Methods for detecting metabolites using microfluidic-based CE-MS systems

Abstract

The present disclosure relates to methods for detecting metabolites using a microfluidic capillary electrophoresis-mass spectrometry (CE-MS) system. These metabolites may be used to diagnose diseases or conditions, and to monitor the therapeutic efficacy of compounds used to treat such diseases or conditions.

Description

Technical Field

[0001] The present disclosure relates to methods for detecting metabolites using a capillary electrophoresis-mass spectrometry (CE-MS) system. These metabolites can be used to diagnose diseases or conditions, including airway inflammatory diseases, cough, heart disease, eye disease, neurodegenerative diseases, mental illness, neuropathic pain, chronic inflammatory diseases, metabolic diseases or cancer, and to monitor the therapeutic efficacy of compounds used to treat these diseases or conditions. Background Art

[0002] Many metabolites are known to be associated with the etiology of disease. For example, abnormal nucleotide concentrations are associated with many diseases. Adenosine 5'-triphosphate (ATP), especially extracellular ATP (eATP), is not only associated with airway inflammatory diseases and cough, but blocking its extracellular release has been shown to produce therapeutic benefits. These data support the view that eATP may be a key driver of the symptoms and pathogenesis of airway diseases. However, the detection methods of eATP remain problematic.

[0003] ATP is a complex nucleoside triphosphate consisting of the nitrogenous base adenine, ribose, and a triphosphate chain. It is easily catalyzed to ADP, AMP, cAMP, adenosine, and other downstream metabolites, such as inosine. Unfortunately, ATP and similar nucleotide analogs are poorly retained by conventional reversed-phase liquid chromatography (RPLC); do not migrate in coated chip capillary electrophoresis applications; are unstable and subject to interconversion from enzymes, pH, and / or temperature; have multiple pKas; and are metal-sensitive analytes.

[0004] Historically, ion-pairing chromatography or passivation have provided solutions for separating challenging compounds such as ATP, however they can be problematic for liquid chromatography / mass spectrometry (LC / MS) systems. Furthermore, common ATP luminescence-based assays measure ATP levels indirectly through enzymatic degradation and lack a simultaneous readout of its analogs. Therefore, analytical techniques for detecting these challenging metabolites remain underdeveloped. Summary of the invention

[0005] The present disclosure relates to a method for detecting metabolites of interest in a sample, the method comprising: (a) contacting a sample containing one or more metabolites of interest with an uncoated capillary electrophoresis (CE) platform; (b) separating the metabolites by molecular weight and / or charge in one or more capillaries using CE; (c) eluting the metabolites from the one or more capillaries; and (d) detecting the eluted metabolites by mass spectrometry.

[0006] The present disclosure also relates to a method for detecting metabolites of interest in a sample, the method comprising: (a) contacting a sample containing one or more metabolites of interest with a capillary electrophoresis (CE) platform having a chemically modified surface; (b) using CE to separate the metabolites by molecular weight and / or charge in one or more capillaries; (c) eluting the metabolites from the one or more capillaries; and (d) detecting the eluted metabolites by mass spectrometry.

[0007] In one aspect, the CE platform is a microchip-based system. In another aspect, the microchip-based CE platform integrates electrophoretic separation and electrospray ionization into a mass spectrometer.

[0008] In one aspect, the metabolites of interest are listed in Table 1. In another aspect, the metabolite of interest is an anionic metabolite. In another aspect, the metabolite is a nucleotide, a nucleotide analog, or a degradation product. In another aspect, the metabolite of interest is adenosine 5'-triphosphate (ATP).

[0009] In one aspect, the sample is blood, plasma, cells or lavage samples. In another aspect, the sample is bronchoalveolar lavage fluid (BALF). In another aspect, the sample comprises a chelating agent. In another aspect, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0010] The present disclosure also relates to a method for diagnosing a disease or condition associated with abnormal nucleotide-dependent signaling in a subject, the method comprising: (a) contacting a sample from the subject with a microchip-based capillary electrophoresis (CE) platform; (b) using CE to separate adenosine 5'-triphosphate (ATP), ATP analogs and / or degradation products by molecular weight and / or charge in one or more capillaries; and (c) eluting the ATP, ATP analogs and / or degradation products from the one or more capillaries; and (d) detecting the eluted ATP, ATP analogs and / or degradation products by mass spectrometry; wherein the microchip-based CE platform integrates electrophoretic separation and electrospray ionization into a mass spectrometer; and wherein the presence of ATP, ATP analogs and / or degradation products indicates a disease or condition associated with abnormal nucleotide-dependent signaling in the subject.

[0011] The present disclosure also relates to a method for monitoring the therapeutic benefit of a compound on a disease or condition associated with aberrant nucleotide-dependent signaling in a subject treated with the compound, the method comprising: (a) contacting a sample from the subject with a microchip-based capillary electrophoresis (CE) platform; (b) using CE to separate the ATP, ATP analogs and / or degradation products by molecular weight and / or charge in one or more capillaries; and (c) eluting the ATP, ATP analogs and / or degradation products from the one or more capillaries; and (d) detecting the eluted ATP, ATP analogs and / or degradation products by mass spectrometry; wherein the microchip-based CE platform integrates electrophoretic separation and electrospray ionization into a mass spectrometer, and wherein the presence of ATP, ATP analogs and / or degradation products indicates a disease or condition associated with aberrant nucleotide-dependent signaling in the subject.

[0012] In one aspect, the disease or condition is associated with an increase in extracellular ATP (eATP) levels. On the other hand, the disease or condition is an inflammatory disease of the airways, a cough, a heart disease, an eye disease, a neurodegenerative disease, a mental illness, a neuropathic pain, a chronic inflammatory disease, a metabolic disease, or a cancer. On the other hand, the cough is a chronic idiopathic cough. On the other hand, the chronic inflammatory disease is systemic lupus erythematosus or Crohn's disease.

[0013] In one aspect, the sample is blood, plasma, cells or a lavage sample. In another aspect, the sample is BALF. In another aspect, the sample comprises a chelating agent. In another aspect, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0014] In one aspect, the microchip is a ZipChip TM In another aspect, the microchip comprises a chemically modified surface. In another aspect, the microchip does not comprise a surface modification.

[0015] In one aspect, the method further comprises adjusting the pH of the background electrolyte (BGE) relative to the metabolite of interest prior to mass spectrometry analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Some aspects of the invention are herein described, by way of example only, with reference to the accompanying drawings.With specific reference now to the drawings in detail, it should be emphasized that the details shown are by way of example and for purposes of illustrative discussion of aspects of the invention.

[0017] Figure 1 Shown with ZipChip TM Capillary electrophoresis parameters for use with the platform.

[0018] Figure 2 Shown with ZipChip TMMass spectrometer parameters used with the platform.

[0019] Figure 3 The electropherogram showing the separation of ATP and metabolite species is shown. DETAILED DESCRIPTION

[0020] I. General Definitions

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure belongs. In the event of a conflict, the present application (including definitions) shall prevail. Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0022] Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present disclosure, suitable methods and materials are described below. Materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the specific embodiments and claims.

[0023] To further define the present disclosure, the following terms and definitions are provided.

[0024] Unless the context clearly dictates otherwise, the singular forms "a / an" and "the" include plural referents. The terms "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In some aspects, the terms "a / an" mean "single". In other aspects, the terms "a / an" include "two / or more" or "multiple".

[0025] The term "about" as used herein means approximately, roughly, about, or within a range thereof. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower limits of the numerical values ​​set forth. Generally speaking, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variation of 10% or more (higher or lower).

[0026] Throughout this disclosure, various aspects of the present invention are presented in range format. It should be understood that the description using range format is only for convenience and clarity, and should not be construed as a rigid limitation on the scope of the present invention. Therefore, the description of the range should be considered as having clearly disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, a description of a range such as from 1 to 6 should be considered as having a specifically disclosed sub-range, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the range, this applies. The numerical ranges listed include the numbers defining the range, and include each integer and fraction within the defined range.

[0027] Units, prefixes and symbols are expressed in their international system of units (Système International de Unites) (SI) acceptable form. Numerical ranges include numbers that limit the range. In the case of listing a series of values, it should be understood that each intermediate integer value and each fraction thereof between the upper and lower limits of the range, and each subrange between such values ​​are also specifically disclosed. The upper and lower limits of any range can be independently included in the range or excluded from the range, and each range including one limit, two limits, none or two limits is also included in the present disclosure. Therefore, the ranges listed herein should be understood as shorthand for all values ​​within the range, including the endpoints listed. For example, a range of 1 to 10 should be understood as including any number, combination of numbers or subranges from the following group, and the group is composed of: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0028] In the case of explicit enumeration of values, it should be understood that the value of the quantity or amount approximately the same as the enumeration value is also within the scope of the present disclosure. In the case of disclosing a combination, each sub-combination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. In contrast, in the case of separately disclosing different elements or groups of elements, their combinations are also disclosed. In the case of any element of the present disclosure being disclosed as having multiple alternatives, examples of the present disclosure in which each alternative is excluded individually or in any combination with other alternatives are also disclosed; more than one element of the present disclosure may have such exclusions, and all combinations of elements with such exclusions are hereby disclosed.

[0029] The term "and / or" when used herein should be regarded as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in this article is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0030] It should be understood that wherever herein an aspect is described using the language "comprising," other similar aspects described using "consisting of" and / or "consisting essentially of" are also provided.

[0031] II. Detection Methods

[0032] The present disclosure relates to methods for detecting metabolites of interest in a sample. In some aspects, the metabolites of interest are associated with a disease state. Therefore, in some aspects, the present disclosure relates to methods for diagnosing a disease or condition by detecting a metabolite of interest, or monitoring the therapeutic efficacy of a compound for a disease or condition.

[0033] In one aspect, the present disclosure relates to a method for detecting a metabolite of interest in a sample, the method comprising: (a) contacting a sample containing one or more metabolites of interest with an uncoated capillary electrophoresis (CE) platform; (b) using CE to separate the metabolites by molecular weight and / or charge in one or more capillaries; (c) eluting the metabolites from the one or more capillaries; and (d) detecting the eluted metabolites by mass spectrometry (MS) analysis. In some aspects, the method includes a CE platform based on an uncoated microchip. On the other hand, a microchip-based CE platform integrates electrophoretic separation and electrospray ionization into a mass spectrometer.

[0034] CE / MS systems combine capillary electrophoresis and mass spectrometry to separate and analyze samples. The CE / MS system works by first separating the ionic components of a sample by applying a voltage to the sample. Due to charge and friction, the ions will move through the capillary at different rates. The separated sample is then sprayed into a mass spectrometer, which produces a spectrum. This spectrum is used to identify the individual components of the sample. In one aspect, the microchip contains the capillary. In another aspect, CE is performed separately from the microchip.

[0035] As used herein, the term "sample" refers to a mixture of components including at least a metabolite of interest (such as ATP), which is manipulated according to the methods of the present invention, including, for example, separation, analysis, extraction or profiling.

[0036] As used herein, "metabolites" refer to endogenous compounds, such as amino acids, lipids, sugars, organic acids, etc., which are usually formed during anabolism or catabolism. Metabolites can have a variety of functions, including energy conversion, signal transduction, epigenetic influences, and cofactor activity, but their presence can also be associated with human diseases or conditions. Exemplary metabolites of the present disclosure are found in Table 1.

[0037] As used herein, "subject" refers to a mammal, such as a dog, cat, horse, or rabbit. In certain embodiments, the subject is a non-human primate, such as a monkey, chimpanzee, or gorilla. In certain embodiments, the subject is a human. "Subject" can be used interchangeably with "patient."

[0038] As used herein, "therapeutic benefit" relates to amelioration of symptoms or slowing of disease progression.

[0039] As used herein, the terms "analysis" or "analyzing" are used interchangeably and refer to any of a variety of methods for isolating, detecting, separating, purifying, solubilizing and / or characterizing metabolites of interest.

[0040] "Detect" and "detection" have their standard meanings and are intended to encompass detection, including the presence or absence, measurement, and / or characterization of a metabolite of interest (eg, ATP).

[0041] Table 1: Exemplary metabolites

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] As used herein, the terms "standard" and / or "internal standard" refer to a well-characterized substance of known quantity and / or identity (e.g., known molecular weight, electrophoretic mobility curve) that can be added to a sample, and both the standard and the molecules in the sample can be characterized by electrophoresis based on molecular weight or isoelectric point. Comparison with the standard then provides a quantitative or semi-quantitative measurement of the amount of an analyte (such as ATP) present in the sample.

[0066] As used herein, "contacting" includes bringing together at least two substances in solution or solid phase.

[0067] "Mass spectrometry" refers to a method of analyzing a sample by generating gas phase ions from the sample and then separating and detecting the gas phase ions according to their mass-to-charge ratio (m / z). Prior to detection, the sample may be subjected to one or more chromatographic separations, e.g., one or more dimensions of liquid chromatography or size exclusion chromatography.

[0068] The sample used in the disclosed method can be heterogeneous, containing multiple components, i.e., different metabolites. Alternatively, the sample can be homogeneous, containing one metabolite or substantially one metabolite of multiple charge or molecular weight species. The sample can be subjected to a pre-analytical treatment prior to detecting the metabolites.

[0069] Historically, the use of microfluidic based CE / MS systems has been a challenge in analyzing anionic substrates. However, using the methods described herein, anionic, neutral and positively charged molecules can be distinguished. In some aspects, a microchip or microfluidic based CE / MS system is used for analysis. In some aspects, the microchip is surface modified to include a substrate. An example of a microchip based CE system that can be used in series with MS is the ZipChip TM (908 Devices, Boston, MA).

[0070] In some aspects, the capillary may include a separation matrix, which may be added in an automated manner by equipment and / or system. In some aspects, the sample is loaded onto a stacker matrix before separation. In one aspect, the separation matrix is ​​a size separation matrix, and has a polymer gel similar to or substantially the same property as used in conventional electrophoresis techniques. The capillary electrophoresis in the separation matrix is ​​similar to the separation in a polymer gel (such as polyacrylamide gel or agarose gel), wherein the molecules are separated based on the size of the molecules in the sample by providing a porous channel through which the molecules can travel. The separation matrix allows separation of analytes by molecular size, because larger molecules will travel more slowly through the matrix compared to smaller molecules. In some aspects, one or more capillaries comprise a separation matrix. In some aspects, samples containing metabolites are separated or split based on molecular weight. In some aspects, the separation matrix comprises a sieving matrix, which is configured to separate proteins by molecular weight. In certain embodiments, the protein component of the sample is separated by molecular weight, and the method is a method for detecting and / or distinguishing the size variants of metabolites and their analogs or degradation products.

[0071] In some aspects, a sample containing metabolites of interest is separated or resolved based on the charge of the sample components. In some aspects, the metabolite components of a sample are separated by charge, and the method is a method of detecting and / or distinguishing charge variants of a metabolite and its analogs or degradation products.

[0072] In some aspects, an internal standard can be used to quantitatively detect a metabolite of interest. The internal standard can be a purified form of the metabolite of interest that can be distinguished from the metabolite of interest in some manner. The distinguishing feature of the internal standard can be any suitable variation, which can include, but is not limited to, dye labeling, stable isotope enrichment, or changing the mobility of the standard during electrophoretic separation to separate it from the metabolite of interest.

[0073] In fact, any method of loading a sample into a capillary can be performed. For example, the sample can be loaded into one end of a capillary. In some aspects, the sample is loaded into one end of a capillary by hydrodynamic flow. For example, in an embodiment where the fluid path is a capillary, the sample can be loaded into one end of the capillary by hydrodynamic flow so that the capillary is used as a micropipette. In some aspects, the sample can be loaded into the capillary by electrophoresis, for example, when the capillary is filled with gel and is therefore more resistant to hydrodynamic flow.

[0074] The capillary can include any microchip structure that allows liquid or dissolved molecules to flow. Therefore, the capillary can include any structure known in the art, as long as the structure is compatible with the method. In some embodiments, the capillary is a hole or channel through which the liquid or dissolved molecules can flow. In some embodiments, the capillary is a channel in a permeable material in which the liquid or dissolved molecules can flow.

[0075] The capillary comprises any material that allows the metabolites of interest to be separated within the capillary. The capillary comprises any convenient material, such as glass, plastic, silicon, fused silica, gel, etc. In some aspects, the method employs a plurality of capillaries. A plurality of capillaries enables the simultaneous analysis of a plurality of samples. In some aspects, the microchip containing the capillaries is coated. In other aspects, the microchip is bare glass.

[0076] Disease or condition target

[0077] The methods described herein can be used to detect metabolites associated with a disease or condition. In one aspect, the disease or condition is shown in Table 1. In another aspect, the disease or condition is associated with increased levels of nucleotides (e.g., ATP) and is an inflammatory disease of the airways, cough, heart disease, eye disease, neurodegenerative disease, psychiatric disease, neuropathic pain, chronic inflammatory disease, metabolic disease, or cancer.

[0078] For example, ATP has all the characteristics of an ideal extracellular messenger: (a) it is virtually absent in the extracellular space under physiological conditions (estimated concentrations are 10–100 nmol / L); (b) it is stored intracellularly in very high amounts (5 to 10 mmol / L); (c) it is water-soluble and freely diffusible in the extracellular space due to negatively charged phosphate residues; (d) it is rapidly degraded by ubiquitous extracellular nucleotidases; and (e) it binds to specific plasma membrane receptors, a feature that confers specificity to its signaling. These properties allow the generation of extracellular messengers that are characterized by (a) very low background noise and, therefore, high signal-to-noise ratios; (b) rapid diffusion through the aqueous tissue interstitium; and (c) rapid signaling shutdown to avoid overstimulation or receptor desensitization.

[0079] The role of eATP in several different physiological and pathological conditions (airway inflammation diseases, cough, heart disease, eye disease, neurodegenerative diseases, mental illness, neuropathic pain, chronic inflammatory diseases, metabolic diseases or cancer) has been identified. For example, eATP plays an important role in lung physiology, including epithelial ciliary sodium and water transport and mucin secretion. eATP is rapidly degraded into adenosine 5'-diphosphate, adenosine 5'-monophosphate and adenosine by extracellular enzymes (mainly CD39 and CD73). Although the rapid degradation of eATP leads to generally low extracellular ATP levels, specific microenvironments and pathophysiological conditions (such as airway inflammation diseases) are associated with increased local concentrations of eATP. ATP also enhances cough reflexes. eATP and P2X2 / 3R are related to the cough mechanism of patients with chronic idiopathic cough.

[0080] Thus, in one aspect, the disclosure relates to a method for detecting an airway inflammatory disease, cough, heart disease, eye disease, neurodegenerative disease, psychiatric disorder, neuropathic pain, chronic inflammatory disease, metabolic disease, or cancer in a subject by detecting the presence of ATP and / or its nucleotide analogs or degradation products in a sample. In another aspect, the presence of ATP and / or its nucleotide analogs or degradation products can be used to assess the therapeutic benefit of a compound for treating an airway inflammatory disease, cough, heart disease, eye disease, neurodegenerative disease, psychiatric disorder, neuropathic pain, chronic inflammatory disease, metabolic disease, or cancer.

[0081] Examples

[0082] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0083] Material

[0084] LC-MS grade water, methanol, and ammonium hydroxide were purchased from Fisher Scientific (Hampton, NH). Adenosine 13 C5 was obtained from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). Ammonium formate, adenosine- 13 C10, 15 N55'-monophosphate, adenosine- 15 N55'-diphosphate, adenosine- 13 C10, 15 N55'-triphosphate and the corresponding unlabeled ATP, ADP, AMP, and adenosine standards were purchased from Millipore Sigma (Tewksbury, MA).TM X-AW33 um polymeric weak anionic solid phase extraction (SPE) columns were purchased from Phenomenex (Torrance, CA).

[0085] Specimen collection

[0086] After participants signed informed consent and enrollment, human blood and plasma blood-derived samples were collected through the Research Specimen Collection Program (available from AstraZeneca, Gaithersburg, MD). HeLa cells and primary Wistar rat plasma and BALF (bronchoalveolar lavage fluid) samples were provided by AstraZeneca collaborators. EDTA was used during the collection of biological samples to prevent ATP hydrolysis.

[0087] Sample preparation

[0088] Metabolites, including ATP and its decomposition products, were extracted using 100% methanol, internal standards were spiked to produce a final concentration of 1 μM, and the ratios of blood, plasma, BALF, and HeLa cells were 1:20, 1:8, 1:3, and 1000 cells:100 μL, respectively. To precipitate the proteins, several cycles of vortexing and sonication were performed on ice, followed by a final centrifugation at 14,000 × g for 10 min at 4 °C. The collected supernatant was concentrated by speed vac, placed at room temperature, and reconstituted in 25 μL LC-MS grade water before CE-MS analysis. Specifically, for BALF samples, an additional desalting step was performed using a polymer weak anion SPE column: the concentrated BALF sample was diluted 1:1 with acidified (pH 4) ammonium formate (10 mM) and then passed through an SPE column that was previously activated with methanol and equilibrated with acidified ammonium formate (10 mM). After first washing the column with acidified ammonium formate (10 mM) (wash 1) and then with methanol (wash 2), the metabolites were eluted with 5% ammonium hydroxide in methanol. The eluted fractions and wash 2 fractions were combined and concentrated by speedvac and then resuspended in 25 μL LC-MS grade water before CE-MS analysis. Stock solutions of unlabeled ATP and its decomposition products were serially diluted in LC-MS grade water to prepare different calibration curve points and three quality control (QC) solutions. The preparation of curve points and QCs followed the same procedure as described for biological samples.

[0089] ZipChip Supplies

[0090] HRB chips from the cartridge package (5 packs) of catalog number 810-0023 and BGE from the native antibody kit (catalog number 850-00048) were purchased from 908 Devices (Boston, MA, USA). The pH of the native antibody BGE used to perfuse the autosampler was 5.5. For this application, a pH of about 8.6 was required by adjusting with ammonium hydroxide.

[0091] CE Method

[0092] All analyses were performed using a ZipChip from 908 Devices (Boston, MA). TM The microfluidic chip was set as follows: initial field strength 500 V / cm, injection volume 1.00 nL, viscosity 1.04 cP, pressure-assisted start time 0 min, and replication delay 10 sec. Figure 1 The capillary electrophoresis parameters used are summarized.

[0093] MS method

[0094] This protocol was demonstrated using the Thermo Scientific IDX. Figure 2 The mass spectrometer parameters used are summarized. The optimal settings for other mass spectrometers may differ. TM The tune page completes data acquisition. The analysis run time lasts 6 minutes.

[0095] Data analysis

[0096] Using Thermo Xcalibur TM Quan Browser software processes the data obtained from targeted analysis. Figure 3 Shown are electropherograms of ATP, ADP, AMP, adenosine, dATP, dGTP, dCTP, and dTTP using the above method.

[0097] All publications, patents, and patent applications mentioned in this application are hereby incorporated by reference in their entirety into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. In addition, the citation or identification of any reference in this application should not be understood as an admission that the reference is available as prior art to the present invention. In the sense that section headings are used, they should not be interpreted as necessarily limiting.

Claims

1. A method for detecting a metabolite of interest in a sample, the method comprising: (a) contacting a sample comprising one or more metabolites of interest with an uncoated capillary electrophoresis (CE) platform; (b) separating the metabolites by molecular weight and / or charge using CE in one or more capillaries; (c) eluting the metabolite from the one or more capillaries; and (d) Detection of eluted metabolites by mass spectrometry.

2. A method for detecting a metabolite of interest in a sample, the method comprising: (a) contacting a sample containing one or more metabolites of interest with a capillary electrophoresis (CE) platform having a chemically modified surface; (b) separating the metabolites by molecular weight and / or charge using CE in one or more capillaries; (c) eluting the metabolite from the one or more capillaries; and (d) Detection of eluted metabolites by mass spectrometry.

3. The method of claim 1 or claim 2, wherein the CE platform is a microchip-based system.

4. The method of claim 3, wherein the microchip-based CE platform integrates electrophoretic separation and electrospray ionization into a mass spectrometer.

5. The method of any one of claims 1 to 4, wherein the metabolite of interest is listed in Table 1.

6. The method of any one of claims 1 to 5, wherein the metabolite of interest is an anionic metabolite.

7. The method of any one of claims 1 to 6, wherein the metabolite is a nucleotide, a nucleotide analogue or a degradation product.

8. The method of any one of claims 1 to 7, wherein the metabolite of interest is adenosine 5'-triphosphate (ATP).

9. The method of any one of claims 1 to 8, wherein the sample is blood, plasma, cells or a lavage sample.

10. The method of claim 9, wherein the sample is bronchoalveolar lavage fluid (BALF).

11. The method of any one of claims 1 to 10, wherein the sample comprises a chelating agent.

12. The method of claim 11, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

13. A method for diagnosing a disease or condition associated with aberrant nucleotide-dependent signaling in a subject, the method comprising: (a) contacting a sample from the subject with a microchip-based capillary electrophoresis (CE) platform; (b) separating adenosine 5'-triphosphate (ATP), ATP analogs and / or degradation products by molecular weight and / or charge using CE in one or more capillaries; and (c) eluting the ATP, ATP analogs and / or degradation products from the one or more capillaries; and (d) detecting the eluted ATP, ATP analogs and / or degradation products by mass spectrometry; wherein the microchip-based CE platform integrates electrophoretic separation and electrospray ionization into a mass spectrometer; and Wherein the presence of ATP, ATP analogs and / or degradation products is indicative of a disease or disorder associated with aberrant nucleotide-dependent signaling in the subject.

14. A method of monitoring the therapeutic benefit of a compound for a disease or condition associated with aberrant nucleotide-dependent signaling in a subject treated with the compound, the method comprising: (a) contacting a sample from the subject with a microchip-based capillary electrophoresis (CE) platform; (b) separating the ATP, ATP analogs and / or degradation products by molecular weight and / or charge in one or more capillaries using CE; and (c) eluting the ATP, ATP analogs and / or degradation products from the one or more capillaries; and (d) detecting the eluted ATP, ATP analogs and / or degradation products by mass spectrometry; The microchip-based CE platform integrates electrophoretic separation and electrospray ionization into a mass spectrometer. Wherein the presence of ATP, ATP analogs and / or degradation products is indicative of a disease or disorder associated with aberrant nucleotide-dependent signaling in the subject.

15. The method of claim 13 or 14, wherein the disease or disorder is associated with increased extracellular ATP (eATP) levels.

16. The method of any one of claims 13 to 15, wherein the disease or condition is an airway inflammatory disease, cough, heart disease, eye disease, neurodegenerative disease, psychiatric disease, neuropathic pain, chronic inflammatory disease, metabolic disease, or cancer.

17. The method of claim 16, wherein the cough is a chronic idiopathic cough.

18. The method of claim 16, wherein the chronic inflammatory disease is systemic lupus erythematosus or Crohn's disease.

19. The method of any one of claims 13 to 18, wherein the sample is blood, plasma, cells or a lavage sample.

20. The method of claim 19, wherein the sample is BALF.

21. The method of any one of claims 13 to 20, wherein the sample comprises a chelating agent.

22. The method of claim 21, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

23. The method of any one of claims 13 to 22, wherein the microchip is a ZipChip TM .

24. The method of any one of claims 3 to 23, wherein the microchip comprises a chemically modified surface.

25. The method of any one of claims 3 to 23, wherein the microchip does not comprise surface modification.

26. The method of any one of claims 1 to 25, further comprising adjusting the pH of the background electrolyte (BGE) relative to the metabolite of interest prior to mass spectrometry analysis.

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