, the lysed contents of the cell would then be separated in a first dimension according to a physicochemical property.

[0056] The second goal can be a planar goal.

[0057] Furthermore, the process allows a matrix to be applied to the sample before the sample is ionized and mass analyzed.

[0058] Furthermore, in this method an electric field can be maintained over at least part of the sample in order to cause at least some parts of the sample to be separated by electromigration.

[0059] Furthermore, the method allows one or more areas of the sample to be ionized using a matrix-assisted laser desorption ionization (“MALDI”) ion source.

[0060] Furthermore, the procedure allows one or more areas of the sample to be ionized using a fast atom bombardment (“FAB”) ion source.

[0061] Furthermore, the process can be used to ionize one or more areas of the sample using a surface-assisted laser desorption ionization (“SALDI”) ion source.

[0062] Furthermore, the method allows one or more areas of the sample to be ionized using a desorption electrospray ionization (“DESI”) ion source.

[0063] The one or more physicochemical properties may include surface binding, surface affinity, solubility, bonding affinity, or polarity.

[0064] According to another aspect, a mass spectrometry method is provided, which includes a method described above.

[0065] According to another aspect, a device is provided which has the following features: a target that has a metallic target surface, a first device which is set up and designed to apply a liquid sample to the target surface and to separate the sample according to one or more physicochemical properties in a first dimension and in a second dimension by mechanical, hydrodynamic and / or aerodynamic means, and a second device which is set up and designed to ionize several separate areas of the sample and to perform a mass analysis on them in order to generate an ion map of at least a part of the sample applied to the target surface.

[0066] According to another aspect, a mass spectrometer is provided which has the device described above.

[0067] Although the present embodiment relates to a method for ion mapping, other embodiments are also considered according to the invention in which the method more generally relates to the generation of a map of a sample and wherein the sample is analyzed using imaging techniques such as chemical staining techniques, UV illumination techniques or absorption techniques.

[0068] According to another aspect not in the invention, a method for mapping a sample is provided which comprises the following: Applying a sample to a target surface, Separation of the sample on the target surface according to a first physicochemical property in a first dimension and according to a second physicochemical property in a second dimension and Analyzing several separate areas of the sample to generate a map of at least a portion of the sample applied to the target surface, wherein the sample is applied to the target surface and separated by mechanical, hydrodynamic and / or aerodynamic means.

[0069] According to another aspect not in accordance with the invention, a device is provided which has the following features: a goal, a first device which is set up and designed to apply a sample to the surface of the target and to separate the sample according to a first physicochemical property in a first dimension and according to a second physicochemical property in a second dimension by mechanical, hydrodynamic and / or aerodynamic means, and a second device which is set up and designed to analyze several separate areas of the sample in order to generate a map of at least a part of the sample applied to the target surface.

[0070] The various embodiments enable very fast pseudo-separation on a surface that can be optimal for imaging by MALDI mass spectrometry.

[0071] According to one embodiment, complex biological samples can be deposited onto a metallic surface, whereupon they are subjected to rapid two-dimensional pseudo-separation, for example, by purely mechanical, hydrodynamic, and / or aerodynamic means, without requiring the application of an electric field. The resulting separations can then be directly analyzed using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry imaging.

[0072] Although the various embodiments relate to a method in which a sample is applied to a target surface, other embodiments are not considered according to the invention in which a sample is already present on a target surface (i.e., provided on a target surface) and the sample is separated on the target surface.

[0073] According to another aspect, a method for ion mapping is provided which has the following features: Providing a sample on a target surface, Separation of the sample on the target surface according to one or more physicochemical properties in a first dimension and in a second dimension and Ionizing and mass-analyzing several separate areas of the sample to generate an ion map of at least a part of the sample on the target surface, the sample is separated on the target surface by mechanical, hydrodynamic and / or aerodynamic means.

[0074] In this process, a liquid solvent can also be applied to the sample on the target surface.

[0075] According to another aspect, a device is provided which has the following features: a goal, a first device which is set up and designed to separate a sample provided on a surface of the target according to one or more physicochemical properties in a first dimension and in a second dimension by mechanical, hydrodynamic and / or aerodynamic means, and a second device which is set up and designed to ionize several separate areas of the sample and to perform a mass analysis on it in order to generate an ion map of at least a part of the sample on the surface of the target.

[0076] According to one aspect, a method for ion mapping is provided which has the following features: Separation of a sample on a target surface according to one or more first physicochemical properties in a first dimension and according to one or more second physicochemical properties in a second dimension by mechanical, hydrodynamic and / or aerodynamic means and Ionizing and mass-analyzing several separate areas of the sample to generate an ion map of at least a part of the sample on the target surface.

[0077] The approach according to various embodiments is particularly advantageous in that the method for separating the sample on the target is quick and simple, and the sample can also be separated on a target that is particularly suitable for subsequent MALDI analysis. This contrasts with conventional arrangements.

[0078] The one or more first physicochemical properties and the one or more second physicochemical properties can be the same or different.

[0079] According to another aspect, a device is provided which has the following features: a goal, a first device which is set up and designed to separate a sample on a surface of the target according to one or more first physicochemical properties in a first dimension and according to one or more second physicochemical properties in a second dimension by mechanical, hydrodynamic and / or aerodynamic means, and a second device which is set up and designed to ionize several separate areas of the sample and to perform a mass analysis on it in order to generate an ion map of at least a part of the sample on the surface of the target.

[0080] According to one aspect, a method for ion mapping is provided which has the following features: Applying a sample to a target such that the sample is separated according to a physicochemical property in a first dimension and according to a physicochemical property in a second, preferably orthogonal, dimension, and Ionizing and mass-analyzing several separate areas of the sample to generate an ion map of at least a portion of the sample applied to the target.

[0081] The target can comprise a metallic target. However, other embodiments are also considered according to the invention, in which the target comprises a semiconductor target or an insulator target.

[0082] According to one embodiment, the target can have one or more elongated grooves for guiding a sample applied to the target.

[0083] In this process, the sample can also be applied to the target using an atomizer.

[0084] In this procedure, the sample can also be applied to the target using a sample syringe or a sample spotter.

[0085] The target may consist of a plate.

[0086] Alternatively, the target can have a staff.

[0087] Furthermore, in this method, a sample applied to the target can be spread using a spreader or other device.

[0088] The sample and / or the target can also be heated during this process.

[0089] In this process, the target can also be shifted, moved, rotated, vibrated, or shaken.

[0090] According to one embodiment, the target has a rod covered with one or more layers, wherein in the method, after the step of applying the sample to the rod target, so that the sample is separated according to one or more physicochemical properties in a first dimension and in a second dimension, which may be orthogonal to the first dimension, the one or more layers are further transferred to a second target.

[0091] The step of ionizing and mass-analyzing several separate areas of the sample to generate an ion map of at least a part of the sample applied to the target can be carried out after the one or more layers have been transferred to the second target.

[0092] According to one embodiment, cells in biological samples, either bound to the target or moving along the target surface, can undergo cell lysis under extreme conditions of turbulence, microvorticity, and ultrasound, etc. Accordingly, the lysed contents of the cell can then be separated in a first dimension according to a physicochemical property.

[0093] The second goal can be a planar goal.

[0094] Furthermore, the process allows a matrix to be applied to the sample before the sample is ionized and mass analyzed.

[0095] Furthermore, in this method an electric field can be maintained over at least part of the sample in order to cause at least some parts of the sample to be separated by electromigration.

[0096] Furthermore, the method allows one or more areas of the sample to be ionized using a matrix-assisted laser desorption ionization (“MALDI”) ion source.

[0097] Furthermore, the procedure allows one or more areas of the sample to be ionized using a fast atom bombardment (“FAB”) ion source.

[0098] Furthermore, the method allows one or more areas of the sample to be ionized using a surface-assisted laser desorption ionization (“SALDI”) ion source.

[0099] Furthermore, the method allows one or more areas of the sample to be ionized using a desorption electrospray ionization (“DESI”) ion source.

[0100] The one or more physicochemical properties can include surface binding, surface affinity, solubility, bonding affinity, and polarity.

[0101] According to another aspect, a mass spectrometry method is provided, which includes a method described above.

[0102] According to another aspect, a device is provided which has the following features: a goal, a first device which is set up and designed to apply a sample to the target such that the sample is separated according to a physicochemical property in a first dimension and according to a physicochemical property in a second, preferably orthogonal, dimension, and a second device which is set up and designed to ionize several separate areas of the sample and to perform a mass analysis on them in order to generate an ion map of at least a part of the sample applied to the target.

[0103] According to another aspect, a mass spectrometer is provided which has the device described above.

[0104] Although the various embodiments relate to a method for ion mapping, other embodiments are also considered according to the invention in which the method relates more generally to the generation of a map of a sample and in which the sample is analyzed using imaging techniques such as chemical staining techniques, UV illumination techniques or absorption techniques.

[0105] According to another aspect not in the invention, a method for mapping a sample is provided which comprises the following: Applying a sample to a target such that the sample is separated according to a physicochemical property in a first dimension and according to a physicochemical property in a second orthogonal dimension, and Analyzing multiple separate areas of the sample to generate a map of at least a portion of the sample applied to the target.

[0106] According to another aspect not in accordance with the invention, a device is provided which has the following features: a goal, a first device which is set up and designed to apply a sample to the target such that the sample is separated according to a physicochemical property in a first dimension and according to a physicochemical property in a second orthogonal dimension, and a second device which is set up and designed to analyze several separate areas of the sample in order to produce a map of at least a part of the sample applied to the target.

[0107] The spectrometer may include an ion source selected from the group consisting of: (i) an electrospray ionization (“ESI”) ion source, (ii) an atmospheric pressure photoionization (“APPI”) ion source, (iii) an atmospheric pressure chemical ionization (“APCI”) ion source, (iv) a matrix-assisted laser desorption ionization (“MALDI”) ion source, (v) a laser desorption ionization (“LDI”) ion source, (vi) an atmospheric pressure ionization (“API”) ion source, (vii) a desorption ionization on silicon (“DIOS”) ion source, (viii) an electron impact (“El”) ion source, (ix) a chemical ionization (“Cl”) ion source, (x) a field ionization (“Fl”) ion source. (xi) a field desorption (“FD”) ion source, (xii) an inductively coupled plasma (“ICP”) ion source, (xiii) a fast atom bombardment (“FAB”) ion source, (xiv) a liquid secondary ion mass spectrometry (“LSIMS”) ion source,(xv) a desorption electrospray ionization (“DESI”) ion source, (xvi) a radioactive nickel-63 ion source, (xvii) an atmospheric pressure matrix-assisted laser desorption ionization ion source, (xviii) a thermospray ion source, (xix) an atmospheric sampling glow discharge ionization (“ASGDI”) ion source, (xx) a glow discharge (“GD”) ion source, (xxi) an impactor ion source, (xxii) a direct analysis in real time (“DART”) ion source, (xxii) a laser spray ionization (“LSI”) ion source, (xxiv) a sonic spray ionization (“SSI”) ion source, (xxv) a matrix-assisted Intake ionization (“MAII”) ion source, (xxvi) a solvent-assisted intake ionization (“SAII”) ion source, (xxvii) a desorption electrospray ionization (“DESI”) ion source and (xxviii) a laser ablation electrospray ionization (“LAESI”) ion source.

[0108] The spectrometer can have one or more continuous or pulsed ion sources.

[0109] The spectrometer can have one or more ion guides.

[0110] The spectrometer may include one or more ion mobility separation devices and / or one or more field asymmetric ion mobility spectrometer devices.

[0111] The spectrometer can have one or more ion traps or one or more ion confinement regions.

[0112] The spectrometer may include one or more collision, fragmentation, or reaction cells selected from the group consisting of: (i) a collision-induced dissociation (“CID”) fragmentation device, (ii) a surface-induced dissociation (“SID”) fragmentation device, (iii) an electron transfer dissociation (“ETD”) fragmentation device, (iv) an electron capture dissociation (“ECD”) fragmentation device, (v) an electron impact or collision dissociation fragmentation device, (vi) a photo-induced dissociation (“PlD”) fragmentation device, (vii) a laser-induced dissociation fragmentation device, (viii) an infrared radiation-induced dissociation device, (ix) a Ultraviolet radiation-induced dissociation device, (x) a nozzle-skimmer interface fragmentation device, (xi) an in-source fragmentation device,(xii) an in-source impact-induced dissociation fragmentation device, (xiii) a thermal or temperature source fragmentation device, (xiv) an electric field-induced fragmentation device, (xv) a magnetic field-induced fragmentation device, (xvi) an enzyme digestion or enzyme degradation fragmentation device, (xvii) an ion-ion reaction fragmentation device, (xviii) an ion-molecule reaction fragmentation device, (xix) an ion-atom reaction fragmentation device, (xx) an ion-metastable ion reaction fragmentation device, (xxi) an ion-metastable molecule reaction fragmentation device, (xxii) an ion-metastable atom reaction fragmentation device, (xxiii) an ion-ion reaction device for reacting ions to form adducts or productions, (xxiv) an ion-molecule reaction device for reacting ions to form adducts or productions,(xxv) an ion-atom reaction device for reacting ions to form adducts or productions, (xxvi) an ion-metastable ion reaction device for reacting ions to form adducts or productions, (xxvii) an ion-metastable molecule reaction device for reacting ions to form adducts or productions, (xxviii) an ion-metastable atom reaction device for reacting ions to form adducts or productions, and (xxix) an electron ionization dissociation ("EID") fragmentation device.

[0113] The spectrometer may include a mass analyzer selected from the group consisting of: (i) a quadrupole mass analyzer, (ii) a two-dimensional or linear quadrupole mass analyzer, (iii) a Paul or three-dimensional quadrupole mass analyzer, (iv) a Penning trap mass analyzer, (v) an ion trap mass analyzer, (vi) a magnetic sector mass analyzer, (vii) an ion cyclotron resonance (ICR) mass analyzer, (viii) a Fourier transform ion cyclotron resonance (FTICR) mass analyzer, (ix) an electrostatic mass analyzer configured to generate an electrostatic field with a quadrologarithmic potential distribution, (x) an electrostatic Fourier transform mass analyzer, (xi) a Fourier transform mass analyzer, (xii) a time-of-flight mass analyzer,(xiii) an orthogonal acceleration time-of-flight mass analyzer and (xiv) a linear acceleration time-of-flight mass analyzer.

[0114] The spectrometer can have one or more energy analyzers or electrostatic energy analyzers.

[0115] The spectrometer can have one or more ion detectors.

[0116] The spectrometer may include one or more mass filters selected from the group consisting of: (i) a quadrupole mass filter, (ii) a two-dimensional or linear quadrupole ion trap, (iii) a Paul or three-dimensional quadrupole ion trap, (iv) a Penning ion trap, (v) an ion trap, (vi) a magnetic sector mass filter, (vii) a time-of-flight mass filter, and (viii) a Wien filter.

[0117] The spectrometer may include a device or ion gate for pulsing ions and / or a device for converting an essentially continuous ion beam into a pulsed ion beam.

[0118] The spectrometer can have a C-trap and a mass analyzer with an outer tubular electrode and a coaxial inner spindle-shaped electrode forming an electrostatic field with a quadrologarithmic potential distribution, wherein in a first operating mode ions are transferred to the C-trap and then injected into the mass analyzer, and wherein in a second operating mode ions are transferred to the C-trap and then transferred to a collision cell or electron transfer dissociation device where at least some ions are fragmented into fragment ions, and wherein the fragment ions are then transferred to the C-trap before being injected into the mass analyzer.

[0119] The spectrometer can have a ring-stack ion guide with multiple electrodes, each having an opening through which ions are allowed to pass during use, wherein the distance between the electrodes increases along the ion path, and wherein the openings in the electrodes in an upstream section of the ion guide have a first diameter, and wherein the openings in the electrodes in a downstream section of the ion guide have a second diameter that is smaller than the first diameter, and wherein opposite phases of an alternating or RF voltage are applied to successive electrodes during use.

[0120] The spectrometer may include a device that is set up and designed to supply an alternating or RF voltage to the electrodes. The AC or RF voltage optionally has an amplitude selected from the following group: (i) approximately < 50 V peak-to-peak, (ii) approximately 50–100 V peak-to-peak, (iii) approximately 100–150 V peak-to-peak, (iv) approximately 150–200 V peak-to-peak, (v) approximately 200–250 V peak-to-peak, (vi) approximately 250–300 V peak-to-peak, (vii) approximately 300–350 V peak-to-peak, (viii) approximately 350–400 V peak-to-peak, (ix) approximately 400–450 V peak-to-peak, (x) approximately 450–500 V peak-to-peak and (xi) approximately > 500 V peak-to-peak.

[0121] The alternating or RF voltage can have a frequency selected from the following group: (i) < about 100 kHz, (ii) about 100–200 kHz, (iii) about 200–300 kHz, (iv) about 300–400 kHz, (v) about 400–500 kHz, (vi) about 0.5–1.0 MHz, (vii) about 1.0–1.5 MHz, (viii) about 1.5–2.0 MHz, (ix) about 2.0–2.5 MHz, (x) about 2.5–3.0 MHz, (xi) about 3.0–3.5 MHz, (xii) about 3.5–4.0 MHz, (xiii) about 4.0–4.5 MHz, (xiv) about 4.5–5.0 MHz, (xv) approximately 5.0 - 5.5 MHz, (xvi) approximately 5.5 - 6.0 MHz, (xvii) approximately 6.0 - 6.5 MHz, (xviii) approximately 6.5 - 7.0 MHz, (xix) approximately 7.0 - 7.5 MHz, (xx) approximately 7.5 - 8.0 MHz, (xxi) approximately 8.0 - 8.5 MHz, (xxii) approximately 8.5 - 9.0 MHz, (xxiii) approximately 9.0 - 9.5 MHz, (xxiv) approximately 9.5 - 10.0 MHz and (xxv) > approximately 10.0 MHz.

[0122] The ion guidance can be maintained at a pressure selected from the group consisting of the following: (i) < about 0.0001 mbar, (ii) about 0.0001 - 0.001 mbar, (iii) about 0.001 - 0.01 mbar, (iv) about 0.01 - 0.1 mbar, (v) about 0.1 - 1 mbar, (vi) about 1 - 10 mbar, (vii) about 10 - 100 mbar, (viii) about 100 - 1000 mbar and (ix) > about 1000 mbar.

[0123] Analyte ions can be subjected to electron transfer dissociation (ETD) fragmentation in an electron transfer dissociation fragmentation device. Analyte ions can be caused to interact with ETD reagents within an ion guide or fragmentation device.

[0124] Optionally, to effect electron transfer dissociation, either: (a) analyte ions are fragmented or dissociated to form product or fragment ions after interacting with reagent ions, and / or (b) electrons are transferred from one or more reagent anions or negatively charged ions to one or more multiply charged analyte cations or positively charged ions, whereupon at least some of the multiply charged analyte cations or positively charged ions are dissociated to form product or fragment ions, and / or (c) analyte ions are fragmented or dissociated to form product or fragment ions after interacting with neutral reagent gas molecules or atoms or a non-ionic reagent gas.and / or (d) electrons are transferred from one or more neutral non-ionic or uncharged starting gases or vapors to one or more multiply charged analyte cations or positively charged ions, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused to dissociate and form product or fragment ions, and / or (e) electrons are transferred from one or more neutral non-ionic or uncharged superbase reagent gases or vapors to one or more multiply charged analyte cations or positively charged ions, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused to dissociate and form product or fragment ions, and / or (f) electrons are transferred from one or more neutral,(a) electrons are transferred from one or more non-ionic or uncharged alkali metal gases or vapors to one or more multiply charged analyte cations or positively charged ions, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused to dissociate and form product or fragment ions, and / or (g) electrons are transferred from one or more neutral, non-ionic or uncharged gases, vapors or atoms to one or more multiply charged analyte cations or positively charged ions, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused to dissociate and form product or fragment ions, wherein the one or more neutral, non-ionic or uncharged gases, vapors or atoms are selected from the group consisting of: (i) sodium vapor or atoms, (ii) lithium vapor or atoms, (iii) potassium vapor or atoms,(iv) rubidium vapor or atoms, (v) cesium vapor or atoms, (vi) francium vapor or atoms, (vii) C, 60 -vapor or atoms and (viii) magnesium vapor or atoms.

[0125] The multiply charged analyte cations or positively charged ions can include peptides, polypeptides, proteins, or biomolecules.

[0126] Optionally, to effect electron transfer dissociation: (a) the reagent anions or negatively charged ions are derived from a polyaromatic hydrocarbon or a substituted polyaromatic hydrocarbon and / or (b) the reagent anions or negatively charged ions are derived from the group consisting of: (i) anthracene, (ii) 9,10-diphenylanthracene, (iii) naphthalene, (iv) fluorine, (v) phenanthrene, (vi) pyrene, (vii) fluoranthene, (viii) chrysene, (ix) triphenylene, (x) perylene, (xi) acridine, (xii) 2,2'-dipyridyl, (xiii) 2,2'-biquinoline, (xiv) 9-anthracenecarbonitrile, (xv) dibenzothiophene, (xvi) 1,10'-phenanthroline, (xvii) 9'-Anthracene carbonitrile and (xviii) anthraquinone and / or (c) contain the reagents or negatively charged ions azobenzene anions or azobenzene radical anions.

[0127] The process of electron transfer dissociation fragmentation can involve the interaction of analyte ions with reagents, where the reagents include dicyanobenzene, 4-nitrotoluene, or azulene.

[0128] The spectrometer can be operated in various modes, including a mass spectrometry (“MS”) mode, a tandem mass spectrometry (“MS / MS”) mode, an operating mode in which starting or precursor ions are alternatively fragmented or reacted to produce fragments or productions, and are not fragmented or reacted or are fragmented or reacted to a lesser extent, a multiple reaction monitoring (“MRM”) mode, a data-dependent analysis (“DDA”) mode, a data-independent analysis (“DIA”) mode, a quantification mode, or an ion mobility spectrometry (“IMS”) mode. BRIEF DESCRIPTION OF THE DRAWING

[0129] Various embodiments are now described only as examples and with reference to the accompanying drawing. It shows: Fig. 1 one embodiment wherein a sample is applied to a metallic target plate using an atomizer and the sample is subjected to two-dimensional separation, Fig. 2 an embodiment wherein a sample spotter is used to apply a sample to a metallic target plate and wherein the sample is then subjected to two-dimensional separation, Fig. 3 one embodiment wherein the target has a cylindrical rod, Fig. 4 one embodiment wherein a liquid sample is applied to a cylindrical rod target by means of a syringe, Fig. 5 an alternative embodiment wherein a liquid sample is applied to an inclined target plate and wherein the target plate is then moved by ultrasound, Fig. 6 an embodiment wherein a scraper is used to spread a sample liquid that has been applied to a target plate, Fig. 7 a flowchart showing the processes according to one embodiment, and Fig. 8A a picture of a target rod attached to a plate before the applied sample was covered with a matrix, Fig. 8B a MALDI ion image intensity map for ions with a mass / charge ratio of 259.9277, Fig. 8C a MALDI ion image intensity map for ions with a mass / charge ratio of 229.1548 and Fig. 8D a MALDI ion image intensity map for ions with a mass / charge ratio of 341.0302. DETAILED DESCRIPTION

[0130] Matrix-assisted laser desorption ionization (“MALDI”) mass spectrometry analysis of complex samples such as blood or urine usually involves a time-consuming sample preparation stage in which the sample undergoes processes such as dilution, desalting and protein precipitation prior to sample application, matrix application and subsequent mass spectrometry analysis.

[0131] A well-known attempt to increase the specificity and simplicity of this process is to combine two-dimensional (“2D”) separation techniques such as thin-layer chromatography (“TLC”) with MALDI mass spectrometry analysis. For example, it is known to separate two-dimensional fractions from a thin-layer chromatography substrate, dilute them in a suitable solvent, and then subject the sample to LC / MS analysis.

[0132] It is known that MALDI / MS data can be obtained directly from thin-film chromatography plates after the application of a suitable matrix coating. This improves the speed of analysis. However, a disadvantage is that the porous and uneven surface of the thin-film chromatography substrate leads to a broadening of spectral mass peaks and a deterioration of mass accuracy when combined with axial time-of-flight mass analysis.

[0133] From a clinical diagnostics perspective, it would be advantageous to further simplify the sample preparation process for complex biological samples. To be suitable for medical diagnostics in the care setting, a sample preparation technique is desired that is fast, free of complex procedures, uses non-toxic reagents, and applies samples to media that are optimal for subsequent imaging experiments using matrix-assisted laser desorption / ionization mass spectrometry.

[0134] Various embodiments relate to a device and a method, combining a two-dimensional spatial separation of sample components on a target suitable for subsequent imaging by matrix-assisted laser desorption ionization mass spectrometry, thereby enabling rapid analysis of complex samples with increased specificity.

[0135] Various techniques can be used to achieve pseudo-spatial separation of components in a complex sample by employing mechanical, hydrodynamic, and / or aerodynamic means, according to different embodiments. For example, according to one embodiment, a sample can be introduced into a high-velocity gas spray, which can then be directed toward a metallic target. This process involves droplet breakup, surface turbulence / microvorticity, liquid separation, and schlieren formation on the liquid surface. The spatial separation of different components occurs as a result of their physicochemical properties, such as surface affinity and solubility in solution.

[0136] This form of separation can be observed with a number of physical arrangements, including, but not limited to, the following: (i) a pneumatically atomized spray directed at a static or moving target, (ii) a pneumatically atomized spray directed at a cylindrical target in a cross-flow arrangement, (iii) an applied liquid sample forced to travel along a target surface under the influence of a gas flow, (iv) an applied liquid sample forced to disperse along an inclined target under the influence of ultrasonic stirring, or (v) an applied liquid sample forced to disperse along a rod target under the influence of a high axial rotation frequency.

[0137] In these examples, analyte separation depends on the basic affinity for the two phases (i.e., the solid or the liquid phase) and can be influenced by physicochemical properties such as surface binding, liquid surface affinity, solubility, and ion pairing affinity. Samples can be dissolved in liquids that include, but are not limited to, reversed-phase chromatography solvents such as water, methanol, and acetonitrile, and normal-phase solvents such as hexane and chloroform, or any mixtures of these solvents.

[0138] Various embodiments relate to a method for ion mapping, wherein a sample is applied to a target surface and components of the sample are separated on the target surface according to one or more physicochemical properties in a first dimension and in a second dimension. In the method, several separate regions of the sample can further be ionized and mass-analyzed to generate an ion map of at least a section of the sample applied to the target surface. According to various embodiments, the sample is applied to the target surface and separated by mechanical, hydrodynamic, and / or aerodynamic means.

[0139] The approach according to various embodiments is particularly advantageous in that the method for applying the sample to the target and for separating the sample is quick and simple, and furthermore, the sample can be applied to a target that is particularly suitable for subsequent MALDI analysis. This contrasts with conventional arrangements.

[0140] The first and second physicochemical properties can be the same or different.

[0141] According to various embodiments, a sample can be applied to a target surface in two dimensions and separated. The separation of the sample can occur, for example, while the sample is in contact with the target surface and / or after the sample has been in contact with the target surface.

[0142] According to various embodiments, the sample can be applied and separated by different mechanical, hydrodynamic, and / or aerodynamic means. In other words, the method can provide mechanical, hydrodynamic, and / or aerodynamic means for applying and separating the sample, and / or the sample can be applied to and separated from the target surface by these mechanical, hydrodynamic, and / or aerodynamic means. This contrasts with conventional arrangements where a sample is separated by chromatographic means.

[0143] For example, according to various embodiments, the deposition and separation of the sample may comprise one or more of the following (the sample may be applied and separated by mechanical, hydrodynamic and / or aerodynamic means to perform the following): (i) introducing the sample into a gas spray and allowing the spray to impact the target surface, (ii) forcing the sample to travel along the target surface under the influence of a gas and / or liquid flow, (iii) dispersing the sample along the target surface under the influence of gravity, (iv) spreading the sample on the target surface, optionally using a scraper or other device, (v) heating the sample and / or the target, and / or (vi) shifting, moving, rotating, vibrating, and / or stirring the target.

[0144] The spatial separation of different components of the sample occurs according to their one or more physicochemical properties in two dimensions in the type of surface bonding, surface affinity, solubility, bonding affinity, polarity, other physicochemical properties and any combination thereof.

[0145] According to various embodiments, several separate areas of the sample can be ionized and mass-analyzed to generate an ion map (i.e., a mass spectrometry image) of at least a part of the sample on the target surface.

[0146] According to various embodiments, in the steps of depositing and separating the sample, the sample can further be applied to the target surface such that the sample separates in the first dimension and in the second dimension according to one or more physicochemical properties by mechanical, hydrodynamic, and / or aerodynamic means. The sample can be applied to the target surface in such a way that it is caused to separate in two dimensions on the target surface.

[0147] For example, according to one embodiment, a sample can be introduced into a gas spray, which can then be directed onto a target surface. This process involves droplet breakup, surface turbulence / microvorticity, liquid deposition, and schlieren formation on the liquid surface. The spatial separation of different components of the sample occurs according to one or more of their physicochemical properties, such as surface affinity and solubility in a solution.

[0148] According to various embodiments, the sample can be applied to the target surface before the separation begins. For example, according to one embodiment, urine or blood samples are applied to the target surface before being separated in two dimensions on the target surface.

[0149] According to various embodiments, the first dimension can be orthogonal to the second dimension, and both the first and second dimensions can be spatial dimensions. The sample can therefore be divided into two spatial dimensions, which are optionally orthogonal to each other.

[0150] The sample can be a liquid. According to one embodiment, the sample can, for example, comprise blood, urine, or another liquid. Additionally or alternatively, the sample can be dissolved in a liquid solvent such as reversed-phase chromatography solvents like water, methanol, acetonitrile, and normal-phase solvents like hexane, chloroform, or any mixture of these solvents. A liquid solvent can also be applied to the sample on the target surface, in which case the sample can then be dissolved in the liquid solvent applied to the sample on the target surface. It will be understood that the sample need not be a liquid.

[0151] The target surface can be substantially non-porous and / or substantially flat, thus providing separation on a target surface suitable for subsequent matrix-assisted laser desorption ionization (“MALDI”) mass spectrometry analysis. For example, according to embodiments, the target surface comprises a metallic target surface, a polished metallic target surface, a stainless steel target surface, or a polished stainless steel target surface.

[0152] According to various embodiments, the sample can already be present on the target surface (i.e., provided there) before the separation begins. For example, according to one embodiment, fingerprints or bloodstains are provided on the target surface before being separated in two dimensions on the target surface. The fingerprints or bloodstains can be dissolved by a solvent applied to the target surface before being separated in two dimensions on the target surface.

[0153] According to various embodiments, a sample can be separated according to one or more first physicochemical properties in a first dimension and according to one or more second physicochemical properties in a second dimension by mechanical, hydrodynamic, and / or aerodynamic means. The one or more first physicochemical properties and the one or more second physicochemical properties can be the same or different.

[0154] The sample can be separated, for example, by introducing the sample into a gas spray and allowing the spray to impact the target surface, forcing the sample to move along the target surface under the influence of a gas and / or liquid flow, dispersing the sample along the target surface under the influence of gravity, spreading the sample on the target surface optionally using a scraper or other device, heating the sample and / or the target, and moving, shifting, rotating, vibrating, and / or stirring the target.

[0155] The one or more first physicochemical properties and / or the one or more second physicochemical properties may include surface binding, surface affinity, solubility, bonding affinity and / or polarity.

[0156] According to various embodiments, a sample is separated on a metallic target surface according to one or more first physicochemical properties in a first dimension and according to one or more second physicochemical properties in a second dimension.

[0157] Various embodiments will now be described in more detail.

[0158] Fig. Figure 1 shows an embodiment wherein a device is provided to effect two-dimensional separation of a complex sample. A liquid sample is infused into an atomizer 1 by a solvent supply pump 2. The liquid stream is then atomized by a high-velocity gas stream generated by a pressurized gas supply 3. The atomized spray is then directed onto a target 4, which has a polished stainless steel plate. The pump 2 may comprise a syringe infusion pump or another pump that may have an injection port for introducing a small amount of the liquid sample into the device.

[0159] The in Fig. The atomizer 1 shown in Figure 1 is in a vertical position. However, according to other embodiments, the atomizer 1 can also be inclined at an angle between 0 and 90° with respect to the target plate 4.

[0160] A heater 5 can be provided and powered by a heating power supply 6 to indirectly heat the target plate 4 and / or the spray to dry the sample as it is dispersed onto the target plate 4 under the influence of the gas flow from the atomizer. The heater 5 can also have an independent gas flow or incorporate a radiation type such as an infrared (IR) lamp. During the sample deposition process, the target plate 4 can either be held statically relative to the atomizer 1 or, alternatively, it can be moved in any direction by a target plate displacement device 7.

[0161] According to this embodiment, the sample is applied to the target surface by mechanical, hydrodynamic, and / or aerodynamic means. The target comprises a plate; the sample is introduced into a gas spray, and the spray is directed onto the target surface. The sample and the target are heated, and the target can be displaced and / or moved. The sample applied to the target surface is separated in a first dimension or direction and in a second dimension or direction according to one or more physicochemical properties, thus achieving a two-dimensional separation. The one or more physicochemical properties can be the same or different.

[0162] Fig. Figure 2 shows an embodiment in which a sample is applied directly onto a target plate 4 in the form of a discrete droplet, a number of droplets, or a liquid line. The sample is applied by a sample syringe 8 or a sample spotter and can initially be applied with or without a gas or liquid flow from an atomizer 1. The sample can be applied, and the atomizing gas can then be applied from the atomizer 1 without a liquid flow. Under these conditions, the sample disperses in the form of rolling droplets or elongated liquid streaks. Alternatively, the sample can be applied before the start of a ballistic (rapid) solvent gradient from the solvent supply pump 2, either in a static or moving target mode. All other experimental parameters are as above with respect to the above with respect to Fig. 1. The embodiment shown and described has been described.

[0163] According to this embodiment, the sample is applied to the target surface by mechanical, hydrodynamic, and / or aerodynamic means. The sample is forced to move along the target surface under the influence of a gas and / or liquid flow. The sample applied to the target surface is separated in a first dimension or direction and in a second dimension or direction according to one or more physicochemical properties, thus achieving a two-dimensional separation. The one or more physicochemical properties can be the same or different.

[0164] Fig. Figure 3 shows an alternative embodiment in which the target plate has been replaced by a cylindrical rod target 9. The cylindrical rod 9 is arranged perpendicular to the gas flow from the atomizer 1. This embodiment features a cross-flow arrangement that generates counter-rotating surface vortices oriented in the direction of the gas flow. The surface microvortices exhibit mass-dependent deposition effects, enabling two-dimensional separation of complex sample components carried along in the atomized spray from the atomizer 1. According to another embodiment, a liquid sample can be applied to the cylindrical rod target 9 with or without an atomizer liquid flow.

[0165] According to this embodiment, the sample is applied to the target surface by mechanical, hydrodynamic, and / or aerodynamic means. The target comprises a rod; the sample is introduced into a gas spray, and the spray is directed onto the target surface. The sample applied to the target surface is separated in a first dimension or direction and in a second dimension or direction according to one or more physicochemical properties, thus achieving a two-dimensional separation. The one or more physicochemical properties can be the same or different.

[0166] Fig. Figure 4 shows another embodiment in which a liquid sample is applied to the cylindrical rod target 9 by means of a sample syringe 8. The cylindrical target 9 is then rapidly rotated about its axis by a motor 10 capable of rotational speeds in the range of, for example, 10–10,000 rpm. In the case of a horizontally oriented target 9, the liquid sample is dispersed longitudinally along the axis of rotation and in both directions from the starting point of deposition. During this process, heat can be applied from a heater 5, which may have a flow of a drying gas.

[0167] According to this embodiment, the sample is applied to the target surface by mechanical, hydrodynamic, and / or aerodynamic means. The target comprises a rotating rod. The sample applied to the target surface is separated in a first dimension or direction and in a second dimension or direction according to one or more physicochemical properties, thus achieving a two-dimensional separation. The one or more physicochemical properties can be the same or different.

[0168] Fig. Figure 5 shows an alternative embodiment in which a liquid sample is applied from a syringe 8 onto an inclined target plate 4. The inclined target plate 4 is moved by a transducer 11, for example, using ultrasonic frequencies (e.g., kHz to GHz). The liquid sample is dispersed along the target plate 4 when the gravitational and ultrasonic forces overcome the surface tension of the initially deposited sample droplet. During this process, heat can be applied from a heater 5, which may include a flow of a drying gas.

[0169] According to this embodiment, the sample is applied to the target surface by mechanical, hydrodynamic, and / or aerodynamic means. The sample is dispersed along the target surface under the influence of gravity, and the target is vibrated and moved. The sample applied to the target surface is separated in a first dimension or direction and in a second dimension or direction according to one or more physicochemical properties, thus achieving two-dimensional separation. The one or more physicochemical properties can be the same or different.

[0170] Fig. Figure 6 shows an alternative embodiment in which a liquid sample is applied from a syringe 8 to a planar target 4. A scraper 12 is positioned to touch the target surface or at least approach a point on it, such that the gap between the scraper 12 and the planar target 4 is significantly smaller than the height of the initial sample droplet(s). A target displacement device 7 is used to move the droplets against the scraper 12, so that the liquid is spread along the target 4. A heater 5 can be used to assist in evaporating the solvent during the spreading process.

[0171] According to this embodiment, the sample is applied to the target surface by mechanical, hydrodynamic, and / or aerodynamic means. The sample is spread on the target surface using a spreader. The sample applied to the target surface is separated in a first dimension or direction and in a second dimension or direction according to one or more physicochemical properties, thus achieving a two-dimensional separation. The one or more physicochemical properties can be the same or different.

[0172] According to another embodiment, rapid separation can be achieved by applying, for example, 2-3 µl of a liquid to a planar target and subsequently spreading the spot over an area of ​​typically 0.3-0.5 cm². 2The process is carried out before the liquid is heated by a hot gas stream (200 °C, 1.5 m / s, orthogonal to the plate with a static target).

[0173] This process typically results in a series of concentric tidal markings (rings) as analytes are deposited according to their solubility in the liquid, whose composition changes rapidly over time. Accordingly, for a 1:1 methanol / water solution, samples of lower polarity are deposited in the outer rings, while the polar analytes are retained in the shrinking liquid as the water concentration increases over time. Consequently, the polar compounds are deposited in the rings closest to the center of the original liquid region.

[0174] According to this embodiment, the sample is applied to the target surface by mechanical, hydrodynamic, and / or aerodynamic means. The sample is spread out on the target surface and heated. The sample applied to the target surface is separated in a first dimension or direction and in a second dimension or direction according to one or more physicochemical properties, thus achieving a two-dimensional separation. The one or more physicochemical properties can be the same or different.

[0175] According to all embodiments described above, the specificity of the two-dimensional separation can be improved by: (i) chemically or physically modifying the target surface to increase or decrease its binding affinity for a particular target analyte or for particular groups of analytes, and / or (ii) modifying the chemical or physical properties of the solvent to support the differential separation of analytes, and / or (iii) performing the separation in the presence of an electric field to promote separation based on electromigration processes.

[0176] The latter modification can be implemented, for example, by increasing the potential of the target plate or target rod to a high voltage while the other components, etc., are grounded. According to this embodiment, an electric field is maintained over at least part of the sample to cause at least some parts of the sample to be separated by electromigration.

[0177] Various embodiments involve combining two-dimensional sample separation with matrix-assisted laser desorption / ionization mass spectrometry imaging for the rapid analysis of complex samples such as blood or urine. In most cases, the implementation of the technique can proceed according to the steps described in Fig. The flowchart shown in section 7 illustrates this.

[0178] The method, according to various embodiments, consists of two-dimensional sample separation on a target using one or more of the techniques described above. This is followed by the application of matrix material directly onto the target; that is, a matrix can be applied to the sample prior to ionization and mass spectrometry. Matrix-assisted laser desorption / ionization mass spectrometry imaging of the target or a relevant portion of the target is then performed, followed by post-processing (data acquisition) of the obtained mass spectrometry data. The matrix coating stage is not essential for obtaining mass spectrometry data from the target.Accordingly, the method is not limited to matrix-assisted laser desorption ionization mass spectrometry analysis and can be used in combination with rapid atom bombardment (“FAB”), surface-assisted laser desorption ionization (“SALDI”), desorption electrospray ionization (“DESI”) or other mass spectrometry (“MS”) imaging techniques.

[0179] According to these various embodiments, one or more areas of the sample are ionized using a matrix-assisted laser desorption ionization (“MALDI”) ion source, a fast atom bombardment (“FAB”) ion source, a surface-assisted laser desorption ionization (“SALDI”) ion source, a desorption electrospray ionization (“DESI”) ion source, or another suitable ion source.

[0180] To demonstrate the potential usefulness of the present analytical technique, pure urine was sprayed from a pneumatically assisted atomizer onto a stainless steel rod positioned perpendicular to the flow, as essentially described in Fig. 3 is shown.

[0181] The nebulizer consisted of a central liquid sample capillary (stainless steel, 127 µm inner diameter, 229 µm outer diameter) and an outer gas flow capillary (stainless steel, 330 µm inner diameter). This concentric arrangement created a gas flow-restricting path over a length of 25 mm, beyond which a nitrogen gas reservoir was pressurized to 7 bar. In this configuration, the nebulizer was surrounded by an annular heater that supplied nitrogen gas at a temperature of 200 °C and a flow rate of 1000 l / h. The target comprised a polished stainless steel rod with a diameter of 1.6 mm, held approximately 3 mm from the nebulizer tip and positioned orthogonally to the spray axis. The urine was sprayed for 7.5 seconds at a flow rate of 200 µl / min (25 µl of the sample were used).The rod was mounted flat on a standard stainless steel target plate for matrix-assisted laser desorption ionization and coated with α-cyano-4-hydroxycinnamic acid (“CHCA”). The CHCA matrix was sprayed at a concentration of 5 mg / ml in a 70 / 30 acetonitrile / water (0.2% trifluoroacetic acid) solution at a flow rate of 20 µl / min. The spray pattern was repeated over 30 passes to ensure uniform coating.

[0182] The matrix-coated rod / plate was then subjected to matrix-assisted laser desorption / ionization mass spectrometry imaging in a vacuum using an orthogonal acceleration time-of-flight mass spectrometer. Following this example, several separate regions of the sample were ionized and mass-analyzed to generate matrix-assisted laser desorption / ionization ion image intensity maps (i.e., an ion map) of at least a portion of the sample deposited onto the target surface.

[0183] Fig. Figure 8A shows an image of a rod and corresponding plate taken before the CHCA coating stage. Fig. Figure 8A shows an impact spot A where the spray first came into contact with the rod, and a series of fine streaks B where the sample material migrated radially from the impact spot under the influence of the high-velocity gas flow.

[0184] The Fig. Figures 8B-D show resulting matrix-assisted laser desorption ionization ion image intensity maps obtained for three different mass / charge ratio values: m / z 259.9277, 229.1548, and 341.0302. In all images, the ion intensity increases from black to white.

[0185] Fig. Figure 8B shows the detection of radial schlieren, where the ion intensity increases with the radial distance from the impact spot. Conversely, it shows Fig. 8C an ion intensity distribution that decreases with the radial distance from the impact spot. Fig. 8D shows a more even distribution, but it still shows a significant empty area along the edge of the impact spot.

[0186] The in the Fig. The data shown in Figures 8B-8D demonstrate that compound-dependent two-dimensional separations can be rapidly obtained for complex samples without the need for time-consuming sample preparation or conventional chromatographic methods. Furthermore, the separations can be performed on surfaces that are optimal for matrix-assisted laser desorption / ionization mass spectrometry or alternative MS imaging techniques.

[0187] According to another embodiment, tracer molecules can be incorporated into a spray or applied to the surface of structural components exposed to high-velocity gas flows. If the component's geometry is suitable for matrix-assisted laser desorption / ionization mass spectrometry imaging, the resulting images reveal surface flow patterns that could be used to validate computational fluid dynamics (CFD) model data. An example would be the surface flow on a scaled aircraft wing.

[0188] Embodiments are being considered that incorporate a combination or combinations of the various embodiments described above, which can be used to effect a pseudo-separation of analyte mixtures on a target prior to MALDI-MS analysis.

[0189] Furthermore, any of the various embodiments described and considered above can also be implemented by adding a MALDI matrix or matrices to the analyte mixture prior to the pseudo-separation step. With regard to the tide-marking effect described above, embodiments are considered in which a mixture of matrices is added to cover a broad polarity range. This embodiment eliminates the second stage of the process described above with respect to the Fig. The preferred process shown in section 7 was described.

Claims

[1] Method for ion mapping of a sample, which has the following features: Application of a liquid sample to a metallic target surface, Separation of components of the liquid sample on the metallic target surface according to one or more physicochemical properties in a first spatial dimension and in a second spatial dimension and Ionizing and mass-analyzing multiple separated areas of the separated sample to generate an ion map of at least a portion of the sample separated on the target surface, wherein the components of the sample are applied to the target surface and separated by mechanical, hydrodynamic and / or aerodynamic means, and wherein the one or more physicochemical properties include one or more from the group comprising surface binding, surface affinity, solubility, binding affinity and polarity. [2] Method according to claim 1, comprising applying the liquid sample to the metallic target surface in such a way that the components of the liquid sample separate according to one or more physicochemical properties in the first spatial dimension and the second spatial dimension. [3] Method according to claim 1, comprising applying the liquid sample to the metallic target surface and subsequently separating the components of the liquid sample on the metallic target surface according to one or more physicochemical properties in the first spatial dimension and in the second spatial dimension. [4] Method according to any of the preceding claims, wherein the liquid sample comprises a sample dissolved in a liquid solvent. [5] Method according to one of the preceding claims, wherein a liquid solvent is further applied to the liquid sample on the metallic target surface. [6] Method according to any of the preceding claims, wherein the metallic target surface comprises a polished metallic target surface. [7] Method according to any of the preceding claims, wherein the metallic target surface comprises a stainless steel target surface. [8] Method according to any of the preceding claims, comprising applying the liquid sample to the metallic target surface using an atomizer, a sample syringe or a sample spotter. [9] Method according to any of the preceding claims, comprising introducing the liquid sample into a gas spray, wherein the spray is directed onto the metallic target surface. [10] Method according to any of the preceding claims, comprising forcing the liquid sample applied to the target surface to move along the metallic target surface under the influence of a gas and / or liquid flow. [11] Method according to any of the preceding claims, comprising dispersing the liquid sample applied to the target surface under the influence of gravity along the metallic target surface. [12] Method according to any of the preceding claims, comprising spreading the liquid sample applied to the metallic target surface using a spreader or other device. [13] Method according to any of the preceding claims, comprising heating the liquid sample and / or the target and / or moving, shifting, rotating and / or vibrating the target. [14] A method for mass spectrometry comprising a method according to any of the preceding claims. [15] Device which features: a target that has a metallic target surface, a first device which is set up and designed to apply a liquid sample to the metallic target surface and to separate components of the sample on the metallic target surface according to one or more physicochemical properties in a first spatial dimension and in a second spatial dimension by mechanical, hydrodynamic and / or aerodynamic means, and a second device which is set up and designed to ionize several separate areas of the separated sample and to perform a mass analysis on them in order to generate an ion map of at least a part of the sample separated on the metallic target surface, wherein the one or more physicochemical properties include one or more from the group comprising surface binding, surface affinity, solubility, binding affinity and polarity.

Images