Mass spectrometry imaging

Abstract

A method of analyzing a sample includes analyzing the sample in a first mode of operation, thereby generating a spatially resolved data set representing the sample, and analyzing the data set to identify one or more regions of the sample. For each of the one or more identified regions of the sample, a tandem mass spectrometry (MS / MS) data set for the region is generated by determining a path through the region, and analyzing the sample along the path using desorption electrospray ionization ("DESI") in a tandem mass spectrometry (MS / MS) mode of operation.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to UK Patent Application No. 2004678.5, filed on March 31, 2020. The entire contents of this application are incorporated herein by reference. Technical Field

[0003] The present invention generally relates to analytical instruments such as mass spectrometers and / or ion mobility spectrometers, and more particularly to methods for imaging samples using analytical instruments such as mass spectrometers and / or ion mobility spectrometers. Background Technology

[0004] Mass spectrometry imaging (MSI) can provide very rich datasets, with hundreds or thousands of peaks per pixel out of tens of thousands of pixels. However, this presents challenges for analysts in interpreting the results, as molecular identification can be extremely difficult.

[0005] The applicant believes that there is still room for improvement in mass spectrometry imaging technology. Summary of the Invention

[0006] According to one aspect, a method for analyzing samples is provided, the method comprising:

[0007] The sample is analyzed in the first operating mode to generate a spatially resolved dataset representing the sample;

[0008] Analyze the dataset to identify one or more regions of the sample; and

[0009] For each identified region in one or more identified regions of the sample, a tandem mass spectrometry (MS / MS) dataset of the region is generated by the following operation:

[0010] Determine the path through the area; and

[0011] The sample is analyzed along the path by analyzing the sample along at least a portion of the path in tandem mass spectrometry (MS / MS) operating mode.

[0012] Various implementations relate to a method for analyzing a sample, wherein the sample is analyzed in a first operating mode (e.g., mass spectrometry imaging (MSI) operating mode) to generate a spatially resolved dataset representing the sample, such as a mass spectrometric image of the sample. This may include, for example, moving the analysis probe relative to the sample in line-by-line mode or moving the sample relative to the analysis probe, and repeatedly sampling (and analyzing) the sample to build the spatially resolved dataset.

[0013] The dataset is analyzed to identify one or more regions of a sample. The analysis can identify multiple (chemically) distinct regions of the sample. Therefore, each identified region can be a substantially (chemically) homogeneous region of the sample, and each identified region can be (chemically) different from every other identified region.

[0014] In various implementations, for each of one or more identified regions of a sample, a tandem mass spectrometry (MS / MS) dataset for said region is generated. In other words, for each region, a dataset comprising the product ion spectra of each of a plurality of different precursor ions is generated.

[0015] This is accomplished by determining a path through the region (where the path is constrained to remain within the region) and then analyzing the sample along the path in tandem mass spectrometry (MS / MS) operating mode. This may include moving the analytical probe relative to the sample or moving the sample relative to the analytical probe to follow the path and repeatedly sampling (and analyzing) the sample to generate a tandem mass spectrometry (MS / MS) dataset. As the probe or sample moves along the path, each of a plurality of different precursor ions can be selected and analyzed (in turn) to generate a corresponding product ion dataset.

[0016] Determining and using a path configured to make the molecular composition of a sample relatively constant along the path in this way can provide sufficient time for analysis to generate detailed tandem mass spectrometry (MS / MS) datasets, such as “full” tandem mass spectrometry (MS / MS) datasets, which include product ion spectra of each of the multiple precursor ions of interest.

[0017] The resulting tandem mass spectrometry (MS / MS) dataset for each region can then be correlated with the spatially resolved data for that region.

[0018] In this way, spatially resolved datasets (mass spectrometry images) can complement tandem mass spectrometry (MS / MS) datasets for each (chemically) homogeneous region of a sample. Providing this additional information for each region can correspondingly facilitate and improve the confidence of the identification of each region.

[0019] Therefore, it should be understood that various implementation schemes provide improved methods for analyzing samples.

[0020] The first operating mode can be the Data Independent Analysis (DIA) operating mode.

[0021] Analyzing samples in the first operating mode may include using desorption electrospray ionization (“DESI”) to analyze the samples.

[0022] The first operating mode can be a mass spectrometry (MS) operating mode or an operating mode in which the precursor ion is alternately activated, fragmented, or reacted to produce product ions, and is not activated, fragmented, or reacted, or is activated, fragmented, or reacted to a lesser extent.

[0023] Spatial resolution datasets can include mass spectrometry images of samples.

[0024] Spatial-resolved datasets can include optical, IR, or UV images of the samples, fluorescence images of the samples, or Raman spectral images of the samples.

[0025] Analyzing a dataset can include analyzing the dataset to identify one or more chemically distinct regions of a sample.

[0026] One or more identified regions can be substantially homogeneous regions of the sample.

[0027] Path-based analysis samples can be included in the path-based analysis sample operation mode.

[0028] Path-based sample analysis can include using desorption electrospray ionization (“DESI”) to analyze samples along the path.

[0029] Analyzing samples along a path can include:

[0030] The sample is analyzed along the first portion of the path in mass spectrometry operation mode; then

[0031] The sample is analyzed along a second different portion of the path in the tandem mass spectrometry (MS / MS) operating mode.

[0032] Analyzing samples along a path can include:

[0033] The sample is analyzed along the first portion of the path in the mass spectrometry operation mode to generate mass spectrometry data;

[0034] Based on the mass spectrometry data, one or more precursor ions of interest are identified; then...

[0035] The sample is analyzed along the second different portion of the path by analyzing each of one or more identified precursor ions of interest in the tandem mass spectrometry (MS / MS) operating mode.

[0036] Analyzing the sample along the path may include switching between mass spectrometry (“MS”) and tandem mass spectrometry (“MS / MS”) operating modes multiple times while analyzing the sample along the path.

[0037] The method may include:

[0038] Based on the results of each corresponding mass spectrometry (“MS”) operating mode, determine one or more precursor ions of interest to be analyzed in the subsequent tandem mass spectrometry (“MS / MS”) operating mode; and

[0039] Each of the identified precursor ions of interest is analyzed in the subsequent tandem mass spectrometry (“MS / MS”) operation mode to generate a product ion spectrum for each precursor ion.

[0040] The method may include excluding precursor ions of interest that have already been analyzed in a previous tandem mass spectrometry (“MS / MS”) operating mode from any subsequent analysis in any tandem mass spectrometry (“MS / MS”) operating mode.

[0041] The method may include:

[0042] Based on the spatially resolved dataset, identify one or more precursor ions of interest; and

[0043] The sample is analyzed along the path by analyzing each of one or more identified precursor ions of interest in the tandem mass spectrometry (MS / MS) operating mode.

[0044] Mass spectrometry (MS) operation mode can be a mode in which (by mass analyzer) the (parent) ions generated from the sample (by ion source) are analyzed to determine the mass-to-charge ratio of the (parent) ions.

[0045] Tandem mass spectrometry (MS / MS) can be an operation mode in which product ions generated from the precursor ions from the sample (generated by the ion source) are analyzed (by a mass analyzer) to determine the mass-to-charge ratio of the product ions.

[0046] Analyzing samples in tandem mass spectrometry (MS / MS) mode can include:

[0047] Select at least one parent ion of interest;

[0048] Activating, fragmenting, or reacting the at least one precursor ion to produce a product ion; and

[0049] The product ions are analyzed to generate a product ion dataset for the at least one precursor ion of interest.

[0050] Analyzing samples in tandem mass spectrometry (MS / MS) mode can include:

[0051] Each of the multiple precursor ions of interest is selected sequentially.

[0052] (In sequence) each of the multiple precursor ions is activated, fragmented, or reacted, thereby producing a product ion for each precursor ion; and

[0053] The product ions are analyzed sequentially to generate a product ion dataset for each of the multiple precursor ions of interest.

[0054] The method may include associating the tandem mass spectrometry (MS / MS) dataset of each region with spatially resolved mass spectrometry data of the region to generate a spatially resolved mass spectrometry dataset representing the sample, the spatially resolved mass spectrometry dataset being supplemented with tandem mass spectrometry (MS / MS) datasets for each of the one or more chemically distinct regions of the sample.

[0055] The method may include analyzing mass spectrometry data and tandem mass spectrometry (MS / MS) data of the region to classify the region.

[0056] According to one aspect, an analytical instrument is provided, the analytical instrument comprising:

[0057] One or more analyzers, the analyzers being configured to analyze samples; and

[0058] The control system is configured to cause the analytical instrument to:

[0059] The sample is analyzed in the first operating mode to generate a spatially resolved dataset representing the sample;

[0060] Analyze the dataset to identify one or more regions of the sample; and

[0061] For each identified region in one or more identified regions of the sample, a tandem mass spectrometry (MS / MS) dataset of the region is generated by the following operation:

[0062] Determine the path through the area; and

[0063] The sample is analyzed along the path by analyzing the sample along at least a portion of the path in tandem mass spectrometry (MS / MS) operating mode.

[0064] The analytical instrument may include a desorption electrospray ionization (“DESI”) ion source, a matrix-assisted laser desorption / ionization (“MALDI”) ion source, a rapid evaporation ionization mass spectrometry (“REIMS”) ion source, or a laser-assisted rapid evaporation ionization mass spectrometry (“LA-REIMS”) ion source, said ion source being configured to generate ions from a sample.

[0065] Analytical instruments may include quality analyzers.

[0066] Analytical instruments may include filters and activation, impact, fragmentation or reaction devices.

[0067] Analytical instruments may include a sample stage and analytical probes.

[0068] The sample stage may be configured to move relative to the analytical probe, and / or the analytical probe may be configured to move relative to the sample stage.

[0069] The control system can be configured to cause the analytical instrument to analyze the sample along the path by:

[0070] The sample is analyzed along the first portion of the path in mass spectrometry operation mode; then

[0071] The sample is analyzed along a second different portion of the path in the tandem mass spectrometry (MS / MS) operating mode.

[0072] The control system can be configured to cause the analytical instrument to analyze the sample along the path by:

[0073] The sample is analyzed along the first portion of the path in the mass spectrometry operation mode to generate mass spectrometry data;

[0074] Based on the mass spectrometry data, one or more precursor ions of interest are identified; then...

[0075] The sample is analyzed along the second different portion of the path by analyzing each of one or more identified precursor ions of interest in the tandem mass spectrometry (MS / MS) operating mode.

[0076] The control system can be configured to:

[0077] Based on the spatially resolved dataset, identify one or more precursor ions of interest; and

[0078] The analytical instrument analyzes the sample along the path by analyzing each of one or more identified precursor ions of interest in the tandem mass spectrometry (MS / MS) operating mode.

[0079] Analytical instruments may include mass spectrometers and / or ion mobility spectrometers. Attached Figure Description

[0080] Various implementation schemes will now be described by way of example only, with reference to the accompanying drawings, in which:

[0081] Figure 1 The analytical instruments according to various implementation schemes are illustrated schematically;

[0082] Figure 2 Desorption electrospray ionization (“DESI”) sources according to various embodiments are shown;

[0083] Figure 3A A first workflow according to various implementation schemes is shown, and Figure 3B The second workflow according to various implementation schemes is shown;

[0084] Figure 4 Workflows according to various implementation schemes are shown; and

[0085] Figure 5 Workflows based on various implementation schemes are shown. Detailed Implementation

[0086] Figure 1 Analytical instruments, including mass spectrometers and / or ion mobility spectrometers, are schematically illustrated according to various embodiments.

[0087] like Figure 1 As shown, the analytical instrument includes an ion source 10, a filter 20 disposed downstream of the ion source 10, an activation, collision, fragmentation or reaction device 30 disposed downstream of the filter 20, and an analyzer 40 disposed downstream of the activation, collision, fragmentation or reaction device 30.

[0088] Ion source 10 is configured to generate ions from a sample. Ion source 10 may include any ion source suitable for mass spectrometry imaging, such as: (i) electrospray ionization (“ESI”) ion source; (ii) atmospheric pressure photoionization (“APPI”) ion source; (iii) atmospheric pressure chemical ionization (“APCI”) ion source; (iv) matrix-assisted laser desorption / ionization (“MALDI”) ion source; (v) laser desorption / ionization (“LDI”) ion source; (vi) atmospheric pressure ionization (“API”) ion source; (vii) silicon-on-silicon desorption / ionization (“DIOS”) ion source; (viii) electron bombardment (… (ix) Chemical ionization (“CI”) ion source; (x) Field ionization (“FI”) ion source; (xi) Field desorption (“FD”) ion source; (xii) Inductively coupled plasma (“ICP”) ion source; (xiii) Fast atomic bombardment (“FAB”) ion source; (xiv) Liquid phase secondary ion mass spectrometry (“LSIMS”) ion source; (xv) Desorption electrospray ionization (“DESI”) ion source; (xvi) Nickel-63 radioactive ion source; (xvii) Atmospheric pressure matrix-assisted laser desorption / ionization ion source. Sources: (xviii) Thermal spray ion source; (xix) Atmospheric sampling glow discharge ionization (“ASGDI”) ion source; (xx) Glow discharge (“GD”) ion source; (xxi) Impactor ion source; (xxii) Real-time direct analysis (“DART”) ion source; (xxiii) Laser spray ionization (“LSI”) ion source; (xxiv) Ultrasonic spray ionization (“SSI”) ion source; (xxv) Matrix-assisted inlet ionization (“MAII”) ion source; (xxvi) Solvent-assisted inlet ionization (“SAII”) ion source. Ion sources; (xxvii) Desorption electrospray ionization (“DESI”) source; (xxviii) Laser ablation electrospray ionization (“LAESI”) source; (xxix) Surface-assisted laser desorption ionization (“SALDI”) source; (xxx) Low-temperature plasma (“LTP”) source; (xxxi) Helium plasma ionization (“HePI”) source; (xxxii) Rapid evaporation ionization mass spectrometry (“REIMS”) source; and / or Laser-assisted rapid evaporation ionization mass spectrometry (“LA-REIMS”) source.

[0089] In various specific embodiments, the ion source 10 includes a desorption electrospray ionization (“DESI”) ion source, a matrix-assisted laser desorption / ionization (“MALDI”) ion source, a rapid evaporation ionization mass spectrometry (“REIMS”) ion source, or a laser-assisted rapid evaporation ionization mass spectrometry (“LA-REIMS”) ion source.

[0090] Figure 2 A desorption electrospray ionization (“DESI”) ion source according to an embodiment is shown. Figure 2 As shown, the atomizer includes a solvent capillary 12 and a gas capillary 13. The solvent capillary 12 (dispenser) is coaxially arranged within the gas capillary 13, with the solvent outlet or tip 12A of the solvent capillary 12 extending beyond the distal end of the gas capillary 13. The solvent flow 14 supplied to the solvent capillary 12 is charged by means of a high-voltage source 18 and guided toward the sample 1 with the assistance of the atomizing gas flow 15 supplied to the gas capillary 13.

[0091] The resulting (primary) charged droplet 11 spray can desorb the analyte material from the surface of sample 1, and the (secondary) droplets carrying the desorbed ionized analyte can then travel via transfer capillary 21 to the atmospheric pressure interface 22 of an analytical instrument such as a mass spectrometer and / or ion mobility spectrometer.

[0092] like Figure 2 As shown, the ion source 10 may include a sample stage configured to hold the sample 1. The sample stage may be configured to move in two (horizontal) dimensions (relative to the nebulizer and transfer capillary 21), allowing analysis of different regions of the sample.

[0093] In the case where the ion source 10 includes a matrix-assisted laser desorption / ionization (“MALDI”) ion source, the sample stage can be configured to move in two (horizontal) dimensions relative to the laser probe of the matrix-assisted laser desorption / ionization (“MALDI”) ion source.

[0094] More generally, the sample stage can be configured to be movable relative to the ion source (e.g., in two (horizontal) dimensions), and / or the analytical probe can be configured to be movable relative to the sample stage (e.g., in two (horizontal) dimensions), allowing different regions of the sample to be analyzed.

[0095] In these implementations, the sample stage or analytical probe may have a fixed position (and only the analytical probe or sample stage may be movable), or both the sample stage and analytical probe may be movable. For example, one of the stage and probe may be configured to move in a first (x) (horizontal) direction, and the other of the stage and probe may be configured to move in a second (y) orthogonal (horizontal) direction, allowing different regions of the sample to be analyzed.

[0096] Back Figure 1 Downstream of ion source 10 is filter 20. Filter 20 may include a mass filter, such as a quadrupole mass filter.

[0097] Filter 20 can be configured to filter ions generated by ion source 10, thereby selecting ions with a specific mass-to-charge ratio. For example, filter 20 can be configured to select ions corresponding to the parent ion of interest by filtering the ions according to the mass-to-charge ratio of the ions received from ion source 10.

[0098] For this purpose, filter 20 can be operated such that ions having a desired mass-to-charge ratio (corresponding to the mass-to-charge ratio of at least one precursor ion) or a mass-to-charge ratio within a desired mass-to-charge ratio range (which may be centered on the mass-to-charge ratio of at least one precursor ion) will be retained and / or forward-transmitted by filter 20. Ions with mass-to-charge ratio values ​​different from the desired mass-to-charge ratio or outside the desired mass-to-charge ratio range may be lost and / or significantly attenuated. Therefore, filter 20 can be configured to select (separate) ions within a mass-to-charge ratio window or range corresponding to (centered on the mass-to-charge ratio) a (single) precursor ion of interest (or a group of several precursor ions of interest).

[0099] In various embodiments, where it is desired to sequentially (one after another) select ions corresponding to each of a plurality of precursor ions of interest, the filter 20 can be operated to sequentially select and transport each of the plurality of precursor ions of interest. This may involve changing the set quality of the filter 20 (i.e., the center of the mass-to-charge ratio or the mass-to-charge ratio range within which ions are selected and / or transported by the filter 20) to sequentially select and transport each of the plurality of precursor ions of interest.

[0100] The filter can also operate in a transport (non-filtering) mode or a relatively wide passband operation mode, such that a relatively large number (most) of the ions generated by the ion source 10 are transported by the filter 20 without being filtered according to their mass-to-charge ratio (selective).

[0101] Refer again Figure 1 Downstream of filter 20 is activation, collision, fragmentation, or reaction device 30. Activation, collision, fragmentation, or reaction device 30 can be configured to activate, fragment, or react the (parent) ions received from filter 20 to produce product ions.

[0102] The activation, collision, fragmentation, or reaction device 30 may include any suitable activation, collision, fragmentation, or reaction device. For example, the activation, collision, fragmentation, or reaction device 30 may be selected from the group consisting of: (i) collision-induced dissociation (“CID”) fragmentation devices; (ii) surface-induced dissociation (“SID”) fragmentation devices; (iii) electron transfer dissociation (“ETD”) fragmentation devices; (iv) electron capture dissociation (“ECD”) fragmentation devices; (v) electron collision or impact dissociation fragmentation devices; (vi) light-induced dissociation (“PID”) fragmentation devices; and (vii) laser-induced dissociation. Fragmentation equipment; (viii) Infrared radiation-induced dissociation equipment; (ix) Ultraviolet radiation-induced dissociation equipment; (x) Nozzle-separator interface fragmentation equipment; (xi) In-source fragmentation equipment; (xii) In-source collision-induced dissociation fragmentation equipment; (xiii) Heat source or temperature source fragmentation equipment; (xiv) Electric field-induced fragmentation equipment; (xv) Magnetic field-induced fragmentation equipment; (xvi) Enzyme digestion or enzyme degradation fragmentation equipment; (xvii) Ion-ion reaction fragmentation equipment; (xviii) Ion... Ion-molecule reaction fragmentation apparatus; (xix) ion-atom reaction fragmentation apparatus; (xx) ion-metastable ion reaction fragmentation apparatus; (xxi) ion-metastable molecular reaction fragmentation apparatus; (xxii) ion-metastable atomic reaction fragmentation apparatus; (xxiii) ion-ion reaction apparatus for causing ions to react to form adducts or product ions; (xxiv) ion-molecule reaction apparatus for causing ions to react to form adducts or product ions; (xxv) ion-atom reaction apparatus for causing ions to react to form adducts or product ions; (xxvi) ion-metastable ion reaction apparatus for causing ions to react to form adducts or product ions; (xxvii) ion-metastable molecular reaction apparatus for causing ions to react to form adducts or product ions; (xxviii) ion-metastable atomic reaction apparatus for causing ions to react to form adducts or product ions; and / or (xxix) electron ionization dissociation (“EID”) fragmentation apparatus.

[0103] Downstream of the activation, collision, fragmentation, or reaction device 30 is an analyzer 40, such as a mass analyzer. The analyzer 40 can be configured to analyze ions received from the activation, collision, fragmentation, or reaction device 30 to determine one or more physicochemical properties of the ions, such as their mass-to-charge ratio and / or ion mobility.

[0104] In various embodiments, analyzer 40 includes an orthogonal acceleration time-of-flight mass analyzer. However, more generally, the mass analyzer may include any suitable mass analyzer selected from the group consisting of: (i) a quadrupole mass analyzer; (ii) a 2D or linear quadrupole mass analyzer; (iii) a Paul or 3D 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 arranged to generate an electrostatic field with a fourth logarithmic potential distribution; (x) a Fourier transform electrostatic 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.

[0105] It should be noted that Figure 1 This is merely illustrative, and the analytical instrument may (and indeed does) include in various implementations. Figure 1 The components, equipment, and functional elements shown.

[0106] For example, analytical instruments may include one or more separators (such as ion mobility separators), one or more ion directors, one or more ion traps, etc.

[0107] like Figure 1 As shown, the analytical instrument may include a control system 50, which may be configured to control the operation of the analytical instrument, for example, in the manner described herein. The control system may include a suitable control circuitry (controller) configured to operate the instrument in the manner described herein. The control system may include a suitable processing circuitry (processor) configured to perform any one or more of the necessary processing and / or post-processing operations for the various embodiments described herein. In various embodiments, the control system may include suitable computing devices, microprocessor systems, programmable FPGAs (Field-Programmable Gate Arrays), etc.

[0108] The analytical instrument can operate in various operating modes, including mass spectrometry (“MS”) operating mode and tandem mass spectrometry (“MS / MS”) operating mode.

[0109] In mass spectrometry (“MS”) operating mode, the precursor ions generated by the ion source can be analyzed, for example, by mass analysis by analyzer 40. In this operating mode, ions generated by ion source 10 can bypass activation, collision, fragmentation, or reaction equipment 30, or the activation, collision, fragmentation, or reaction equipment 30 can operate in a mode where the ions are not activated, fragmented, or reacted (or where the ions are activated, fragmented, or reacted to a relatively small extent). In this operating mode, filter 20 can operate in its transport (non-filtering) mode or a relatively wide passband operating mode (as described above), such that a relatively large number (most) of the ions generated by ion source 10 are analyzed by analyzer 40. In this way, one or more mass spectra of the (precursor) ions generated by ion source 10 can be generated.

[0110] In tandem mass spectrometry (“MS / MS”) operating mode, product ions generated by activation, collision, fragmentation, or reaction device 30 can be analyzed by analyzer 40. In this operating mode, ions generated by ion source 10 can be activated, fragmented, or reacted to produce product ions. In this operating mode, filter 20 can operate in its filtering (selection) mode (as described above) to select the precursor ion of interest. Filter 20 can be configured to sequentially select ions corresponding to each of a plurality of precursor ions of interest (as described above), such that analyzer 40 sequentially analyzes product ions from each of the plurality of precursor ions of interest. In this way, mass spectra of product ions for each precursor ion of interest can be generated.

[0111] In this operating mode, filter 20 can be configured to select a single precursor ion at any given time. Alternatively, filter 20 can be configured to select a subset of multiple precursor ions of interest at some or all times. Intentionally selecting multiple precursor ions simultaneously will produce a mixed tandem mass spectrometry (“MS / MS”) spectrum and can increase the duty cycle of the tandem mass spectrometry (“MS / MS”) operating mode.

[0112] Alternatively, in tandem mass spectrometry (“MS / MS”) operating mode, filter 20 can be configured to sequentially fill the ion trap with subsets of multiple precursor ions of interest. These precursor ions can then be co-fragmented, and the resulting product ions can be subjected to mass analysis. This can be particularly advantageous for instruments with relatively slow mass analysis steps, such as Fourier transform-MS (FT-MS) instruments. In these embodiments, the selection step can occur in parallel with the previous mass analysis of the ion cluster.

[0113] Various implementations involve a method in which a sample is analyzed in a first operating mode to generate a spatially resolved dataset representing the sample, the dataset is analyzed to identify one or more regions of the sample, and for each identified region of the one or more identified regions of the sample, a tandem mass spectrometry (MS / MS) dataset of said region is generated. This is accomplished by determining a path through said region and analyzing the sample along said path in tandem mass spectrometry (MS / MS) operating mode.

[0114] The sample can include any suitable sample, such as a sample with a surface to be imaged. For example, the sample can include a biological sample, such as a tissue slice.

[0115] The samples are analyzed in the first operating mode to generate a spatially resolved dataset representing the samples.

[0116] The first operating mode can be any suitable operating mode, such as the Data Independent Analysis (DIA) operating mode.

[0117] In various implementation schemes, the first operating mode is the mass spectrometry (MS) operating mode, which is the operating mode that generates a mass spectrum (of the precursor ion of the sample) (as described above).

[0118] In various other embodiments, the first operating mode can be an operating mode in which the precursor ion is alternately activated, fragmented, or reacted to produce the product ion, and is not activated, fragmented, or reacted, or is activated, fragmented, or reacted to a lesser extent (such as MSE, HDMSE, or "sonar" operating modes). Thus, the first operating mode can be a mass spectrometry operating mode that produces both precursor and product ions.

[0119] In various specific implementations, the first operating mode is a mass spectrometry imaging (MSI) operating mode, that is, an operating mode that generates mass spectrometry data (such as the mass spectrum of the parent ion of the sample and, optionally, the mass spectrum of the product ion) for each of multiple locations (pixels) on the sample.

[0120] Thus, a spatially resolved dataset representing a sample can include a dataset containing mass spectrometry data (one or more mass spectra) for each of multiple locations (pixels) on the sample. In other words, a spatially resolved dataset representing a sample can include a mass spectrometry image of the sample.

[0121] In these implementations, multiple locations (pixels) on the sample can comprise an array (grid) of locations on the sample. However, other arrangements are also possible.

[0122] Spatial-resolved datasets can be generated, for example, by moving the analysis probe relative to the sample in line-by-line mode or by moving the sample relative to the analysis probe, and by repeatedly sampling (and analyzing) the sample to establish the spatial-resolved dataset (using the analysis instrument in the manner described above).

[0123] However, in various other implementations, the spatially resolved dataset may include another type of spatially resolved dataset representing the sample. For example, the spatially resolved dataset may include (optical, IR, or UV) images of the sample, fluorescence images of the sample, Raman spectral images of the sample, etc.

[0124] According to various implementation schemes, a spatially resolved dataset is analyzed to identify one or more regions of the sample. Where the spatially resolved dataset includes mass spectrometry images, this may include analyzing the mass-to-charge ratio and / or intensity of peaks appearing within each mass spectrum to identify one or more regions of the sample.

[0125] The analysis can identify multiple chemically distinct regions of a sample. One or more of the identified regions, or each identified region, can be substantially (chemically) homogeneous regions of the sample (and each identified region can be (chemically) different from each other).

[0126] Samples can be analyzed in this manner using any suitable data processing techniques. For example, when the spatially resolved data is a mass spectrometry image, data processing techniques, including data reduction techniques such as principal component analysis (PCA), uniform manifold approximation and projection (UMAP), and t-distributed random neighborhood embedding (t-SNE), can be used.

[0127] This analysis can be performed after the entire spatially resolved dataset has been generated, or it can be performed "instantly" while the spatially resolved data is being generated.

[0128] Optionally, the spatially resolved dataset can also be analyzed to (provisionally) determine the chemical composition of each identified region. This may include, for example, (optionally deconvolution and / or deisotopeing, etc.) comparing the (optionally deconvolution and / or deisotopeing) mass spectrometry data of the region with a classification library, as appropriate. Again, this analysis can be performed after the entire spatially resolved dataset has been generated, or "on the fly" while the spatially resolved data is being generated.

[0129] Optionally, spatially resolved datasets can also be analyzed to determine one or more parameters or settings for subsequent analysis of each region. For example, one or more parameters or settings for activation, collision, fragmentation, or reaction devices can be determined based on the analysis of spatially resolved datasets. For example, an ion mode can be selected (from positive or negative ion modes), and / or a fragmentation technique can be selected (from any of the activation, collision, fragmentation, or reaction devices described herein). This determination can be based on or derived from any suitable aspect of the spatially resolved dataset, such as mass-to-charge ratio (m / z), charge state, presumed identification, etc.

[0130] In various implementations, for each of one or more identified regions of a sample, a tandem mass spectrometry (MS / MS) dataset of said region is generated. In other words, for each of the one or more regions, a dataset of product ion spectra including each of a plurality of different (interested) precursor ions is generated.

[0131] This is accomplished by determining a path through the region and then analyzing the sample along that path in tandem mass spectrometry (MS / MS) operating mode.

[0132] In other words, sub-regions of each region in the form of a path (which have already been analyzed in the first operating mode) are reanalyzed in tandem mass spectrometry (MS / MS) operating mode. It should be noted in this respect that desorption electrospray ionization (“DESI”) is particularly suitable for various implementation schemes because DESI analysis allows for resampling of the same surface with a comparable response.

[0133] A single analytical instrument can be used to generate both a spatially resolved dataset and a tandem mass spectrometry (MS / MS) dataset. Alternatively, a first analytical instrument can be used to generate a spatially resolved dataset, and a second, different analytical instrument can be used to generate a tandem mass spectrometry (MS / MS) dataset.

[0134] For example, quadrupole time-of-flight (Q-TOF) instruments can be used to generate spatially resolved datasets, and tandem quadrupole instruments can be used to generate tandem mass spectrometry (MS / MS) datasets. Using different instruments in this way can create high-throughput workflows.

[0135] A path may include sub-regions of a region (in the form of a path). A path may include a subset of sample locations (pixels) corresponding to the region. A path may include continuous or discontinuous paths traversing the region, wherein the path is constrained to remain within the region.

[0136] Analyzing samples along a path may include moving the analytical probe relative to the sample or moving the sample relative to the analytical probe to follow the path and repeatedly sampling (and analyzing) the sample to produce a tandem mass spectrometry (MS / MS) dataset (using the analytical instrument as described above).

[0137] Sample analysis along a path can use all defined paths. Alternatively, fewer than all defined paths can be used. For example, when enough data has been collected for the region, the analysis of the region can be terminated (so that fewer than all defined paths are (re)analyzed).

[0138] As the analysis (probe or sample) moves along the path, each of the multiple different precursor ions can be selected by the filter 20 (in sequence) and analyzed by the analyzer 40, thereby generating a product ion dataset (mass spectrometry) for each precursor ion.

[0139] The precursor ion to be selected by filter 20 can be determined in any suitable manner.

[0140] Path-based analysis samples can be included in the path-based analysis sample operation mode.

[0141] In various implementations, when analyzing samples along the path, the analytical instrument can switch between mass spectrometry (“MS”) operating mode (as described above) and tandem mass spectrometry (“MS / MS”) operating mode (as described above) once or multiple times.

[0142] Therefore, the method may include analyzing a sample along a path by: analyzing the sample along a first portion of the path in mass spectrometry operation mode, and then analyzing the sample along a second different (adjacent) portion of the path in tandem mass spectrometry (MS / MS) operation mode. The method may also include analyzing the sample along a third different (adjacent) portion of the path in mass spectrometry operation mode, and then analyzing the sample along a fourth different (adjacent) portion of the path in tandem mass spectrometry (MS / MS) operation mode, etc.

[0143] The results of each mass spectrometry (“MS”) operating mode (i.e., the mass spectrum of ions generated by ion source 10) can be analyzed to identify one or more precursor ions of interest, and then each of the identified one or more precursor ions of interest can be analyzed in the subsequent tandem mass spectrometry (“MS / MS”) operating mode (by selecting and activating, fragmenting or reacting the precursor ion) to generate a product ion spectrum for each precursor ion of interest (using the analytical instrument as described above).

[0144] In these implementations, an exhaustive or fully tandem mass spectrometry (MS / MS) dataset can be generated for each region, for example, by switching the analyzer multiple times between mass spectrometry (“MS”) and tandem mass spectrometry (“MS / MS”) operating modes while analyzing samples along the path. The results of each corresponding mass spectrometry (“MS”) operating mode (i.e., the mass spectrum of ions produced by ion source 10) can be analyzed to determine one or more precursor ions of interest to be analyzed in the subsequent tandem mass spectrometry (“MS / MS”) operating mode, and each of the identified precursor ions of interest can then be analyzed in the subsequent tandem mass spectrometry (“MS / MS”) operating mode to generate a product ion spectrum for each precursor ion.

[0145] For example, the results of an initial mass spectrometry (“MS”) operating mode (i.e., the mass spectrum of ions generated by ion source 10) can be analyzed to determine a first group of one or more precursor ions of interest to be analyzed in an initial tandem mass spectrometry (“MS / MS”) operating mode. Each precursor ion of interest in the first group of one or more identified precursor ions of interest can then be analyzed sequentially in the initial tandem mass spectrometry (“MS / MS”) operating mode, thereby generating a product ion spectrum for each precursor ion in the first group (using the analytical instrument as described above). The results of a second (immediately following) mass spectrometry (“MS”) operating mode (i.e., the mass spectrum of ions generated by ion source 10) can be analyzed to determine a second group of one or more precursor ions of interest to be analyzed in a second tandem mass spectrometry (“MS / MS”) operating mode. Each precursor ion of interest in the second group of one or more identified precursor ions of interest can then be analyzed sequentially in the second tandem mass spectrometry (“MS / MS”) operating mode, thereby generating a product ion spectrum for each precursor ion in the second group (using the analytical instrument as described above). The results of a third (immediately following) mass spectrometry (“MS”) operating mode can be analyzed to identify a third group or more precursor ions of interest to be analyzed in the second tandem mass spectrometry (“MS / MS”) operating mode.

[0146] These implementations may employ an exclusion list approach, whereby precursor ions of interest that have already been analyzed in a previous tandem mass spectrometry (“MS / MS”) operating mode are excluded from analysis (not analyzed) in any subsequent tandem mass spectrometry (“MS / MS”) operating mode (for the specific region in question).

[0147] The parent ions included in each corresponding parent ion group can be selected in any suitable manner.

[0148] In various implementations, the first group of precursor ions may include the precursor ions most closely conforming to one or more criteria identified in the initial mass spectrometry (“MS”) operating mode, and the second group of precursor ions may include the next most closely conforming precursor ions (excluding the first group of precursor ions) identified in the second mass spectrometry (“MS”) operating mode. For example, the first group of precursor ions may include the most abundant precursor ions identified in the initial mass spectrometry (“MS”) operating mode, and the second group of precursor ions may include the most abundant precursor ions (excluding the first group of precursor ions) identified in the second mass spectrometry (“MS”) operating mode.

[0149] Alternatively, there may be one or more precursor ions of particular interest, which may be user-defined, and which may be preferentially used for analysis in tandem mass spectrometry (“MS / MS”) operating mode. Alternatively, there may be one or more precursor ions, which may be user-defined, and which may be excluded from analysis in tandem mass spectrometry (“MS / MS”) operating mode.

[0150] By performing a sufficient number of iterations of mass spectrometry (“MS”) and tandem mass spectrometry (“MS / MS”) operating modes in this manner, for example until all (identifiable) precursor ions of interest have been analyzed in tandem mass spectrometry (“MS / MS”) operating mode (e.g., until precursor ions of interest that have not yet been analyzed in tandem mass spectrometry (“MS / MS”) operating mode can no longer be identified in mass spectrometry (“MS”) operating mode), an exhaustive or full tandem mass spectrometry (MS / MS) dataset can be generated.

[0151] In various other implementations, when analyzing samples along the path, the analytical instrument may operate only in tandem mass spectrometry (“MS / MS”) operating mode.

[0152] In these implementations, a spatially resolved dataset can be analyzed to identify one or more precursor ions of interest (in each region), and each of the identified precursor ions of interest can then be sequentially analyzed in tandem mass spectrometry (“MS / MS”) operation mode to generate a product ion spectrum for each precursor ion of interest (using the analytical instrument as described above).

[0153] Similarly, an exhaustive or full tandem mass spectrometry (MS / MS) dataset can be generated for each region by sequentially analyzing all the precursor ions of interest identified from the spatially resolved dataset in the tandem mass spectrometry (“MS / MS”) operating mode of the region (using the analytical instrument as described above).

[0154] As described above, the spatially resolved dataset includes mass spectra of precursor ions and product ions (e.g., where the first operating mode is an operating mode in which precursor ions are alternately activated, fragmented, or reacted to produce product ions, and in which they are not activated, fragmented, or reacted, or are activated, fragmented, or reacted to a lesser extent (such as MSE, HDMSE, or “sonar” operating mode), and the tandem mass spectrometry (“MS / MS”) operating mode can be used for precursor ions that have not yet been identified with sufficient certainty in the spatially resolved dataset (and the resulting tandem mass spectrometry (“MS / MS”) data can be used to confirm the identity of these precursor ions).

[0155] It should be understood that identifying and using a path configured such that the molecular composition of the sample is relatively constant along the path, as described above, can provide sufficient time for analysis to generate a detailed tandem mass spectrometry (MS / MS) dataset, such as a “full” tandem mass spectrometry (MS / MS) dataset, which includes the product ion spectra of each of the multiple precursor ions of interest.

[0156] The process of determining the path through the identified region and analyzing the sample along the path in tandem mass spectrometry (MS / MS) operation mode can be repeated for one or more of the identified regions.

[0157] Therefore, the method may include: generating a tandem mass spectrometry (MS / MS) dataset for a first region by determining a first path through a first region, and analyzing samples along the first path in MS / MS operating mode (as described above); and then generating a tandem mass spectrometry (MS / MS) dataset for a second (different) region by determining a second (different) path through a second region, and analyzing samples along the second path in MS / MS operating mode (as described above). The method may also include: generating a tandem mass spectrometry (MS / MS) dataset for a third (different) region by determining a third path through a third region, and analyzing samples along the third path in MS / MS operating mode (as described above), etc.

[0158] These implementation schemes may optionally employ an exclusion list approach, whereby one or more precursor ions of interest that have been analyzed for a previous region are excluded from analysis (not analyzed) for one or more subsequent regions.

[0159] The resulting tandem mass spectrometry (MS / MS) dataset for each region can then be correlated with the spatially resolved data for that region.

[0160] In this way, spatially resolved datasets (mass spectrometry images) can complement tandem mass spectrometry (MS / MS) datasets for each (chemically) homogeneous region of a sample. Providing this additional information for each region can correspondingly facilitate and improve the confidence of the identification of each region.

[0161] In various implementations, mass spectrometry and tandem mass spectrometry (MS / MS) data collected for each region can be analyzed to determine (and thus classify) the chemical composition of the region. This may include, for example (optionally deconvolution and / or deisotopeing, etc.), comparing the (optionally deconvolution and / or deisotopeing) mass spectrometry and tandem mass spectrometry (MS / MS) data with a classification library, as appropriate.

[0162] This analysis can be performed after all mass spectrometry and tandem mass spectrometry (MS / MS) data have been generated (e.g., at the end of the experimental acquisition), or "on the fly" when the data is generated (during the experimental acquisition).

[0163] If this analysis is performed when the data is generated, the results (classification) can be used to influence (guide) further analysis.

[0164] In various implementations, the resulting classification for each region can be provided to the user and / or associated with spatially resolved data for each region. In this way, the spatially resolved dataset (mass spectrometry image) can supplement the classification of each (chemically) homogeneous region of the sample.

[0165] As will be understood from the above, various implementation schemes involve automated data-oriented acquisition (DDA) technology for mass spectrometry imaging (MSI). These schemes provide a complete information report for each sample.

[0166] Various implementations complement the very rich datasets generated by mass spectrometry imaging (MSI), which may have hundreds or thousands of peaks per pixel out of tens of thousands of pixels. These implementations contribute to improved understanding of MSI data and molecular identification.

[0167] Precise mass can be provided by mass spectrometry imaging (MSI) data, and can be analyzed to identify candidate molecules with spectral characteristics present in the data. Various implementations can provide further information, such as molecular fragmentation patterns, which can be used for more confident confirmation.

[0168] Various implementation schemes can be automated and can be configured to allow MS / MS data to be collected in the same experimental acquisition as mass spectrometry imaging (MSI) data. This provides a particularly simple procedure for obtaining both MS / MS and mass spectrometry imaging (MSI) data.

[0169] Various implementations allow peaks within spatially resolved data to be annotated with molecular identity. This allows users to directly determine the spatial distribution of a specific compound (e.g., xanthine) in their sample (rather than simply being able to identify a peak with high intensity in a specific region at a specific mass-to-charge ratio (such as m / z 151)).

[0170] Based on various implementation schemes, the applicant has recognized that surface desorption electrospray ionization (DESI) mass spectrometry analysis allows for resampling of the same surface with comparable response. The applicant also recognizes that time-of-flight mass spectrometry can have sufficient mass accuracy and resolution to provide a list of possible molecular identifications based on database matching.

[0171] Therefore, according to various implementation schemes, the time-of-flight mass spectrometer operates in data-directed acquisition (DDA) mode to collect a large number of MS / MS spectra at a high sampling rate (as described above).

[0172] As mentioned above, the MS / MS spectra to be collected can be guided by measurement scans or an externally constructed list. Similarly, as mentioned above, the exclusion list for the DDA algorithm can be constructed in real time.

[0173] Various implementation schemes provide imaging experiment workflows that include the following steps.

[0174] Before sample imaging, known compounds can be analyzed as lock-masses. These known compounds can be located, for example, at a reference point on the imaging stage. Alternatively, they can be introduced into the DESI spray solvent. Other lock-mass arrangements are also possible.

[0175] The sample can then be imaged using DESI (or MALDI, REIMS, etc.) mass spectrometry imaging, and locked mass acquisition can be performed periodically as needed.

[0176] Optionally, a lightweight representation of the data can be created during the analysis so that it can be evaluated when the main analysis is completed.

[0177] After imaging, a lock-on quality analysis can be performed again.

[0178] The data can then be processed and evaluated to prepare for subsequent DDA analysis.

[0179] Based on the results of the processing and evaluation steps, DDA analysis can be performed on the same sample.

[0180] Before performing DDA analysis, the data can be automatically statistically analyzed, and decisions can be made about the sample.

[0181] For example, there may be an integer number of distinct regions in the sample, and each region can be analyzed using an exhaustive exclusion list method (as described above), in which multiple sets of measurements and MS / MS analyses are performed until no new (interesting) peaks are found.

[0182] Figure 3A An exemplary workflow based on this implementation scheme is described. For example... Figure 3A As shown, samples were analyzed in the imaging experiment, and the resulting data were analyzed to identify three regions, R1, R2, and R3.

[0183] Then, each of the three regions undergoes Directional Analysis (DDA). This is done by calculating the analytical path for each region and then repeatedly switching between precursor ion measurement scan (mass spectrometry) and tandem mass spectrometry (MS / MS) operating modes (as described above) to analyze the sample along said path. An exclusion list method is used, thereby adding an exclusion list after each measurement scan.

[0184] Alternatively, there may be an integer number of highly interesting peaks identified through statistical analysis of the imaging data, and these peaks can be used to create a list of DDAs (as described above).

[0185] Figure 3B An exemplary workflow based on this implementation scheme is described. For example... Figure 3B As shown, the sample was analyzed in an imaging experiment, and the resulting data was analyzed to identify three regions, R1, R2, and R3. The data from the imaging experiment was then used to determine the precursor ions for which product ion data were needed, and these precursor ions were then analyzed to obtain MS / MS data.

[0186] exist( Figure 3B In the second method of generating a peak list from imaging data, it is expected that all possible peaks of interest are present in the imaging data. A spectrum can be generated for each identified region (e.g., by reducing the imaging data), and a peak list can be generated for each region from the spectrum of said region. Any suitable method can be used to create a peak list for each region for MS / MS analysis, as there may be sufficient quality and time for data processing.

[0187] However, in cases where imaging data needs to be processed at high spatial resolution, it may be desirable to acquire the imaging data at a high scan rate. In such cases, even when data from all pixels in a region are summed, some composite peaks may be lost due to noise. Therefore, in these situations, it may be desirable to use the first method ( Figure 3ASimilarly, if the region has already been identified by optical or spectroscopic methods (as described above), then the first "true DDA" method can be used because there is no prior knowledge of the mass spectrometric response of the sample.

[0188] Figure 4 Exemplary data processing workflows according to various implementation schemes are shown. For example... Figure 4 As shown, data can be reduced, for example, by using adjustable principal component analysis (PCA) (step 100), followed by adjustable uniform manifold approximation and projection (UMAP) or T-distributed random neighborhood embedding (t-SNE) (step 110). For each identified region (step 120), a DDA analysis can be performed on each region using an exclusion list method (step 130).

[0189] Alternatively, a logarithmic multiple change graph can be generated (step 140), and DDA analysis can be performed using the highest multiple change peak (step 150).

[0190] DDA analysis can be performed by moving the sample under the analytical probe, while ensuring that the same chemically distinct regions of the sample are analyzed (as described above). The control system 50 can define the coordinates of the route to be taken and can synchronize the stage mode with the MS analysis.

[0191] It should be understood that the additional time for DDA analysis will be a fraction of the total time required for analysis, and the data size of the DDA file will be much smaller than that of the complete imaging data. Therefore, the implementation method produces minimal overhead while providing significant benefits.

[0192] This is due to Figure 5 Explanation. For example... Figure 5 As shown, the initial imaging experiment may take approximately tens of minutes or several hours (e.g., about 1 hour). However, for each region, the subsequent DDA analysis may take approximately a few minutes (e.g., about 7 minutes).

[0193] Similarly, Figure 5 As explained, various implementation schemes ensure that users can not only submit mass-to-charge ratio (m / z) values ​​of interest to match with known molecular databases, but they will also have MS / MS data of these peaks to help identify specific molecules attributed to the MSI distribution.

[0194] Although the invention has been described with reference to preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the invention as set forth in the appended claims.

Claims

1. A method for analyzing a sample, the method comprising: The sample is analyzed in the first operating mode to generate a spatially resolved dataset representing the sample; Analyze the dataset to identify one or more chemically distinct and substantially homogeneous regions of the sample; and For each identified region in one or more identified regions of the sample, a tandem mass spectrometry (MS / MS) dataset of the region is generated by the following operation: Determine the path through the area; and Analyzing the sample along the path by analyzing the sample using desorption electrospray ionization ("DESI") along at least a portion of the path in tandem mass spectrometry (MS / MS) operating mode, wherein analyzing the sample along the path includes: The sample is analyzed along the first part of the path in mass spectrometry operation mode to generate mass spectrometry data; Based on the mass spectrometry data, one or more precursor ions of interest are identified; then... The sample is analyzed by analyzing ions corresponding to one or more identified precursor ions of interest along a second, different portion of the path in the tandem mass spectrometry (MS / MS) operating mode.

2. A method for analyzing a sample, the method comprising: The sample is analyzed in the first operating mode to generate a spatially resolved dataset representing the sample; Analyze the dataset to identify one or more chemically distinct and substantially homogeneous regions of the sample; For each of one or more identified regions of the sample, multiple precursor ions of interest are determined based on the spatially resolved dataset; For each identified region in one or more identified regions of the sample, a tandem mass spectrometry (MS / MS) dataset of the region is generated by the following operation: Determine the path through the area; and The sample is analyzed along the path by using desorption electrospray ionization ("DESI") along at least a portion of the path in tandem mass spectrometry (MS / MS) operating mode. Analyzing the sample along the path includes analyzing the sample along the path in the tandem mass spectrometry (MS / MS) operating mode by analyzing ions corresponding to a plurality of precursor ions of interest determined for the region, and wherein, as the analysis of the sample moves along the path, different precursor ions from the plurality of precursor ions of interest are sequentially selected for analysis.

3. The method of claim 1, further comprising, for each of the one or more identified regions of the sample, determining a plurality of precursor ions of interest, wherein as the analysis of the sample moves along the path, different precursor ions from the plurality of precursor ions of interest are sequentially selected for analysis.

4. The method according to claim 1 or 2, wherein the first operating mode is a data independent analysis (DIA) operating mode.

5. The method according to claim 1 or 2, wherein the spatially resolved dataset comprises a mass spectrometry image of the sample.

6. The method according to claim 1 or 2, wherein the spatially resolved dataset comprises an optical image, IR image, or UV image of the sample.

7. The method according to claim 1 or 2, wherein the spatially resolved dataset comprises a fluorescence image of the sample or a Raman spectral image of the sample.

8. The method of claim 1 or 2, wherein analyzing the sample along the path comprises: When analyzing the sample along the path, the operation mode is switched multiple times between mass spectrometry ("MS") and tandem mass spectrometry ("MS / MS") mode. Based on the results of each corresponding mass spectrometry ("MS") operating mode, determine one or more precursor ions of interest to be analyzed in the subsequent tandem mass spectrometry ("MS / MS") operating mode; and Each of the identified precursor ions of interest is analyzed in the subsequent tandem mass spectrometry ("MS / MS") operation mode to generate a product ion spectrum for each precursor ion.

9. The method of claim 8, wherein the precursor ion of interest that has been analyzed in a previous tandem mass spectrometry ("MS / MS") operating mode is excluded from analysis in any subsequent tandem mass spectrometry ("MS / MS") operating mode.

10. The method according to claim 1 or 2, wherein analyzing the sample in tandem mass spectrometry (MS / MS) operation mode comprises: Select at least one parent ion of interest; The at least one parent ion is activated, fragmented, or reacted to generate a product ion; and The product ions are analyzed to generate a product ion dataset for the at least one precursor ion of interest.

11. The method of claim 1 or 2, the method further comprising associating the tandem mass spectrometry (MS / MS) dataset of each region with spatially resolved mass spectrometry data of the region to generate a spatially resolved mass spectrometry dataset representing the sample, the spatially resolved mass spectrometry dataset being supplemented with a tandem mass spectrometry (MS / MS) dataset of each of the one or more identified regions of the sample.

12. The method according to claim 1 or 2, the method further comprising analyzing mass spectrometry data and tandem mass spectrometry (MS / MS) data of the region to classify the region.

13. An analytical instrument, the analytical instrument comprising: A desorption electrospray ionization ("DESI") ion source, the desorption electrospray ionization ion source being configured to generate ions from a sample; One or more analyzers configured to analyze samples; and The control system is configured to cause the analytical instrument to: The sample is analyzed in the first operating mode to generate a spatially resolved dataset representing the sample; Analyze the dataset to identify one or more chemically distinct and substantially homogeneous regions of the sample; and For each identified region in one or more identified regions of the sample, a tandem mass spectrometry (MS / MS) dataset of the region is generated by the following operation: Determine the path through the area; and The sample is analyzed along the path by using a desorption electrospray ionization ("DESI") ion source along at least a portion of the path in tandem mass spectrometry (MS / MS) operating mode. The control system is configured to cause the analytical instrument to analyze the sample along the path by: The sample is analyzed along the first part of the path in mass spectrometry operation mode to generate mass spectrometry data; Based on the mass spectrometry data, one or more precursor ions of interest are identified; Then The sample is analyzed by analyzing ions corresponding to one or more identified precursor ions of interest along a second, different portion of the path in the tandem mass spectrometry (MS / MS) operating mode.

14. An analytical instrument, the analytical instrument comprising: A desorption electrospray ionization ("DESI") ion source, the desorption electrospray ionization ion source being configured to generate ions from a sample; One or more analyzers configured to analyze samples; and The control system is configured to cause the analytical instrument to: The sample is analyzed in the first operating mode to generate a spatially resolved dataset representing the sample; Analyze the dataset to identify one or more chemically distinct and substantially homogeneous regions of the sample; For each of the one or more identified regions of the sample, multiple precursor ions of interest are determined based on the spatially resolved dataset; and For each of the one or more identified regions of the sample, a tandem mass spectrometry (MS / MS) dataset of the region is generated by the following operation: Determine the path through the area; and The sample is analyzed along the path by analyzing the sample using a desorption electrospray ionization ("DESI") ion source along at least a portion of the path in tandem mass spectrometry (MS / MS) operating mode, wherein the control system is configured to cause the analytical instrument to analyze the sample along the path in the tandem mass spectrometry (MS / MS) operating mode by analyzing ions corresponding to a plurality of precursor ions of interest determined for the region, and wherein as the analysis of the sample moves along the path, different precursor ions among the plurality of precursor ions of interest are sequentially selected for analysis.

15. The analytical instrument of claim 13, wherein the control system is configured to determine a plurality of precursor ions of interest for each of the one or more identified regions of the sample, and wherein, as the analysis of the sample moves along the path, different precursor ions of interest are sequentially selected for analysis.

16. The analytical instrument according to claim 13 or 14, wherein the analytical instrument includes a mass analyzer.

17. The analytical instrument according to claim 13 or 14, wherein the analytical instrument further comprises a filter and activation, impact, fragmentation or reaction devices.

18. The analytical instrument according to claim 13 or 14, further comprising a sample stage and analytical probes; wherein: The sample stage is configured to move relative to the analytical probe, and / or the analytical probe is configured to move relative to the sample stage.

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