Method for detecting metabolites using a microfluidics-based CE-MS system

Abstract

The present disclosure relates to a method for detecting metabolites using a microfluidic capillary electrophoresis-mass spectrometry (CE-MS) system. Metabolites can be useful for diagnosing diseases or disorders and for monitoring the therapeutic efficacy of compounds used to treat these diseases or disorders.

Description

Technical Field

[0001] The present disclosure relates to a method for detecting metabolites using a capillary electrophoresis-mass spectrometry (CE-MS) system. Metabolites can be useful for diagnosing diseases or disorders such as airway inflammatory diseases, cough, heart diseases, eye diseases, neurodegenerative diseases, mental diseases, neuropathic pain, chronic inflammatory diseases, metabolic diseases, or cancer, and for monitoring the therapeutic effectiveness of compounds used to treat the diseases or disorders.

Background Art

[0002] Many metabolites are known to be associated with the etiology of diseases. For example, abnormal nucleotide concentrations are associated with several diseases. Adenosine 5'-triphosphate (ATP), specifically extracellular ATP (eATP), has been shown to be involved not only in airway inflammatory diseases and cough, but also to provide therapeutic utility by blocking its extracellular release. These data support the concept that eATP can be an important driver of the symptoms and etiology of airway diseases. However, problems remain with the methods for detecting eATP.

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

[0004] Historically, ion pair formation chromatography or passivation has provided solutions for separating difficult compounds such as ATP. However, this can be a problem for liquid chromatography / mass spectrometry (LC / MS) systems. Additionally, common ATP luminescence-based assays indirectly measure ATP levels by enzymatic degradation, without simultaneous readout for its analogs. Therefore, analytical techniques for detecting these difficult metabolites remain undeveloped. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0005] The present disclosure relates to a method for detecting a metabolite of interest in a sample, 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 a metabolite of interest in a sample, 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 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.

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

[0009] In one aspect, the sample is a blood sample, plasma sample, cell sample, or wash sample. In another aspect, the sample is bronchoalveolar lavage fluid (BALF). In a further aspect, the sample contains a chelating agent. In another aspect, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0010] Also, the present disclosure relates to a method for diagnosing a disease or disorder associated with abnormal nucleotide-dependent signaling in a subject, comprising: (a) contacting a sample derived 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 in one or more capillaries using CE; (c) eluting 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 the presence of ATP, ATP analogs, and / or degradation products indicates a disease or disorder associated with abnormal nucleotide-dependent signaling in the subject.

[0011] Also, the present disclosure is a method for monitoring the therapeutic utility of a compound for a disease or disorder associated with abnormal nucleotide-dependent signaling in a subject treated with the compound, comprising: (a) contacting a sample derived from the subject with a microchip-based capillary electrophoresis (CE) platform; (b) separating ATP, ATP analogs, and / or degradation products by molecular weight and / or charge in one or more capillaries using CE; (c) eluting 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 the presence of ATP, ATP analogs, and / or degradation products indicates a disease or disorder associated with abnormal nucleotide-dependent signaling in the subject.

[0012] In one aspect, the disease or disorder is associated with an increase in extracellular ATP (eATP) levels. In another aspect, the disease or disorder is an airway inflammatory disease, cough, heart disease, eye disease, neurodegenerative disease, mental disease, neuropathic pain, chronic inflammatory disease, metabolic disease, or cancer. In another aspect, the cough is chronic idiopathic cough. In another aspect, the chronic inflammatory disease is systemic lupus erythematosus or Crohn's disease.

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

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

[0015] In one aspect, the method further includes adjusting the pH of the background electrolyte (BGE) for the metabolite of interest prior to mass spectrometry.

[0016] Some aspects of the invention are described herein by way of example only, with reference to the accompanying drawings. Here, referring to the drawings in detail, it is emphasized that the details shown are for the purpose of illustration and for consideration in the explanation of aspects of the invention, and are by way of example only.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0018] I. General Definitions 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. In case of conflict, this specification, including the definitions, will control. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. 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.

[0019] Suitable methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice and testing of this disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of this disclosure will become apparent from the detailed description and the claims.

[0020] To further define this disclosure, the following terms and definitions are provided.

[0021] The singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" can be used interchangeably herein. In certain embodiments, the term "a" or "an" means "singular". In other embodiments, the term "a" or "an" includes "two or more" or "plural".

[0022] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in combination with a numerical range, "about" modifies the range by extending the boundaries above and below the indicated numerical values. Generally, the term "about" can be used herein to modify the numerical values above and below the indicated value by a variation of plus or minus 10 percent.

[0023] Throughout this disclosure, various aspects of the invention are presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. It should be understood that the description of a range is to be regarded as specifically disclosing all possible sub-ranges and individual numerical values within that range. For example, a range description such as 1 to 6 is to be regarded as specifically disclosing sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within the range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range. The recited numerical ranges include the numbers defining the range and each integer and decimal within the defined range.

[0024] Units, prefixes, and symbols are expressed in the forms recognized in these International System of Units (SI). Numerical ranges include the numbers defining the range. When a range of values is recited, it should be understood that each intervening integer value, and each of its decimals, between the recited upper and lower limits of the range, as well as each sub-range between such values, are specifically disclosed. The upper and lower limits of any range may be independently included in or excluded from the range, and each range where either, neither, or both of the upper and lower limits are included is also encompassed within the disclosure. Thus, the ranges described herein are to be understood as a shorthand for all values within the range including the recited endpoints. For example, a range of 1 to 10 is to be understood as including any number, combination of numbers, or sub-range selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0025] When values are expressly recited, values that are substantially the same quantity or amount as the recited values should also be understood to be within the scope of the present disclosure. When combinations are disclosed, each sub-combination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, when different elements or groups of elements are disclosed individually, their combinations are also disclosed. If any element of the present invention is disclosed as having a plurality of alternatives, examples of the present disclosure in which each alternative is excluded, either alone or in any combination with other alternatives, are also disclosed herein; a plurality of elements of the present disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0026] As used herein, the term "and / or" should be construed to mean each specific disclosure of two specified features or components, including or excluding the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein 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 include 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).

[0027] When an aspect is described herein using the language "comprising", it is always understood that other similar aspects are also provided, described by the terms "consisting of" and / or "consisting essentially of".

[0028] II. Detection Method The present disclosure relates to a method for detecting a metabolite of interest in a sample. In some aspects, the metabolite of interest is associated with a pathological condition. Thus, in some aspects, the present disclosure relates to a method for diagnosing a disease or disorder by detecting a metabolite of interest, or a method for monitoring the therapeutic effect of a compound on a disease or disorder.

[0029] In one aspect, the present disclosure is a method for detecting a metabolite of interest in a sample, comprising: (a) contacting a sample containing one or more metabolites of interest with an uncoated capillary electrophoresis (CE) platform; (b) separating the metabolites in one or more capillaries using CE by molecular weight and / or charge; (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 an uncoated microchip-based CE platform. In another aspect, the microchip-based CE platform integrates electrophoretic separation and electrospray ionization into a mass spectrometer.

[0030] A CE / MS system combines capillary electrophoresis and mass spectrometry to separate and analyze a sample. The CE / MS system functions by first applying a voltage to the sample to separate the ionic components of the sample. The ions will move through the capillary at different speeds due to their charge and frictional forces. The separated sample is then sprayed into a mass spectrometer to generate a spectrum. The spectrum is used to identify the individual components of the sample. In one aspect, the microchip contains a capillary. In another aspect, CE is performed separately from the microchip.

[0031] As used herein, the term "sample" refers to a mixture of components containing at least a metabolite of interest, such as ATP, which is subjected to an operation according to the method of the present invention, such as separation, analysis, extraction, or profiling.

[0032] As used herein, "metabolite" refers to endogenous compounds such as amino acids, lipids, sugars, organic acids, etc. These are routinely formed during the processes of assimilation or catabolism. Metabolites can have a number of functions including energy conversion, signal transduction, epigenetic effects, and cofactor activity, but their presence can also be associated with human diseases or disorders. Exemplary metabolites of the present disclosure are found in Table 1.

[0033] 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".

[0034] As used herein, "therapeutic utility" relates to improvement of symptoms or slowing of disease progression.

[0035] As used herein, the terms "analysis" or "analyzing" are used interchangeably and refer to any of a variety of methods for separating, detecting, isolating, purifying, solubilizing, and / or characterizing a metabolite of interest.

[0036] "Detect" and "detection" have their standard meanings and are intended to include detection where the presence, measurement, and / or characterization of a metabolite of interest, such as ATP, is involved.

[0037] [Table 1]

[0038] [Table 2]

[0039] [Table 3]

[0040] [Table 4]

[0041] [Table 5]

[0042]

Table 6

[0043]

Table 7

[0044]

Table 8

[0045]

Table 9

[0046]

Table 10

[0047]

Table 11

[0048]

Table 12

[0049]

Table 13

[0050]

Table 14

[0051]

Table 15

[0052]

Table 16

[0053]

Table 17

[0054] As used herein, the terms “standard” and / or “internal standard” refer to a well-characterized substance of known amount and / or identity (e.g., known molecular weight, electrophoretic mobility profile) that can be added to a sample, and the molecules in both the standard and the sample can be characterized based on molecular weight or isoelectric point by electrophoresis. Then, comparison with the standard substance provides a quantitative or semi-quantitative measure of the amount of an analyte, such as ATP, present in the sample.

[0055] As used herein, “contacting” includes bringing together at least two substances in solution phase or solid phase.

[0056] “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 the mass-to-charge ratio (m / z). Prior to detection, the sample may be subjected to one or more dimensions of chromatographic separation, such as one or more dimensions of liquid or size-exclusion chromatography.

[0057] Samples used in the disclosed methods can be heterogeneous and contain various components, i.e., various metabolites. In addition, samples can be homogeneous and contain one metabolite or essentially one metabolite of multiple charge species or molecular weight species. A pre-analytical treatment may be performed on the sample prior to detecting the metabolite.

[0058] Historically, the use of microfluidic-based CE / MS systems has been difficult for analyzing anionic substrates. However, using the methods described herein, it is possible to discriminate anionic molecules, neutral molecules, and molecules with a positive charge. In some embodiments, a microchip or microfluidic-based CE / MS system is used for analysis. In some embodiments, the microchip is surface-modified to include a substrate. An example of a microchip-based CE system that can be used in conjunction with MS is ZipChip™ (908 Devices, Boston, MA).

[0059] In some embodiments, the capillary can include a separation matrix, and the separation matrix can be added in an automated fashion by the device and / or system. In some embodiments, the sample is loaded onto a stacking matrix and then separated. The separation matrix, in one embodiment, is a size separation matrix and is similar to or has substantially the same properties as the polymer gels used in conventional electrophoresis techniques. Capillary electrophoresis in the separation matrix is similar to the separation in polymer gels, such as polyacrylamide gels or agarose gels, which provide porous channels through which molecules can move and are separated based on the size of the molecules in the sample. Since larger molecules move more slowly through the matrix than smaller molecules, separation of the analyte by molecular size is enabled. In some embodiments, one or more capillaries include the separation matrix. In some embodiments, a sample containing metabolites is separated or fractionated based on molecular weight. In some embodiments, the separation matrix includes a sieving matrix configured to separate proteins by molecular weight. In some embodiments, the protein component of the sample is separated by molecular weight, and the method is a method for detecting and / or discriminating size changes between metabolites and their analogs or degradation products.

[0060] In some embodiments, a sample containing a metabolite of interest is separated or partitioned based on the charge of the components of the sample. In some embodiments, the metabolite components of the sample are separated by charge, and the method is a method for detecting and / or discriminating charge changes between the metabolite and its analogs or degradation products.

[0061] In some embodiments, an internal standard can be used to quantitatively detect the metabolite of interest. The internal standard can be a purified form of the metabolite of interest that is distinguishable from the metabolite of interest in some way. Distinguishing features of the internal standard can include, but are not limited to, dye labeling, stable isotope enrichment, or modification of the mobility of the standard such that the standard is separated from the metabolite of interest during electrophoretic separation, or any suitable change.

[0062] Virtually any method of loading a sample into a capillary can be performed. For example, the sample can be loaded into one end of the capillary. In some embodiments, the sample is loaded into one end of the capillary by hydrodynamic flow. For example, in embodiments where the fluid path is a capillary, the sample can be loaded into one end of the capillary by hydrodynamic flow such that the capillary is used as a micropipette. In some embodiments, the sample can be loaded into the capillary by electrophoresis, for example, when the capillary is gel-filled and thus more resistant to hydrodynamic flow.

[0063] The capillary can include any microchip structure that allows for the flow of liquids or dissolved molecules. Thus, the capillary can include any structure known in the art, so long as it is compatible with the method. In some embodiments, the capillary is a bore or channel through which liquids or dissolved molecules can flow. In some embodiments, the capillary is a passageway within a permeable material through which liquids or dissolved molecules can flow.

[0064] The capillary includes any material that enables the separation of the metabolite of interest within the capillary. The capillary includes any convenient material such as glass, plastic, silicon, fused silica, gel, etc. In some embodiments, the method uses a plurality of capillaries. The plurality of capillaries enables the simultaneous analysis of a plurality of samples. In some embodiments, the microchip containing the capillary is coated. In other embodiments, the microchip is bare glass.

[0065] Disease target or disorder target The methods described herein are useful for the detection of metabolites associated with a disease or disorder. In one embodiment, the disease or disorder is shown in Table 1. In another embodiment, the disease or disorder is associated with an increase in the level of a nucleotide, such as ATP, and is any of an airway inflammatory disease, cough, heart disease, eye disease, neurodegenerative disease, mental disease, neuropathic pain, chronic inflammatory disease, metabolic disease, or cancer.

[0066] For example, ATP has all the characteristics of an ideal extracellular messenger: (a) it is substantially absent from the extracellular space under physiological conditions (estimated concentration 10 - 100 nmol / L); (b) it is stored in large amounts (5 - 10 mmol / L) intracellularly; (c) it is water-soluble due to negatively charged phosphate residues and can diffuse freely within the extracellular space; (d) it is rapidly degraded by extracellular nucleotidases present everywhere; (e) it ligates specific plasma membrane receptors (a characteristic that confers specificity to signal transduction). These properties enable the generation of an extracellular messenger characterized by (a) very low background noise and thus a high signal-to-noise ratio; (b) rapid diffusion through the aqueous tissue interstitial space; (c) rapid signal termination to avoid overstimulation or receptor desensitization.

[0067] The role of eATP has been identified in several different physiological and pathological conditions, including airway inflammatory diseases, cough, heart diseases, eye diseases, neurodegenerative diseases, mental diseases, neuropathic pain, chronic inflammatory diseases, metabolic diseases, or cancer. For example, eATP plays an important role in lung physiological functions such as sodium and water transport and mucin secretion in the ciliary body epithelium. eATP is rapidly degraded by ectoenzymes, mainly CD39 and CD73, into adenosine 5'-diphosphate, adenosine 5'-monophosphate, and adenosine. The rapid degradation of eATP generally results in low levels of extracellular ATP, but certain microenvironments and pathophysiological conditions, such as airway inflammatory diseases, are associated with increased local concentrations of eATP. Also, ATP enhances the cough reflex. eATP and P2X2 / 3R are involved in the mechanism of cough in patients with chronic idiopathic cough.

[0068] Accordingly, in one aspect, the present disclosure relates to a method for detecting airway inflammatory diseases, cough, heart diseases, eye diseases, neurodegenerative diseases, mental diseases, neuropathic pain, chronic inflammatory diseases, metabolic diseases, 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 evaluate the therapeutic utility of a compound used to treat airway inflammatory diseases, cough, heart diseases, eye diseases, neurodegenerative diseases, mental diseases, neuropathic pain, chronic inflammatory diseases, metabolic diseases, or cancer.

Examples

[0069] Here, together with the above description, reference is made to the following examples that non-limitingly illustrate some embodiments of the present invention.

[0070] Materials LC-MS grade water, methanol, and ammonium hydroxide were purchased from Fisher Scientific (Hampton, NH). Adenosine 13C5 was obtained from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). Ammonium formate, adenosine- 13 C10, 15 N5 5'-monophosphate, adenosine- 15 N5 5'-diphosphate, adenosine- 13 C10, 15 N5 5'-triphosphate, as well as the corresponding unlabeled ATP, ADP, AMP, and adenosine standards were purchased from Millipore Sigma (Burlington, MA). Strata (trademark) X-AW 33μm Polymeric Weak Anion solid-phase extraction (SPE) columns were purchased from Phenomenex (Torrance, CA).

[0071] Sample collection After obtaining the signed informed consent and registration of the participants, human blood and plasma-derived samples were collected by the Research Specimen Collection Program available at AstraZeneca (Gaithersburg, MD). HeLa cells and naive Wistar rat plasma and bronchoalveolar lavage fluid (BALF) samples were provided by co-researchers at AstraZeneca. EDTA was used while collecting the biological samples to prevent ATP hydrolysis.

[0072] Sample preparation Metabolites including ATP and its degradation products were extracted using 100% methanol with added internal standard, resulting in a final concentration of 1 μM at ratios of 1:20 for blood, 1:8 for plasma, 1:3 for BALF, and 1000 cells:100 μL for HeLa cells. To enable protein precipitation, several cycles of vortexing and sonication on ice were performed, followed by a final centrifugation at 4 °C, 14,000×g for 10 minutes. The collected supernatant was concentrated by SpeedVAC, set to room temperature, reconstituted in 25 μL of LC-MS grade water, and then CE-MS analysis was performed. Specifically, for BALF samples, an additional desalting step was continued using a polymeric weak anion SPE column: the concentrated BALF sample was diluted 1:1 with 10 mM ammonium formate acidified (pH 4), then passed through an SPE column pre-activated with methanol and equilibrated with 10 mM acidified ammonium formate. The column was washed first with 10 mM acidic ammonium formate (wash 1), followed by methanol (wash 2), and then the metabolites were eluted with 5% ammonium hydroxide in methanol. The elution and wash 2 fractions were combined, concentrated by speed vac, resuspended in 25 μL of LC-MS grade water, and then CE-MS analysis was performed. Stock solutions of unlabeled ATP and its degradation 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 QC followed the same procedure as described for biological samples.

[0073] ZipChip consumables The HRB chip from Cartridge Package (5-pack) of catalog #810-0023 and the BGE from Native Antibodies kit (catalog #850 00048) were purchased from 908 Devices (Boston, MA, USA). The native antibody BGE used to prime the autosampler has a pH of 5.5. For this application, it was necessary to achieve a pH of approximately 8.6 by adjustment with ammonium hydroxide.

[0074] CE method All analyses were performed using a ZipChip™ device and an autosampler from 908 Devices (Boston, MA). The microfluidic chip settings were: initial electric field strength 500 V / cm, injection volume 1.00 nL, viscosity 1.04 cP, pressure assist start time 0 min, and replicate delay 10 s. Figure 1 summarizes the capillary electrophoresis parameters used.

[0075] MS method This protocol was demonstrated using a Thermo Scientific IDX. Figure 2 summarizes the mass spectrometer parameters used. Optimal settings for other mass spectrometers may vary. Data acquisition was achieved using the Thermo Xcalibur™ TUNE page triggered by ZipChip software. The analysis runtime lasted 6 minutes.

[0076] Data analysis Data obtained from targeted analysis was processed using Thermo Xcalibur™ Quan Browser software. Figure 3 shows the electrophoregrams of ATP, ADP, AMP, adenosine, dATP, dGTP, dCTP, and dTTP using the above method.

[0077] All publications, patents, and patent applications mentioned in this application are hereby incorporated by reference in their entirety, as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. In addition, any citation or identification of a reference document in this application should not be construed as an admission that such reference document is available as prior art to the present invention. To the extent section headings are used, section headings should not necessarily be construed as limiting.

Claims

**Claim 1** A method for detecting a metabolite of interest in a sample, comprising: (a) contacting a sample containing one or more metabolites of interest with an uncoated capillary electrophoresis (CE) platform; (b) separating the metabolites in one or more capillaries using CE by molecular weight and / or charge; (c) eluting the metabolites from the one or more capillaries; and (d) detecting the eluted metabolites by mass spectrometry. A method as described above. **Claim 2** A method for detecting a metabolite of interest in a sample, 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 in one or more capillaries using CE by molecular weight and / or charge; (c) eluting the metabolites from the one or more capillaries; and (d) detecting the eluted metabolites by mass spectrometry. A method as described above. **Claim 3** The method according to claim 1 or 2, wherein the CE platform is a microchip-based system. **Claim 4** The method according to claim 3, wherein the microchip-based CE platform integrates electrophoresis separation and electrospray ionization into a mass spectrometer. **Claim 5** The method according to any one of claims 1 to 4, wherein the metabolite of interest is listed in Table 1. **Claim 6** The method according to any one of claims 1 to 5, wherein the metabolite of interest is an anionic metabolite. **Claim 7** The method according to any one of claims 1 to 6, wherein the metabolite is a nucleotide, nucleotide analog, or degradation product. **Claim 8** The method according to any one of claims 1 to 7, wherein the metabolite of interest is adenosine 5'-triphosphate (ATP). **Claim 9** The method according to any one of claims 1 to 8, wherein the sample is a blood sample, plasma sample, cell sample, or wash sample. **Claim 10** The method according to claim 9, wherein the sample is bronchoalveolar lavage fluid (BALF). **Claim 11** The method according to any one of claims 1 to 10, wherein the sample contains a chelating agent. **Claim 12** The method according to claim 11, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA). **Claim 13** A method for diagnosing a disease or disorder associated with abnormal nucleotide-dependent signal transduction in a subject, 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 in one or more capillaries using CE; (c) eluting ATP, ATP analogs, and / or degradation products from the one or more capillaries; (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 the presence of ATP, ATP analogs, and / or degradation products indicates a disease or disorder associated with abnormal nucleotide-dependent signal transduction in the subject.

14. A method for monitoring the therapeutic utility of a compound for a disease or disorder associated with abnormal nucleotide-dependent signal transduction in a subject treated with the compound, 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 in one or more capillaries using CE; (c) eluting ATP, ATP analogs, and / or degradation products from the one or more capillaries; (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 the presence of ATP, ATP analogs, and / or degradation products indicates a disease or disorder associated with abnormal nucleotide-dependent signal transduction in the subject.

15. The method according to claim 13 or 14, wherein the disease or disorder is associated with an increase in extracellular ATP (eATP) levels.

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

17. The method according to claim 16, wherein the cough is chronic idiopathic cough.

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

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

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

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

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

23. The method according to any one of claims 13 to 22, wherein the microchip is ZipChip (trademark).

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

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

26. The method according to any one of claims 1 to 25, further comprising adjusting the pH of a background electrolyte (BGE) for the metabolite of interest before mass spectrometry.

Images