Biological Identification Using Low Resolution Tandem Mass Spectrometry

Abstract

A system and device are disclosed that generate multidimensional mass fingerprints that allow identification on low-resolution mass spectrometers with post-ionization fragmentation. In this approach, rather than interrogating samples processed into peptides using a single high-resolution MS scan as in traditional fingerprinting, the raw raw sample (containing all biochemical species such as lipids, proteins, peptides and metabolites) is analyzed by combining matrix-assisted laser dissociation ionization (MALDI) ionization with low-resolution tandem mass spectrometry. The proposed system combines improvements in MS hardware and software with state-of-the-art machine learning (ML) approaches to pioneer rapid biological detection. The technique does not require prior separation (liquid or gas chromatography) and is therefore rapid (e.g., less than 5 seconds) and amenable to high throughput (e.g., more than 384 samples / hour).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 171,767, filed April 7, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] The past two decades have seen a proliferation of new rapid detection and diagnostic assays that can be used in the field or in the clinic. Most of these technologies are antibody-based, have many false positives and negatives, and require prior knowledge of the causative organism. These detection methods are easily defeated by genetic manipulation, making them a weak link in defenses against biological weapons and emerging pathogens. Modern genomic techniques, such as PCR, address the specificity issue, but are costly to implement, require a significant amount of sample preparation, must be performed in a brick-and-mortar laboratory, and can produce false negatives through genetic manipulation or naturally acquired mutations. Taken together, these constraints limit throughput and are far from real-time, with results not available within 4–5 hours after sampling. Summary of the Invention

[0003] Multidimensional mass fingerprinting is disclosed that allows identification on low-resolution mass spectrometers with post-ionization fragmentation. In this approach, rather than interrogating samples processed into peptides / proteins using a single high-resolution MS scan as in traditional fingerprinting, the raw raw sample (containing all biochemical species such as lipids, proteins, peptides and metabolites) is analyzed by combining ionization with low-resolution tandem mass spectrometry. The proposed system combines improvements in MS hardware and software with state-of-the-art machine learning (ML) approaches to pioneer rapid biological detection. Central to these efforts is the creation of multidimensional mass fingerprints consisting of a parent mass scan (MS1) and a series of fragment mass scans (MS2), all derived from a single sample, ideally with minimal processing to reduce variability due to sample handling. The technique does not require prior separation (liquid or gas chromatography), and is therefore rapid (e.g., less than 5 seconds) and amenable to high throughput (e.g., more than 384 samples / hour).

[0004] As shown in Figure 1, the system and method involves acquiring and recording a mass scan (MS1) of a sample of interest introduced into a mass spectrometer by an ionization technique such as Matrix-Assisted Laser Dissociation Ionization (MALDI), Electrospray Ionization (ESI), Nanospray Ionization (NSI), Desorption Electrospray Ionization (DESI) or Paper Spray Ionization (PSI). This is referred to herein as the "first dimension." An example MS1 scan is shown in Figure 2.

[0005] The systems and methods further include acquiring and recording a series of fragment mass scans (MS2) from graduated predefined mass windows, referred to herein as the "second dimension." These mass windows can vary in size, for example, from a single ion to the entire MS1 mass range. In some embodiments, the windows are about 100-200 m / z. The windows can be uniform or of various sizes. The windows can span the entire spectrum or can be focused on information-rich regions of the MS1 scan. In some embodiments, the windows are non-overlapping, as shown in FIG. 3. In other embodiments, the windows are overlapping, as shown in FIG. 4.

[0006] In embodiments, non-limiting examples of mass spectrometry include secondary ion mass spectrometry, laser desorption mass spectrometry, matrix-assisted laser desorption / ionization mass spectrometry, electrospray mass spectrometry, or desorption electrospray ionization.

[0007] Finally, the system and method further includes combining the MS1 and MS2 spectral data to generate a unique multidimensional fingerprint. The unique mass fingerprint can be composed of all scans made, and once generated, must remain fixed for comparison, as shown in Figure 5. This mass fingerprint becomes part of a library and can be compared to unknown samples for species and strain level identification. Compiling this library is also likely to identify unique features at both the MS1 and MS2 levels that allow for putative identification (e.g., at the genus or species level) of organisms not in the library.

[0008] In some embodiments, these data are analyzed with machine learning to match the fingerprints to specific organisms (species / strains) or biomaterials. Thus, the disclosed systems and devices can further include a database of mass fingerprints generated by the disclosed methods for various organisms and / or biomaterials.

[0009] The disclosed systems and methods can be used to provide species level identification and fingerprints of bacteriological species including proteins, peptides, lipids, and other molecules of interest that directly correlate to growth conditions, as shown in FIG.

[0010] The disclosed methods can be used to detect and identify the presence of any biological or chemical entity, including chemicals, compounds, cells or organisms, in a sample. This can be done for diagnostic purposes in the case of pathogens or diseases (e.g., cancer). The disclosed systems and methods can be used, for example, in conjunction with tissue biopsies. This is useful in that it replaces traditional tissue staining and microscopy techniques that rely on human judgment.

[0011] In some embodiments, the disclosed systems and methods are used to detect and identify harmful chemicals or organisms in samples from subjects or the environment, including pure / cultured samples or clinical samples with little or no prior sample preparation.

[0012] In some embodiments, the disclosed systems and methods are used to identify the presence of harmful biological or chemical agents.

[0013] In some embodiments, the disclosed systems and methods are used to diagnose infection or chemical exposure for point-of-care purposes.

[0014] In some embodiments, the disclosed systems and methods can be used to provide a fingerprint of a tissue sample containing proteins, peptides, lipids, and other molecules of interest that directly correlate with disease states, including but not limited to cancer, diabetes, and infectious diseases.

[0015] In some embodiments, the disclosed systems and methods can incorporate spatial analysis of biological samples with the aim of correlating molecular fingerprints with specific cell types or tissue substructures. This correlation can be achieved in a physician-guided targeted experiment. Alternatively, a regular sample raster can be used to interrogate the entire tissue surface and render images of each component of the molecular fingerprint.

[0016] In embodiments, the methods described herein may further include obtaining a sample from said subject. For example, the subject may be a mammalian subject, such as a human subject. In some embodiments, the mammal may be a dog, cat or horse. In this example, a tissue sample may be obtained from the subject and tested for melanoma biomarkers using the methods described herein. The tissue may be obtained using methods known in the art.

[0017] In embodiments, the methods described herein may further include performing mass spectrometry of a specimen having a known biological state. For example, a known cancerous lesion may be obtained from a subject whose sample tissue is being tested using the embodiments and methods described herein. In embodiments, the methods described herein may further include performing histological analysis on the tissue sample. Additionally, the embodiments described herein may also include performing immunohistochemical analysis on the tissue sample. The embodiments described herein may determine the diagnosis, prognosis, or treatment approach of a subject. The embodiments described herein may be used in combination with other methods known in the art to determine the diagnosis, prognosis, or treatment approach of a subject.

[0018] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 illustrates multidimensional mass fingerprinting. To obtain a multidimensional fingerprint, a primary MS1 scan is acquired very similar to the conventional process. A large mass region of interest (approximately 100-200 m / z) is then isolated and fragmented. The fragments are then "read / scanned" by performing an MS2 scan. This sequence of events can then be repeated for other ion regions of interest within the initial MS1 scan until the sample is exhausted or all desired data has been acquired. All spectral data was then processed and compiled to generate a new fingerprint. By adding the second dimension of information (MS2), the molecular detail acquired increases by at least an order of magnitude, allowing identification of distortion levels using low-resolution portable mass spectrometers. [Diagram 2] FIG. 2 shows an example of a full mass scan obtained on a low resolution instrument. [Diagram 3] FIG. 3 shows an example of non-overlapping isolation windows of an MS2 scan shown on an MS1 ​​scan. [Figure 4] FIG. 4 shows an example of overlapping isolation windows for an MS2 scan. [Diagram 5] Figure 5 shows an example of a final multidimensional mass fingerprint, which is a library of masses acquired at the MS1 and MS2 levels. This library can be indexed and the unique masses at each level can be identified. [Figure 6] Figure 6 shows that two strains each of Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus were analyzed using both the one-dimensional and two-dimensional approaches defined above. The discrimination of each organism was improved by incorporating a second-dimensional scan using unsupervised classification approaches, principal component analysis and hierarchical clustering. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0021] Where a range of values ​​is stated, each intervening value, to one-tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value within that range, unless the context clearly dictates otherwise, is understood to be encompassed within the scope of the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the scope of the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0023] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein as disclosed and described in connection with the methods and / or materials for which the publications are cited. The citation of a publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0024] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. A described method may be carried out in the order of events described or in any other order which is logically possible.

[0025] The embodiments of the present disclosure employ, unless otherwise indicated, chemical, biological and other techniques within the skill of those in the art.

[0026] The following examples are presented to provide a complete disclosure and description to one of ordinary skill in the art of how to carry out the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature is 20°C, and standard pressure is 1 atmosphere.

[0027] Before describing embodiments of the present disclosure in detail, it is to be understood that, unless otherwise indicated, the disclosure is not limited to particular materials, reagents, reactants, manufacturing processes, etc., and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure to carry out steps in differing order where this is logically possible.

[0028] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0029] Mass spectrometry techniques have been demonstrated to have best-in-class sensitivity and specificity for pathogen detection and identification in clinical settings, utilizing an approach known as mass fingerprinting. Briefly, proteins are extracted from clinical samples, processed into peptides, and then broken down by parent mass scanning (MS1) in a high-resolution mass spectrometer to obtain a unique “mass fingerprint.” These FDA-approved instrument platforms are not designed to operate in the harsh environments required for widespread deployment to the frontline or in point-of-care settings. As a direct result, diagnostic mass spectrometry techniques are limited to brick-and-mortar centralized laboratories with highly trained staff. Furthermore, similar to genomic sequencers, the cost of these systems typically exceeds $350,000, preventing large-scale deployment. With current technology, the combination of high cost and complexity has slowed the dissemination of much-needed real-time information on pathogen exposure. Although low-cost portable MS systems exist today, the majority were developed for the detection and quantification of chemical warfare agents. The need for portability necessitated compromises in performance. These compromises have prevented the technology from making inroads in the biological detection field. Despite some technical improvements over the past five years, significant fundamental limitations in instrument mass range and resolution remain. Traditional Mass Fingerprinting However, this remains a barrier to using the approach to detect biomolecules specific to bacteria or viruses.

[0030] Disclosed herein are systems and methods that leverage and improve upon existing portable miniaturized instrument designs, using low-resolution tandem mass spectrometry to obtain high-fidelity data that can be used to perform biological identification in complex environmental and clinical settings without sample preparation, thus replacing the need for expensive high-resolution mass spectrometry.

[0031] To overcome the limitations of current state-of-the-art miniaturized mass spectrometry to achieve bacterial and viral identification, multidimensional mass fingerprints that enable identification can be obtained with low-resolution mass spectrometers with post-ionization fragmentation. In this approach, rather than interrogating samples processed into peptides with a single high-resolution MS scan as in traditional fingerprinting, the disclosed system and method analyzes the raw, unprocessed sample (containing all biochemical species such as lipids, proteins, peptides and metabolites) by combining ionization with low-resolution tandem mass spectrometry. The proposed effort aims to combine improvements in MS hardware and software with machine learning (ML) approaches to usher in rapid biological detection.

[0032] Central to these efforts is the creation of a multidimensional mass fingerprint composed of a parent mass scan (MS1) and a series of fragment mass scans (MS2), all obtained from a single sample. This technique does not require prior separation (liquid or gas chromatography), and is therefore rapid (e.g., less than 5 seconds) and amenable to high throughput (e.g., greater than 384 samples / hour). One embodiment of this approach is shown in Figure 1.

[0033] Mass spectrometry is an analytical technique that can ionize a sample into charged molecules and measure their mass-to-charge ratio (m / z). In some embodiments, the ion source for mass spectrometry is matrix-assisted laser desorption / ionization (MALDI).

[0034] MALDI is a soft ionization technique that uses a laser to irradiate a matrix of small molecules, bringing the analyte molecules into the gas phase without fragmenting or decomposing them. Some biomolecules are too large to be decomposed by heating, and conventional techniques fragment or destroy the macromolecules. MALDI is suitable for the analysis of biomolecules such as peptides, lipids, sugars, or other organic polymers.

[0035] The MALDI process is described in further detail in International Publication No. WO 2009 / 065580 A1 to Ulrich Weller, entitled "Identification of Pathogens in Bodily Fluids," the contents of which are incorporated herein by reference in their entirety.

[0036] In some embodiments, the analytes are embedded in a very large excess of matrix compounds deposited on a solid surface, called a target, which is usually made of a conductive metal and has spots for several different samples to be applied. After a very short laser pulse, the illuminated spot is rapidly heated and vibrationally excited. The matrix molecules are energetically ablated from the sample surface, absorbing the laser energy and carrying the analyte molecules into the gas phase as well. During the ablation process, the analyte molecules are usually ionized by being protonated or deprotonated by nearby matrix molecules. The most common MALDI ionization format is that the analyte molecules carry a single positive charge.

[0037] Lasers used in MALDI come in a variety of types, including lasers of both ultraviolet (UV) and infrared (IR) wavelengths, but UV lasers are the most common light source for analytical MALDI. Of these, nitrogen lasers and frequency tripled or quadrupled Nd:Yag lasers are often useful for the majority of applications. IR-MALDI is dominated by Er:Yag lasers, although TEA-CO2 lasers may also be used.

[0038] The primary function of the matrix is ​​believed to be essentially to dilute and separate the analyte molecules from one another. This occurs during the evaporation of the solvent and the concomitant formation of a solid solution. It then acts as a mediator for the absorption of energy upon laser irradiation. Selection of the appropriate matrix is ​​key to the success of MALDI. In general, highly polar analytes work better with highly polar matrices, and non-polar analytes are preferably combined with non-polar matrices. A variety of matrices such as nicotinic acid, picolinic acid, 3-hydroxypicolinic acid, 3-aminopicolinic acid, 6-aza-2-thiothymine, 2,5-dihydroxybenzoic acid, DHB-based mixtures, 3-aminoquinoline, α-cyano-4-hydroxycinnamic acid, 4-chloro-α-cyano-peroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, 2-(4-hydroxyphenylazo)benzoic acid, 2-mercaptobenzothiazole, 5-chloro-2-mercaptobenzothiazole, 2,6-dihydroxyacetophenone, 2,4,6-trihydroxyacetophenone, dithranol (1,8,9-anthracenetriol), 9-nitroanthracene, benzo[a]pyrene, and 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile are in demand and widely used. Currently, the most commonly used matrices are α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and 2,6-dihydroxyacetophenone.

[0039] In some embodiments, tandem mass spectrometry is used to measure their mass-to-charge ratio (m / z). Tandem mass spectrometry, also known as MS / MS, is a technique in instrumental analysis that couples two or more mass spectrometers using additional reaction steps to increase the ability to analyze chemical samples. Once the analytes are ionized, a first spectrometer (called MS1) separates these ions by their mass-to-charge ratio (often given as m / z or m / Q). Ions of a particular m / z ratio from MS1 are selected and then split into smaller fragment ions, for example by collision-induced dissociation, ion-molecule reactions or photodissociation. These fragments are then introduced into a second mass spectrometer (MS2), which then separates the fragments by their m / z ratio and detects them. The selection-fragmentation-detection sequence can be further extended to first generation product ions. For example, the selected product ions generated in MS2 can be further fragmented to generate another group of product ions (MS3), etc. The fragmentation step makes it possible to identify and separate ions with very similar m / z ratios in a conventional mass spectrometer.

[0040] Tandem MS involves three distinct steps: selection, fragmentation, and detection, and the separation of these three steps can be achieved spatially or temporally. Representative spatial tandem MS instruments include QqQ, QTOF, and hybrid ion trap / FTMS, while representative temporal tandem (Tandem-in-Time) MS / MS instruments include ion trap and FT-ICR MS.

[0041] Precursor ions can be activated (by increasing their internal energy) in a wide variety of ways. The fragmentation pattern depends on how energy is transferred to the precursor ion, how much energy is transferred, and how the transferred energy is distributed internally. Collision-induced dissociation and infrared multiphoton dissociation are "slow heating" techniques that increase the Boltzmann temperature of the ion and therefore preferentially cleave the weakest bonds to produce mainly b and y ions. These techniques are very efficient for peptides, lipids and other relatively small compounds, but may remove post-translational modifications of proteins (e.g., phosphates and sugars). Electron capture dissociation and electron transfer dissociation produce mainly c and z ions while retaining post-translational modifications (PTMs). Thus, ECD and ETD have been widely applied to proteins and peptides with labile PTMs, and ECD / ETD can also produce cross-ring cleavage a and z ions that are important for the localization of glycosidic bonds.

[0042] Tandem mass spectrometry includes triple quadrupole mass spectrometers (QqQ), quadrupole time-of-flight (Q-TOF), and hybrid mass spectrometers. Triple quadrupole mass spectrometers use the first and third quadrupoles as mass filters. As the analyte passes through the second quadrupole, it undergoes fragmentation due to collisions with gas. It is typically used in the pharmaceutical industry. Quadrupole time-of-flight (Q-TOF) mass spectrometers combine TOF and quadrupole instruments, resulting in high mass accuracy of product ions, accurate quantification capabilities, and applicability of fragmentation experiments. It is a mass spectrometry method that determines ion fragmentation (m / z) ratios by time-of-flight measurements. Hybrid mass spectrometers consist of more than two mass analyzers.

[0043] In some embodiments, the spectrometers used in the disclosed systems and methods are low resolution instruments that typically distinguish between ions with mass differences of 0.2 mass units (mu) (for M=200, R≈1000), whereas high resolution instruments can distinguish between ions with mass differences of 0.0001 mu (for M=200, R≈2,000,000).

[0044] The disclosed method includes generating a multidimensional fingerprint from a combination of MS1 mass spectral data and MS2 mass spectral data. In some embodiments, this is done using a machine learning classification model. In some implementations, the machine learning classification model includes a convolutional neural network classifier. In embodiments, the sample includes a desired body tissue, non-limiting examples of which include blood, serum, cerebrospinal fluid, urine, sweat, saliva, skin, skin punch, or solid tissue biopsy. In embodiments, the multidimensional fingerprint is also known as a molecular profile. In embodiments, the multidimensional fingerprint includes one molecule or multiple molecules. Non-limiting examples of types of molecules represented in the mass spectrometry profile include proteins, peptides, lipids, metabolites, nucleic acids, or combinations thereof. In embodiments, the similarity of the sample from the subject to a known profile, e.g., a known molecular profile, is determined using a statistical algorithm or a machine learning algorithm. Non-limiting examples of machine learning algorithms include a genetic algorithm, a support vector machine, or a supervised neural network. In some embodiments, similarity is determined by assessing whether peaks appearing in a mass spectrometry profile obtained from a sample subject overlap with peaks appearing in mass spectrometry profiles obtained from known samples, control samples, and / or reference samples.

[0045] The machine learning methods and statistical approaches used for classification are described in U.S. Pat. No. 10,877,040, U.S. Pat. No. 8,822,159, and U.S. Patent Publication No. 20190391157, which are incorporated herein by reference for the teachings of these methods.

[0046] In some embodiments, the generated fingerprint is matched to known fingerprints to identify a particular entity. In other embodiments, the fingerprint is unknown, but compared to similar fingerprints to provide a classification, such as a family or genus, of the organism. In some embodiments, fingerprints are generated for groups of organisms based on biological similarities. For example, fingerprints can determine whether a bacterium is gram positive or gram negative.

[0047] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated herein by reference.

[0049] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A method for analyzing a biological analyte, comprising: (a) ionizing a biological analyte to produce an ion mixture; (b) acquiring and recording a parent mass spectrum of the ion mixture in a first stage mass analysis (MS1) without fragmentation; (c) acquiring and recording a series of mass spectra of the ion mixture in a second stage mass analysis (MS2) involving fragmentation using a predefined mass window within the parent spectrum; and (d) generating a multidimensional fingerprint from the combination of the MS1 mass spectral data and the MS2 mass spectral data. A method comprising:

2. The method of claim 1 , wherein the biological analyte is derived from a pathogenic bacterium or virus.

3. The method of claim 1 , wherein the biological analyte is derived from a tissue sample.

4. 10. The method of claim 1, wherein the biological analyte is derived from one or more of the following sources: a mammal, a microorganism, or an exogenous compound.

5. 10. The method of claim 1, wherein the biological analytes represent a mixture of one or more of proteins, peptides, lipids, metabolites, nucleic acids, drugs, toxins or other exogenous molecules.

6. The method of claim 1 , wherein the biological analyte comprises a bacterial or viral pathogen.

7. The method of any one of claims 1 to 6, wherein the predefined mass window is approximately in the order of magnitude of 100-200 m / z.

8. The method of any one of claims 1 to 6, wherein the predefined mass windows are non-overlapping.

9. The method of any one of claims 1 to 6, wherein the predefined mass windows overlap.

10. The method of any one of claims 1 to 6, wherein step (c) is repeated for at least five predefined mass windows within the parent spectrum.

11. The method according to any one of claims 1 to 6, wherein steps (a) and (b) are obtained using low resolution tandem mass spectrometry.

12. 1. A method for analyzing a biological analyte, comprising: (a) selectively ionizing spatially defined cell types from a region of interest; (b) ionizing the biological analyte to produce an ion mixture; (c) acquiring and recording a parent mass spectrum of the ion mixture in a first stage mass analysis (MS1) without fragmentation; (d) acquiring and recording a series of mass spectra of the ion mixture in a second stage mass analysis (MS2) involving fragmentation using a predefined mass window within the parent spectrum; and (e) generating a multidimensional fingerprint from the combination of the MS1 mass spectral data and the MS2 mass spectral data. A method comprising:

13. The method of claim 12, wherein the spatial information is used to classify a disease state of cells within the defined region of interest.

14. The method of claim 12 , wherein the spatial information is used to render an image of the tissue based on molecular location or disease state.

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