Methods of detecting chromogranin a by mass spectrometry

Abstract

To provide methods of detecting or determining the amount of chromogranin A (CgA) in a sample by mass spectrometry, including tandem mass spectrometry.SOLUTION: A method of determining the amount of chromogranin A (CgA) in a sample is provided, the method comprising (a) purifying CgA in the sample, (b) ionizing CgA to produce one or more ions of CgA detectable by mass spectrometry, and (c) determining the amount of the ion(s) from the step (b) by mass spectrometry, where the amount of ions is related to the amount of CgA in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application is the benefit of U.S. Provisional Patent Application No. 62 / 644,210, filed March 16, 2018. The benefit of which is claimed and is incorporated herein by reference in its entirety. [Background technology]

[0002] Chromogranin-A (CgA) is a 50 kDa protein expressed in secretory granules of neuroendocrine tissues. Currently, blood levels of CgA are measured by various immunoassays. However, as with any antibody-based assay, This is due to non-specific binding and the need for sample dilution, which reduces the dynamic range. There are several limitations to the implementation of such tests in diagnostic laboratories. causes.

[0003] An accurate and sensitive assay for quantifying chromogranin A is needed. Summary of the Invention

[0004] Mass spectrometry, including tandem mass spectrometry, allows for the determination of the A method for detecting or determining the amount of chromogranin A (CgA) is provided herein. It is served.

[0005] In certain embodiments, the methods provided herein involve the use of chromogranin A (CgA 1. A method for detecting or determining the amount of CgA in a sample, comprising: (a) purifying CgA in a sample; (b) Ionizing CgA to produce ions detectable by mass spectrometry and (c) detecting or quantifying the CgA ion by mass spectrometry. determining the amount of CgA ions in a manner that correlates with the amount of CgA in the sample. be.

[0006] In certain embodiments, the methods provided herein involve administering to a subject a therapeutic target for chromogranin A (CgA 1. A method for detecting or determining the amount of a nucleotide sequence comprising: (a) subjecting a sample to solid phase extraction; (b) enzymatically digesting CgA; (c) subjecting CgA to liquid chromatography. (d) ionizing CgA to produce ions detectable by mass spectrometry; and (e) detecting or quantifying CgA ions by mass spectrometry. determining a quantity of CgA ions in a manner that correlates with the amount of CgA in the sample; It is the law.

[0007] In certain embodiments, the methods provided herein involve selective reaction monitoring ( SRM) Mass Spectrometry.

[0008] In some embodiments, the methods provided herein are fully automated. do.

[0009] In some embodiments, the methods provided herein are antibody-free methods. be.

[0010] In some embodiments, the purifying provided herein can be performed using solid phase extraction (SPE). In some embodiments, SPE involves extraction of serum using anion exchange. In some embodiments, the SPE is a mixed-mode anion exchange solid phase extraction. Extraction. In some embodiments, the extracted sample is concentrated.

[0011] In some embodiments, the purifying provided herein comprises liquid chromatography. In some embodiments, the liquid chromatography includes high performance liquid chromatography. In some embodiments, liquid chromatography (HPLC) This includes high turbulence liquid chromatography (HTLC).

[0012] In some embodiments, the extracted sample is enzymatically digested. In an embodiment, the extracted sample is enzymatically digested with trypsin.

[0013] In some embodiments, ionization includes electrospray ionization (ESI). In some embodiments, ionizing includes ionizing in a positive mode. In some embodiments, ionizing comprises ionizing in negative mode.

[0014] In some embodiments, ionization comprises atmospheric pressure chemical ionization (APCI). In some embodiments, ionizing includes ionizing in a positive mode. In some embodiments, ionizing comprises ionizing in a negative mode.

[0015] In some embodiments, the methods provided herein provide a method for determining the degree of oxidative stress in a patient with a pulmonary artery disease (PAS) of 593.2±0.5. This involves measuring the amount of precursor ions having a mass-to-charge ratio.

[0016] In some embodiments, the methods provided herein provide a method for determining the degree of oxidative stress in a patient with a pulmonary artery disease (PAS) of 729.6±0.5. This involves measuring the amount of precursor ions having a mass-to-charge ratio.

[0017] In some embodiments, the methods provided herein involve fragmenting chromogranin A. In some embodiments, the measured CgA fragment comprises the sequence ELQDLALQGA (SEQ ID NO: 1). In some embodiments, the measured Cg The A fragment contains the sequence RRPEDQELESLSAIEAELEK (SEQ ID NO: 4).

[0018] In some embodiments, the methods provided herein provide a method for detecting 516.3±0.5 mg / mL of erythrocytes. fragment ions with a mass-to-charge ratio of 815.5±0.5 or 815.5±0.5, or both This includes measuring the amount of

[0019] In some embodiments, the methods provided herein provide a method for detecting 831.5±0.5 mg / mL of erythrocytes. fragment ions with a mass-to-charge ratio of 989.5±0.5 or 989.5±0.5, or both This includes measuring the amount of

[0020] In some embodiments, the methods provided herein include adding an internal standard. In some embodiments, the internal standard is isotopically labeled. In some embodiments, the internal standard is C 13 N 15 Contains labeled amino acids. In an embodiment, the internal standard is labeled at leucine (L) or lysine (K). In some embodiments, the internal standard has the sequence ILSILRHQNLLKELQDLAL*Q GAK*ERAHQQK (SEQ ID NO: 2), where * is C 13 N 15 Labeled amino acids In some embodiments, the internal standard has the sequence RRPEDQELESL*SAIE AELEK* (SEQ ID NO: 5), where * is C 13 N15 The labeled amino acid.

[0021] In some embodiments, the methods provided herein provide a method for detecting a β-amyloid in a blood sample containing β-amyloid. Internal standard precursor ions with mass-to-charge ratios of 602.4±0.5, 830. of product ions with mass-to-charge ratios of 6±0.5 or 958.7±0.5. This includes measuring the quantity.

[0022] In some embodiments, the methods provided herein provide a method for detecting a β-amyloid in a blood sample containing β-amyloid. Internal standard precursor ions with mass-to-charge ratios of 734.6±0.5, 839, and / or 734.6±0.5. of product ions with mass-to-charge ratios of 5±0.5 or 997.6±0.5 This includes measuring the quantity.

[0023] In certain embodiments, the limit of quantitation of the method is less than or equal to 100 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 90 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 80 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 70 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 60 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 50 ng / mL.

[0024] In some embodiments, the detection limit of the method is less than or equal to 50 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 40 ng / mL. In some embodiments, the detection limit of the method is less than, or equal to, 35.5 ng / mL. It's nice.

[0025] In some embodiments, the methods provided herein provide a method for treating ulcerative colitis in a patient receiving ... Includes linearity of quantitation over a range of 1,000 ng / mL.

[0026] In some embodiments, the methods provided herein provide an assay with a CV≦15%. Includes inter- and intra-assay reproducibility.

[0027] In some embodiments, the CgA is not derivatized prior to mass spectrometry.

[0028] In certain embodiments, the sample is a body fluid. In some embodiments, the sample is a brain In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva. fluid or urine.

[0029] In some embodiments, the method comprises adding a drug to the sample in an amount sufficient to deproteinize the sample. This may involve adding an agent to the sample.

[0030] In some embodiments, the level of chromogranin A is elevated compared to the reference range. This indicates an increased risk of neuroendocrine tumors (NETs). In embodiments, the quantified level of chromogranin A is indicative of the size of a neuroendocrine tumor. In some embodiments, the quantified level of chromogranin A indicates neuroendocrine differentiation. In some embodiments, quantification of chromogranin A is indicative of tumor burden in a tumor. The level is indicative of a response to treatment of the neuroendocrine tumor. Quantified levels of chromogranin A are prognostic indicators of neuroendocrine tumors.

[0031] As used herein, unless otherwise indicated, the singular forms "a," "an," and "the" are used. " includes plural meaning. Thus, for example, when we say "one protein," we mean multiple proteins. Protein molecules are also included.

[0032] As used herein, the term "purification" or "to purify" refers to the removal of a desired component from a sample. Purification does not mean the removal of all substances other than the analyte of interest. the concentration of the analyte of interest relative to other components in the sample that may interfere with the detection of the analyte of interest. A sample, as used herein, refers to a procedure that enriches the amount of one or more analytes that are present in a sample. is one or more interfering substances, e.g., selected Cg Various means to allow for the removal of one or more substances that interfere with the detection of parent and daughter ions of A It is purified by

[0033] As used herein, the term "test sample" refers to any sample that may contain CgA. As used herein, the term "body fluid" refers to any fluid that can be isolated from the body of an individual. For example, "body fluids" includes blood, plasma, serum, bile, saliva, urine, tears, sweat, etc. Some examples include:

[0034] As used herein, the term "derivatizing" refers to reacting two molecules to form a new Derivatization agents include isothiocyanate groups, dinitrofluoro groups, containing a phenyl group, a nitrophenoxycarbonyl group, and / or a phthalaldehyde group, etc. obtain.

[0035] As used herein, the term "chromatography" refers to the process of separating particles carried by a liquid or gas. When a chemical mixture flows around or above a stationary liquid or solid phase, chemical entities The term "separation" refers to the process of separation into components as a result of differential partitioning of

[0036] As used herein, the term "liquid chromatography" or "LC" refers to the When a fluid permeates uniformly through a column of a substance or through a capillary passage, It refers to the process by which one or more components of a fluid solution are selectively retarded. or between multiple stationary phases and the bulk liquid (i.e., mobile phase), where this fluid is in phase with the stationary phase It is caused by the distribution of components in a mixture as they move relative to one another. Examples of "-" include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), High-performance liquid chromatography (HPLC), and high-turbulence liquid chromatography (HTLC).

[0037] As used herein, the term "high performance liquid chromatography" or "HPLC" means by applying force to the mobile phase under pressure through a stationary phase, typically a densely packed column. This means that the degree of separation is increased by the use of liquid chromatography.

[0038] As used herein, the term "high turbulence liquid chromatography" or "HTLC" means The basic principle for carrying out separation is the turbulent flow of the substance to be assayed through the column packing. HTLC refers to a form of chromatography that uses mass spectrometry. It has been applied to prepare samples containing two unnamed drugs prior to analysis by HPLC. For example, Zimmer et al., J. Chromatogr. A854:23-35 (1999) See U.S. Patent No. 5,968,367, which further describes HTLC; See also Nos. 5,919,368, 5,795,469, and 5,772,874. Those skilled in the art understand "turbulent flow." When a fluid flows slowly and smoothly, For example, the flow moving at a low flow rate through an HPLC column is called laminar flow. The flow is laminar. In laminar flow, the movement of particles in the fluid is orderly and the particles move in straight lines. At higher speeds, the inertial force of the water exceeds the frictional force of the fluid, resulting in turbulence. Fluids that do not come into contact with irregular boundaries are slowed down by friction or flow through uneven surfaces. When a fluid flows in a turbulent state, it "overtakes" the fluid that has changed direction due to the surface. The flow is swirling (or vortex-like), which makes it more "resistant" than if the flow were stratified. There are many references available to help determine whether a fluid flow is laminar or turbulent. (e.g. Turbulent Flow Analysis: Measurement t and Prediction, PS Bernard & J.M.Wallace , John Wiley&Sons, Inc., (2000);An Introdu ction to Turbulent Flow Flow, Jean Mathie u&Julian Scott, Cambridge University Pres s (2001)).

[0039] As used herein, the term "gas chromatography" or "GC" refers to the process of analyzing a sample. The mixture is vaporized and passed through a column containing a stationary phase composed of a liquid or particulate solid. The compound is injected into a flow of carrier gas (nitrogen or helium) that travels through the It refers to chromatography in which a compound is separated into its constituent compounds based on its affinity for the stationary phase. Taste.

[0040] As used herein, the term "large particle column" or "extraction column" refers to a column having a particle size of approximately 35 μm. In this context, the term "chromatography column" refers to a column containing particles with a mean particle size greater than m. When used herein, the term "about" means ±10%. It contains particles approximately 60 μm in diameter.

[0041] As used herein, the term "analytical column" refers to a column that allows the separation of materials in a sample from the column. for sufficient separation to allow determination of the presence, absence, or amount of analyte. means a chromatography column having a chromatographic plate. The system then extracts the retained material to obtain a purified sample for further analysis. Many of these come from "extraction columns" which have the general purpose of separating or extracting substances from others. When used in this context, the term "about" means ±10%. In a preferred embodiment, the analytical column contains particles approximately 4 μm in diameter.

[0042] As used herein, the term "online automated method" or "online extraction" refers to The terms "online" or "inline" as used herein mean a system that requires no operator intervention. In contrast, the term "offline" refers to a procedure that is performed without the need for a When used in this context, it refers to a procedure that requires manual intervention by an operator. is precipitated and then the supernatant is manually loaded into the autosampler, The precipitation and loading steps are off-line from the subsequent steps. In embodiments, one or more steps may be performed in an online, automated manner.

[0043] As used herein, the term "mass spectrometry" or "MS" refers to the method of analyzing a compound. MS refers to analytical techniques that identify ions by their mass-to-charge ratio, or refers to a method of filtering, detecting, and measuring based on "m / z". MS technology is (1) ionizing a compound to form a charged compound; and (2) ionizing the charged compound. The method generally involves determining the molecular weight of the compound and calculating the mass-to-charge ratio. Ionization and detection may be by any suitable means. A "mass spectrometer" is an ionization device. and an ion detector. Generally, one or more molecules of interest are ionized. The ions are then introduced into a mass spectrometric instrument, where magnetic and electric fields The combination of these causes the ions to follow a path in space that depends on their mass ("m") and charge ("z"). For example, "Mass Spectrometry From Surface No. 6,204,500, entitled "Methods and Apparatus for The same paper, entitled "Abstract for Tandem Mass Spectrometry" No. 6,107,623, “DNA Diagnostics Based On Mass No. 6,268,144, entitled "Surface Spectrometry" -Enhanced Photolabile Attachment And Rel ease For Desorption And Detection Of Ana No. 6,124,137, entitled "Prostate Lytes"; Wright et al., Prostate Lytes Cancer and Prostatic Diseases, Vol. 2: 264–76 pp. (1999); and Merchant and Weinberger, Electro See phoresis, 21:1164-67 (2000).

[0044] As used herein, the term "operating in negative ion mode" refers to a mode in which negative ions are generated and detected. The term "operating in positive ion mode" means a mass spectrometry method in which: As used herein, it refers to a mass spectrometry method in which positive ions are generated and detected. do.

[0045] As used herein, the term "ionization" or "ionizing" refers to one or more The process of producing analyte ions with a net charge equal to 1 electron unit is called An anion is an ion that has a net negative charge of one or more electron units. whereas cations are ions with a net positive charge of one or more electron units. It is.

[0046] As used herein, the term "electron ionization method" or "EI method" refers to a gas phase The way in which the analyte of interest in the gas or vapor phase interacts with the electron flow The impact of electrons on the analyte produces analyte ions, which are then analyzed by mass spectrometry. It can be the subject of technology.

[0047] As used herein, the term "chemical ionization" or "CI" refers to a method using a reagent gas (e.g., The reaction mixture (e.g., ammonia) is bombarded with electrons and reacts with the reagent gas ions and analyte molecules. By "analyte ion" is meant the manner in which the analyte ion is formed.

[0048] As used herein, the term "fast atom bombardment" or "FAB" refers to a method for producing high-energy A beam of atoms (often Xe or Ar) bombards a non-volatile sample, The test sample is a viscous liquid. Matrices, e.g., glycerol, thioglycerol, m-nitrobenzyl alcohol , 18-crown-6-crown ether, 2-nitrophenyl octyl ether, sulfur It is soluble in ethanolamine, diethanolamine, and triethanolamine. The selection of a suitable matrix for pulling is an empirical process.

[0049] As used herein, the term "matrix-assisted laser desorption / ionization" or "MA" refers to a method for detecting a nucleophilic substance. "LDI" refers to various processes including photoionization, protonation, deprotonation, and cluster decay. The non-volatile sample is then ionized by a suitable ionization pathway, which desorbs and ionizes the analytes in the sample. In MALDI, the sample is exposed to laser radiation that induces the analyte It is mixed with an energy absorbing matrix that promotes molecular desorption.

[0050] As used herein, the term "surface-enhanced laser desorption ionization" or "SELDI" " refers to various ionization processes including photoionization, protonation, deprotonation, and cluster decay. The ionization pathway converts a non-volatile sample into a laser that desorbs and ionizes the analytes in the sample. In SELDI, the sample is exposed to one or more laser beams. Generally, a number of analytes of interest are bound to a surface that preferentially retains them. This process may also utilize an energy absorbing material to facilitate ionization.

[0051] As used herein, the term "electrospray ionization" or "ESI" refers to A solution is passed along a short length of capillary tube, and a positive or negative high voltage is attached to the end of the capillary tube. This refers to the way in which an electric potential is applied. When the solution reaches the end of the tube, it is vaporized (atomized) and Within the solvent vapor, a jet or spray of very small droplets of the solution forms. The mist passes through a slightly heated evaporation chamber to prevent condensation and evaporate the solvent. As the droplets become smaller, the surface charge density increases, and eventually the droplets move spontaneously between the same charge. The natural repulsion causes the ejection of ions as well as neutral molecules.

[0052] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to ES It refers to a mass spectrometry technique similar to I, but APCI occurs in a plasma at atmospheric pressure. Ions are produced by ion-molecule reactions. The plasma is generated between the spray capillary and the counter electrode. The ions are then pumped through a series of differentially pumped skimmers. It is typically extracted into the mass spectrometer by the use of a dry and preheated N2 Countercurrent gas flow can be used to improve solvent removal. In some cases, gas phase ionization in APCI can be more effective than ESI.

[0053] The term "atmospheric pressure photoionization" or "APPI" as used herein refers to the process for determining the molecular M The mechanism for photoionization of M+ is photon absorption and electron emission to form the molecular ion M+. The photon energy is generally just above the ionization potential, so Molecular ions are less prone to dissociation and often require no chromatography. It is believed that samples can be analyzed without the need for a separate sample, thus saving considerable time and expense. In the presence of water vapor or a protic solvent, the molecular ion removes an H to form MH+. This tends to occur when M has a high proton affinity. This does not affect the accuracy of the quantification, as the sum of M+ and MH+ is constant. Drug compounds in solvents are usually observed as MH+, while non-polar compounds, e.g., naphtha Ren or testosterone usually takes the M+ form. Robb, DB, Covey , TR and Bruins, AP (2000): See, e.g., Robb et al., Atmo spherical pressure photoionization: An ioni zation method for liquid chromatography- Mass spectrometry. Anal. Chem. Vol. 72(15):36 See pages 53-3659.

[0054] As used herein, the term "inductively coupled plasma" or "ICP" refers to At a sufficiently high temperature that the elements are atomized and ionized, the sample is partially ionized. It refers to the way in which the electrons interact with the ionized gas.

[0055] As used herein, the term "field desorption" refers to a method in which a non-volatile test sample is ionized. This refers to a method in which a strong electric field is used to generate analyte ions, which are placed on a surface. do.

[0056] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or means that the analyte is introduced into the gas phase.

[0057] As used herein, the terms "limit of quantification" and "quantitation limit" are used interchangeably. "Limit of quantitation," or "LOQ," means the limit at which a measurement is quantitatively meaningful. The analyte response at this LOQ is distinguishable and unique. It is unique and has a precision of 20% and a reproducibility of 80% to 120% accuracy.

[0058] As used herein, the term "limit of detection" or "LOD" refers to the limit at which a measurement is made. The LOD is the point at which the uncertainty in the concentration is greater than the uncertainty in the concentration. is arbitrarily defined as

[0059] As used herein, the "amount" of CgA in a body fluid sample refers to the amount of CgA detected in a volume of body fluid. Generally, absolute values reflecting the mass of CgA available are used. However, amounts may differ depending on the amount of CgA. For example, the amount of CgA in a body fluid is relative to the amount of CgA normally present. The amount may be above or below the control or normal level of CgA.

[0060] The term "about" as used herein in connection with quantitative measurements, excluding ion mass measurements, is intended to mean any measurement that is within the range of the table. The mass spectrometry instrument determines the mass of a given analyte. The term "about" refers to the mass of an ion or the mass / mass of an ion. In the context of charge ratios, ±0.5 atomic mass units is meant.

[0061] The above summary of the invention is not limiting, and other features and advantages of the invention are set forth below. This will be apparent from the detailed description and from the claims. [Brief explanation of the drawings]

[0062] [Figure 1] Figure 1 shows the analytical workflow for the CgA LC-MS / MS assay. Negatively charged CgA binds to a mixed-mode anion-exchange resin through electrostatic, lipophilic, and hydrophilic interactions under conditions that allow it to be retained while other proteins are washed away. After elution, the isolated CgA is digested with trypsin, and representative peptides are quantified by LC-MS / MS analysis. [Figure 2] Figure 2 shows three calibration curves measured over three different days. The linear range was demonstrated to be 50-50,000 ng / mL. The CV was less than 10%. [Figure 3] FIG. 3 shows chromatograms corresponding to patient samples with normal (top) and elevated (bottom) CgA values. [Figure 4] FIG. 4 shows the mean ELISA immunoassay values compared with those obtained by LC-MS / MS. [Figure 5] FIG. 5 shows a comparison between the CisBio immunoassay and the LC-MS / MS CgA assay (Passing & Bablok curve fitting, 308 samples). [Figure 6] FIG. 6 shows peak area graphs for normal and abnormal CgA levels determined by LC-MS / MS. [Figure 7] FIG. 7 shows an example chromatogram for a chromogranin internal standard. DETAILED DESCRIPTION OF THE INVENTION

[0063] CgA levels are increased in the presence of tumors of neuroendocrine origin (NETs), which may contribute to the CgA These findings suggest that serum markers may be useful for monitoring patients with NETs. Circulating levels of CgA are proportional to tumor burden and provide prognostic information on treatment response. We present a novel, fully automated, antibody-based method for quantifying chromogranin A from serum. We describe an LC-MS / MS assay method that utilizes the acidic properties of CgA and does not contain CgA. Then, 100 μL of serum was extracted using an anion exchange solid phase extraction plate, followed by An internal standard is added. The extracted sample is then concentrated and purified using trypsin. The peptides unique to CgA are then chromatographically separated. The isotope-labeled endonucleases were analyzed by SRM on a Sciex 6500+QTrap. The ratio of the analyte peak area to the standard peak area is used to achieve quantitation. CgA is available in a wide range (50-50,000 ng / mL, R 2 ≧0.99) The assays demonstrate robustness, as well as inter- and intra-assay reproducibility (CV≦15%). CgA serum levels measured by GA-ELISA-US immunoassay method were compared as described A cohort of 300 patient samples was collected for comparison with the LC-MS / MS assay. In this cohort, CgA was analyzed by immunoassay and LC-MS. R of 0.71 when comparing MS / MS measurements 2 was observed and the two assay platforms There was a good correlation between the

[0064] In certain embodiments, the methods provided herein involve administering to a subject a therapeutic target for chromogranin A (CgA 1. A method for detecting or determining the amount of CgA in a sample, comprising: (a) purifying CgA in a sample; (b) Ionizing CgA to produce ions detectable by mass spectrometry and (c) detecting or quantifying the CgA ion by mass spectrometry. determining the amount of CgA ions in a manner that correlates with the amount of CgA in the sample. be.

[0065] In certain embodiments, the methods provided herein involve administering to a subject a therapeutic target for chromogranin A (CgA 1. A method for detecting or determining the amount of a nucleotide sequence comprising: (a) subjecting a sample to solid phase extraction; (b) enzymatically digesting CgA; (c) subjecting CgA to liquid chromatography. (d) ionizing CgA to produce ions detectable by mass spectrometry; and (e) detecting or quantifying CgA ions by mass spectrometry. determining the amount of CgA ions; wherein the amount of CgA ions is related to the amount of CgA in the sample. is.

[0066] In certain embodiments, the methods provided herein involve selective reaction monitoring ( SRM) Mass Spectrometry.

[0067] In some embodiments, the methods provided herein are fully automated. do.

[0068] In some embodiments, the methods provided herein are antibody-free methods. be.

[0069] In some embodiments, the purifying provided herein can be performed using solid phase extraction (SPE). In some embodiments, SPE involves extraction of serum using anion exchange. In some embodiments, the SPE is a mixed-mode anion exchange solid phase extraction. Extraction. In some embodiments, the extracted sample is concentrated.

[0070] In some embodiments, the purifying provided herein comprises liquid chromatography. In some embodiments, the liquid chromatography includes high performance liquid chromatography. In some embodiments, liquid chromatography (HPLC) This includes high turbulence liquid chromatography (HTLC).

[0071] In some embodiments, the extracted sample is enzymatically digested. In this embodiment, the extracted sample is enzymatically digested with trypsin.

[0072] In some embodiments, ionization includes electrospray ionization (ESI). In some embodiments, ionizing includes ionizing in a positive mode. In some embodiments, ionizing comprises ionizing in negative mode.

[0073] In some embodiments, ionization comprises atmospheric pressure chemical ionization (APCI). In some embodiments, ionizing includes ionizing in a positive mode. In some embodiments, ionizing comprises ionizing in a negative mode.

[0074] In some embodiments, the methods provided herein provide a method for determining the degree of oxidative stress in a patient with a pulmonary artery disease (PAS) of 593.2±0.5. This involves measuring the amount of precursor ions having a mass-to-charge ratio.

[0075] In some embodiments, the methods provided herein provide a method for determining the degree of oxidative stress in a patient with a pulmonary artery disease (PAS) of 729.6±0.5. This involves measuring the amount of precursor ions having a mass-to-charge ratio.

[0076] In some embodiments, the methods provided herein involve fragmenting chromogranin A. In some embodiments, the measured CgA fragment comprises the sequence ELQDLALQGA (SEQ ID NO: 1). In some embodiments, the measured Cg The A fragment contains the sequence RRPEDQELESLSAIEAELEK (SEQ ID NO: 4).

[0077] In some embodiments, the methods provided herein provide a method for detecting 516.3±0.5 mg / mL of erythrocytes. fragment ions with a mass-to-charge ratio of 815.5±0.5 or 815.5±0.5, or both This includes measuring the amount of

[0078] In some embodiments, the methods provided herein provide a method for detecting 831.5±0.5 mg / mL of erythrocytes. fragment ions with a mass-to-charge ratio of 989.5±0.5 or 989.5±0.5, or both This includes measuring the amount of

[0079] In some embodiments, the methods provided herein include adding an internal standard. In some embodiments, the internal standard is isotopically labeled. In some embodiments, the internal standard is C 13 N 15 Contains labeled amino acids. In an embodiment, the internal standard is labeled at leucine (L) or lysine (K). In some embodiments, the internal standard has the sequence ILSILRHQNLLKELQDLAL*Q GAK*ERAHQQK (SEQ ID NO: 2), where * is C 13 N 15 Labeled amino acids In some embodiments, the internal standard has the sequence RRPEDQELESL*SAIE AELEK* (SEQ ID NO: 5), where * is C 13 N15 The labeled amino acid.

[0080] In some embodiments, the methods provided herein provide a method for detecting a β-amyloid in a blood sample containing β-amyloid. Internal standard precursor ions with mass-to-charge ratios of 602.4±0.5, 830. Quantity of product ions with mass-to-charge ratios of 6±0.5 or 958.7±0.5 This includes measuring

[0081] In some embodiments, the methods provided herein provide a method for detecting a β-amyloid in a blood sample containing β-amyloid. Internal standard precursor ions with mass-to-charge ratios of 734.6±0.5, 839, and / or 734.6±0.5. Quantity of product ions with mass-to-charge ratios of 5±0.5 or 997.6±0.5 This includes measuring

[0082] In certain embodiments, the limit of quantitation of the method is less than or equal to 100 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 90 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 80 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 70 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 60 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 50 ng / mL.

[0083] In some embodiments, the detection limit of the method is less than or equal to 50 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 40 ng / mL. In some embodiments, the detection limit of the method is less than, or equal to, 35.5 ng / mL. It's nice.

[0084] In some embodiments, the methods provided herein provide a method for treating ulcerative colitis in a patient receiving ... Includes linearity of quantitation over a range of 1,000 ng / mL.

[0085] In some embodiments, the methods provided herein provide an assay with a CV≦15%. Includes inter- and intra-assay reproducibility.

[0086] In some embodiments, the CgA is not derivatized prior to mass spectrometry.

[0087] In certain embodiments, the sample is a body fluid. In some embodiments, the sample is a brain In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva. fluid or urine.

[0088] In some embodiments, the method comprises adding a drug to the sample in an amount sufficient to deproteinize the sample. This may involve adding an agent to the sample.

[0089] In some embodiments, the level of chromogranin A is elevated compared to the reference range. This indicates an increased risk of neuroendocrine tumors (NETs). In embodiments, the quantified level of chromogranin A is indicative of the size of a neuroendocrine tumor. In some embodiments, the quantified level of chromogranin A indicates neuroendocrine differentiation. In some embodiments, quantification of chromogranin A is indicative of tumor burden in a tumor. The level is indicative of a response to treatment of the neuroendocrine tumor. Quantified levels of chromogranin A are prognostic indicators of neuroendocrine tumors.

[0090] Suitable test samples include any test sample that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample; i.e., any biological sample. Samples obtained from biological sources, such as animals, cell cultures, organ cultures, etc. In a preferred embodiment, the sample is obtained from a mammal, such as a dog, cat, horse, etc. Particularly preferred mammals are primates, most preferably humans, male or female. Preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tears, cerebrospinal fluid, or Examples of such samples include tissue samples from patients; i.e., A living organism that presents itself in a clinical setting to diagnose, predict, or treat a disease or condition. The test sample is preferably obtained from a patient, e.g., a human, male or female. For example, serum.

[0091] Sample Preparation for Mass Spectrometry Enrich CgA relative to other components (e.g., proteins) in the sample Methods available for this purpose include, for example, filtration, centrifugation, thin layer filtration, and the like. TLC, electrophoresis including capillary electrophoresis, immunoaffinity affinity separation methods, including isolation methods; extraction methods, including ethyl acetate extraction methods and methanol extraction methods; and the use of chaotropic agents, or any combination of the above.

[0092] Protein precipitation is one preferred method of preparing test samples. Protein purification methods are well known in the art and are described, for example, in Polson et al., Journal of al of Chromatography B, Vol. 785: 263-275 (20 (2003) describes protein precipitation techniques suitable for use in the present method. Protein precipitation removes most of the protein from the sample, leaving CgA in the supernatant. The sample is centrifuged to separate the liquid supernatant from the precipitated proteins. The resulting supernatant can then be subjected to liquid chromatography and subsequent mass spectrometry. In certain embodiments, protein precipitation methods, such as acetaminophen, may be applied to chromatographic analysis. By using methods such as nitrile protein precipitation, HPLC and mass spectrometry can be performed. High turbulence liquid chromatography (HTLC) or other online extraction is performed before Thus, in such an embodiment, the method comprises: (1) determining a sample of interest; and (2) performing on-line extraction or high turbulence liquid chromatography. The supernatant was directly loaded onto the HPLC-mass spectrometer without the need for high-temperature liquid chromatography (HTLC). It is related to doing.

[0093] In some preferred embodiments, HPLC is used alone or in combination with one or more purification steps. This method can be used in conjunction with other methods to purify CgA prior to mass spectrometry. In such an embodiment, the sample is passed through an HPLC extraction cartridge that captures the analyte. The eluate is extracted using HCl, then eluted and chromatographed on a second HPLC column. or can be eluted onto an analytical HPLC column before ionization. The steps involved in the purification procedure can be linked in an automated manner, thus facilitating the purification of the analyte. The need for operator involvement during the process is minimized. This results in time and cost savings and eliminates the opportunity for operator error. can be removed.

[0094] For example, turbulence caused by HTLC columns and methods can enhance mass transfer rates. HTLC columns contain rigid particles, which are believed to improve separation properties. Components are separated by high chromatographic flow rates through a packed column. By using a high flow rate (e.g., 3-5 mL / min), turbulence is generated in the column, and the stationary phase and This results in a near complete interaction between the analytes of interest. As a result, high molecular weight species are not retained under turbulent flow conditions and therefore are associated with the biofluid matrix. The accumulation of linked macromolecules is avoided. The combined HTLC method reduces the need for lengthy sample preparation and significantly increases Such methods offer superior analytical performance to laminar flow (HPLC) chromatography. HTLC also enables direct injection of biological samples (plasma, urine, etc.) When injected directly, denatured proteins and other biological debris rapidly pass through the separation column. This blocking occurs quickly, making it difficult to achieve with conventional chromatography. is less than 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL, preferably 0 It also allows for very small sample volumes of 0.1 mL.

[0095] Examples of HTLC applied to sample preparation prior to analysis by mass spectrometry include: See, for example, Zimmer et al., J. Chromatogr. A854, Vol. :23-35 (1999); U.S. Patent No. 5,968,367; U.S. Patent No. 5,91 See also No. 9,368; No. 5,795,469; and No. 5,772,874. Head In certain embodiments of the method, the sample is subjected to titration as described above before loading onto the HTLC column. In an alternative preferred embodiment, the sample is subjected to protein precipitation. The HTLC extraction column is preferably a column that can be loaded directly onto the HTLC without any precipitation treatment. Preferably, it is a large particle column. In various embodiments, one or more steps of the method This can be performed in an online, automated manner. For example, in one embodiment, Steps (i) to (v) are carried out in an online, automated manner. The steps of quantification and detection are carried out online after steps (i) to (v).

[0096] Liquid chromatography (LC), including high performance liquid chromatography (HPLC), Traditional HPLC analysis is based on a relatively slow laminar flow technique. The laminar flow of the column relies on the column packing material, which is the basis for separating the analytes of interest from the sample. Those skilled in the art understand that separation in such columns is a diffusion process. HPLC has been successfully applied to the separation of compounds in biological samples, but significant amounts of Sample preparation is required before separation and subsequent analysis by mass spectrometry (MS). Furthermore, most HPLC systems are not compatible with mass spectrometry, making the technique labor-intensive. The equipment is not being used to its full potential, with one HPLC system being used for one MS instrument. As a result, it takes a long time to perform multiple assays. Need.

[0097] Regarding the use of HPLC for sample removal prior to mass spectrometry analysis, Various methods have been described, see, e.g., Taylor et al., Therapeutic Drugs. ug Monitoring, 22:608-12 (2000); and Salm et al., Clin. Therapeutics, Vol. 22, Suppl. B: B71-B85 (2 000).

[0098] Those skilled in the art will be able to select suitable HPLC equipment and columns for use with CgA. A chromatographic column is a medium (i.e., a The medium generally includes fine particles. The compound has a binding surface that interacts with various chemical moieties to facilitate separation of the chemical moieties. The bonding surface is a hydrophobic bonding surface, such as an alkyl bonding surface. , C-4, C-8, C-12, or C-18 bonded alkyl groups, preferably C-18 bonded groups The chromatographic column may include an injection port for receiving a sample and a fractionation port. In one embodiment, the sample is passed through a drain port for draining the effluent containing the sample. a sample (or pre-purified sample) is applied to the column at the injection port; The solution is eluted with a solvent or solvent mixture and discharged through the discharge port. The column can be selected to elute the analyte of interest. - Use gradient mode, isocratic mode, or polymorphic (i.e., mixed) mode During chromatography, separation of materials can be achieved by a variety of variables, such as elution time. The effect of the solvent (also known as the "mobile phase"), elution mode, gradient conditions, temperature, etc. be influenced.

[0099] In certain embodiments, the analyte is selected from those in which the analyte of interest is reversibly retained by the column packing material. The sample is then loaded onto the column under conditions where the sample is retained while one or more other substances are not retained. In such an embodiment, the first mobile phase conditions may be: The analyte of interest can be configured to be retained by the column, and the second mobile phase The conditions are then such that the unretained substances are washed out and the retained substances are removed from the column. Alternatively, the analyte may be one or more other substances. Samples are analyzed under mobile phase conditions such that the analytes of interest elute at different rates compared to the Such a procedure may involve the application of one or more of the samples to a column. the possibility of enriching the amount of one or more analytes of interest relative to a number of other components There is.

[0100] In one preferred embodiment, the HTLC is a hydrophobic column chromatography system. In certain preferred embodiments, Cohesive Tech nologies TurboFlow Cyclone P® Polymer The base column (particle size 60 μm, column dimensions 50 × 1.0 mm, pore size 100 Å) was used. In a related preferred embodiment, Phe is used, which has a hydrophilic end-capping. Synergi Polar-RP® Ether from nomenex Inc. Analytical column for bonded phenyl (particle size 4 μm, column dimensions 150 × 2.0 mm, pore size In certain preferred embodiments, HTLC and HPLC are performed using a mobile phase of The analysis is carried out using HPLC-grade ultrapure water and 100% methanol.

[0101] Careful valve selection and connector piping eliminates the need for any manual steps. Two or more chromatographic columns are used so that material passes from one column to the next. In a preferred embodiment, the valve selection and piping The work is managed by a computer that is pre-programmed to carry out the necessary steps. Most preferably, the chromatography system also includes a detector system, e.g. The system is connected to the S system in such an online manner. Simply place the tray of samples into the autosampler and the computer will do the rest. - performed under controlled conditions, so that the purification and analysis of all selected samples is complete .

[0102] In certain preferred embodiments, CgA or fragments thereof in a sample are ionized In a particularly preferred embodiment, the chromatography is carried out by gas chromatography. It's not graphics.

[0103] Detection and quantification by mass spectrometry In various embodiments, CgA or a fragment thereof can be prepared by any method known to those of skill in the art. Mass spectrometry can be used to analyze the fractionated samples. A mass spectrometer having an ion source that ionizes and produces charged molecules for further analysis. For example, sample ionization can be performed using electron ionization, chemical ionization, , electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (AP CI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid Laser desorption ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization Ionization, field desorption, thermospray / plasma spray ionization, surface-enhanced laser desorption Ionization (SELDI), Inductively Coupled Plasma (ICP), and Particle Beam Ionization Those skilled in the art will appreciate that the choice of ionization method depends on the analyte being measured, the type of sample, and the type of ionization method being used. This can be determined based on the type of detector, the choice of positive mode or negative mode, etc. I understand.

[0104] In a preferred embodiment, CgA or a fragment thereof is The sample is ionized by electrospray ionization (ESI) in the active mode.

[0105] After the sample is ionized, the resulting positively or negatively charged ions are The ions may be analyzed to determine their mass-to-charge ratio. Suitable analyzers for this purpose include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Ions are detected using several detection modes. For example, selected ions can be detected, i.e., in selective ion monitoring mode. Ions can be detected using a scanning inversion (SIM) or alternatively, the ions can be detected using a scanning inversion (SIM) mode. For example, using multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio can be determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" device, the A force proportional to the applied DC potential, the amplitude of the RF signal, and the mass / charge ratio acts on the oscillatory The voltage and amplitude are applied to ions in a radio frequency field. Selected so that only on ions travel the length of the quadrupole while all other ions are missed. Therefore, quadrupole instruments can be used to "filter" ions injected into the instrument. It can act as both a "mass detector" and a "mass detector."

[0106] By utilizing "tandem mass spectrometry" or "MS / MS", the resolution of MS technology This technique allows for the enhancement of the precursor ions (parent ions) generated from the target molecules. The precursor ions (also called ions) can be filtered in the MS instrument, and is then fragmented to produce a single molecule that is then subjected to analysis in a second MS step. Or, multiple fragment ions (also called daughter ions or product ions) result. By carefully selecting precursor ions, only ions produced by specific analytes are identified. They pass through a fragmentation chamber where they collide with atoms of an inert gas to form fragment ions. Both precursor ions and fragment ions are generated by ionization / fragmentation. MS / MS technology is an extremely powerful analytical tool because it reproducibly produces nucleotides under a set of defined conditions that catalyze their synthesis. For example, a combination of filtering / fragmentation can provide analytical tools to identify interfering substances. and can be used to remove ions, particularly in complex samples, such as biological samples. It can be useful.

[0107] Mass spectrometers generally perform ion scanning; that is, scanning over a predetermined range (e.g., 100 ~1000 amu), the relative abundance of each ion with a specific mass / charge is calculated using The results of the analyte assay, i.e., mass spectra, are provided to the user. can be correlated with the amount of analyte in the original sample by numerous methods known in the art. For example, provided that sampling and analytical parameters are carefully controlled, The relative abundance of a given ion can be calculated by converting the relative abundance to the absolute amount of the original molecule. Alternatively, molecular standards can be run alongside the samples. A standard curve is then constructed based on the ions generated from the standards. Using a standard curve, the relative abundance of a given ion is converted to the absolute amount of the original molecule. In certain preferred embodiments, an internal standard may be used as a standard for calculating the amount of CgA. Methods for generating and using such standard curves are well known in the art. It is well known in the art and one skilled in the art has the ability to select an appropriate internal standard. An isotope of gA can be used as an internal standard. The amount of the ion is related to the amount of the original molecule. Numerous other methods for linking are known to those skilled in the art.

[0108] One or more steps of the method may be performed using automated machinery. In embodiments, one or more purification steps are performed online, and more preferably In most cases, all of the purification and mass spectrometry steps are performed online. obtain.

[0109] In certain embodiments, such as when precursor ions are isolated for further fragmentation, In MS / MS and other methods, collision activation dissociation is used. It is often used to generate fragment ions for further detection. In D, precursor ions gain energy through collisions with inert gas, but then They fragment through a process called "unimolecular decomposition." The increase in vibrational energy Due to this, sufficient energy is introduced into the precursor ions so that certain bonds within the ions can be broken. It must be accumulated within the

[0110] In a particularly preferred embodiment, CgA is detected and analyzed using MS / MS as follows: The sample is subjected to liquid chromatography, preferably HPLC, and / or quantified. The liquid solvent flow from the chromatographic column is then directed to the heated MS / MS analyzer. The solvent / analyte mixture enters the nebulizer interface, and the The analyte is converted to a vapor in a heated tube at the face. The ions, e.g., precursor ions, pass through an aperture in the device, and Enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, Ions (i.e., "precursors" and "fragments") are classified based on their mass-to-charge ratio (m / z). Quadrupole 2 (Q2) is the collision cell where ions are The first quadrupole (Q1) of the mass spectrometer detects the mass-to-charge ratio of CgA. The ratio is used to select molecules. A precursor ion with the correct mass / charge ratio for CgA is selected by collision. While undesirable species with other mass / charge ratios can be introduced into the chamber (Q2), Any precursor ions entering Q2 collide with the sides of the quadrupole and are removed. It collides with neutral argon gas molecules and fragments. This process is called collisional activation solution. The generated fragment ions are introduced into quadrupole 3 (Q3). , where the fragment ions of CgA are selected while other ions are removed. do.

[0111] The method involves MS / MS performed in either positive or negative ion mode. Using standard methods well known in the art, one skilled in the art can Identify one or more fragment ions of a specific CgA precursor ion available for selection. It has the ability to distinguish.

[0112] When an ion collides with the detector, it emits a pulse of electrons that is converted into a digital signal. The acquired data is plotted as the collected ion counts versus time. The resulting mass chromatogram is then transmitted to a computer that processes the data. The area under the peak corresponding to a particular ion, or The amplitude of such peaks is measured, and the area or amplitude is correlated with the amount of analyte of interest. In certain embodiments, fragment ions and / or precursor ions are The area under the curve or the amplitude of the peak is measured to determine the amount of CgA. using a calibration standard curve based on peaks derived from one or more ions of an internal molecular standard The relative abundance of a given ion can then be converted to the absolute amount of the original analyte.

[0113] The following examples serve to illustrate the invention. These examples do not limit the scope of the present method. There is no intention to impose any restrictions on [Example]

[0114] [Example 1] Quantitation of CgA by mass spectrometry Reagent Summary

[0115] [Table A]

[0116] Equipped with a Sciex 6500+QTrap mass spectrometer and an Agilent pump. Thermo Fisher Aria Cohesive TLX4, Hamil ton Microlab Star, SPEware IP8, to detect CgA was used.

[0117] Patient serum (100 μL) was added to 600 μL of 120 mM ammonium bicarbonate. The entire volume was then extracted using a mixed-mode anion exchange plate (Waters The samples were then diluted with 3% ammonium hydroxide. The column was washed twice with ethanol and water. CgA was then eluted and the internal standard (IS) was added. was added and the sample was evaporated under heated nitrogen.

[0118] After drying down, the samples were reconstituted, reduced, alkylated, and subjected to rapid enzymatic digestion. Trypsin was incubated for 2 hours in a Crowave system (Hudson Technology). Digested.

[0119] After digestion, the samples were acidified and then purified using an Aria TLX-4 Transcend U The analysis was carried out by LC-MS / MS on a PLC. Thermo Accucore C18 100 x 2.1 mm, 2. Peptides were separated using a 6 micron HPLC column. MS detector was Sciex6 500+Qtrap. Quantitation of CgA corresponds to representative tryptic peptides. , fragment ions generated during the MS / MS period and their corresponding heavily labeled ISs Based on chromatographic peaks, the analytical workflow is summarized in Figure 1.

[0120] Multiple reaction monitoring is used to detect both the analyte and the internal standard (IS). Used.

[0121] The concentration of chromogranin A (CgA) was determined using peak area ratio and a calibration curve. The assay for detecting ELQDLALQGAK (SEQ ID NO: 1) uses a labeled winged peptide. The internal standard used was: ILSILRHQNLLKELQDLAL*QGAK*ERA HQQK (SEQ ID NO: 2): *C 13 N 15 After digestion, the resulting peptides The sequence was ELQDLAL*QGAK* (SEQ ID NO: 3). In the assay for detecting RRPEDQELESLSAIEAELEK (SEQ ID NO: 4), the target A recognized winged peptide internal standard was used: EGSANRRPEDQELESL*SA IEAELEK*VAHQL (SEQ ID NO: 5);*C 13 N 15 Labeled amino acids. Digestion After this, the resulting peptide was 6).

[0122] Table 1: CgA levels in samples, as well as precursor and product ions (mass pair charge CgA fragments used to quantify the charge (m / z ratio).

[0123] [Table 1]

[0124] Table 2: CgA levels in samples, as well as precursor and product ions (mass pair charge CgA fragments used to quantify the charge (m / z ratio).

[0125] [Table 2]

[0126] Table 3: The values obtained were compared with those quantified by ELISA immunoassay. See also Figure 4.

[0127] [Table 3]

[0128] Samples from 308 patients were analyzed using the Cisbio CGA-ELISA-US CgA serum levels measured by immunoassay (Codolet, France) were used in our study. The values were compared with those from the LC-MS / MS assay.

[0129] After natural logarithmic transformation of the measurements, a normal distribution was observed in 308 patients. Paired t-tests were performed on the data, and the association between assays was evaluated using the Pearson correlation coefficient. and Passing & Bablok curve fitting. This was performed using SE-IT v2.30.

[0130] The assay was validated and the performance characteristics are shown in Table 4:

[0131] [Table 4]

[0132] Representative results for patient samples containing low and high levels of circulating CgA are shown in Figure 3. show.

[0133] LC-MS / MS measured CgA levels generally comparable to the CisBio assay However, substantial scatter was observed (Pearson correlation 0.76) (Figure 5).

[0134] Conclusion: We have developed a fully automated LC-MS / MS assay for the analysis of serum-derived CgA. The S assay was developed and validated.

[0135] All publications, patents, and patent applications, and other documents described or cited herein and the content of electronically available information, each publication is incorporated by reference. No. 6,027,797, filed Dec. 1, 2004, and entitled "Patent Document No. 10-110049," which is incorporated herein by reference in its entirety to the same extent as if specifically and individually indicated. Applicants reserve the right to modify, translate, translate and distribute any such documents, patents, patent applications, or other physical and electronic materials. The right to physically incorporate into this application any and all materials and information from the document is reserved. do.

[0136] The methods illustratively described herein may be used in combination with any one element not specifically disclosed herein. Alternatively, the practice may be suitably carried out in the absence of one or more elements, one or more limitations. For example, the terms "comprising," "including," and "containing" "Ining" and the like should be interpreted expansively and without limitation. The terms and expressions used are intended to be words of description and not of limitation. and in the use of such terms and expressions, any equivalent of the properties shown and described. There is no intention to exclude any of the foregoing or any portion thereof. Various modifications are intended to be within the scope of the claimed invention. It is recognized that the present invention is therefore directed to preferred embodiments and optional features. Modifications of the invention specifically disclosed by but incorporated into the invention disclosed herein. It is understood that modifications and variations may be made by those skilled in the art, and such modifications and variations are It should be understood that all such modifications are considered to be within the scope of the present invention.

[0137] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings included also form part of the method. The general description of the method with a condition or negative qualification that removes any of the subjects from the genus is The excised material is included whether or not specifically listed herein. do.

[0138] Other embodiments are within the scope of the following claims. is described by a Markush group, one skilled in the art would recognize that the invention is thereby Neither individual members nor subgroups of members of the Kashmiri group are described. Recognize that there is.

Claims

1. 1. A method for determining the amount of chromogranin A (CgA) in a sample, comprising: (a) purifying CgA in said sample; (b) Ionizing CgA to obtain a CgA fragment that is detectable by mass spectrometry. generating one or more ions; (c) determining the abundance of said ions from step (b) by mass spectrometry. To do wherein the amount of said ion is related to the amount of CgA in said sample.

2. 10. The method of claim 1, wherein the purifying comprises extraction by solid phase extraction (SPE).

3. 3. The method of claim 2, wherein the SPE is anion exchange solid phase extraction.

4. 3. The method of claim 2, wherein the SPE is a mixed-mode anion exchange solid phase extraction.

5. The method of claim 2, wherein the extracted sample is enzymatically digested.

6. The method of claim 5 , wherein the digestion comprises trypsin digestion.

7. 10. The method of claim 1, wherein the purifying comprises liquid chromatography.

8. The liquid chromatography comprises high performance liquid chromatography (HPLC). The method of claim 7.

9. The liquid chromatography comprises high turbulence liquid chromatography (HTLC). The method of claim 7.

10. The method of claim 1 , wherein the ionization comprises electrospray ionization (ESI). Law.

11. The method of claim 1 further comprising adding an internal standard.

12. The method of claim 11 , wherein the internal standard is isotopically labeled.

13. The method of claim 1 , wherein the sample is serum.

14. The method of claim 1 , wherein the sample is cerebrospinal fluid (CSF).

15. measuring the amount of precursor ions having a mass-to-charge ratio of 729.6±0.5; The method of claim 1.

16. The method of claim 1, comprising measuring the amount of a fragment of CgA.

17. The measured CgA fragment has the sequence RRPEDQELESLSAIEAELEK (sequence 17. The method of claim 16, comprising:

18. Fragment I having a mass-to-charge ratio of 831.5±0.5 or 989.5±0.5 The method of claim 1 , comprising measuring the amount of ON.

19. The method of claim 1, further comprising adding an internal standard.

20. 20. The method of claim 19, wherein the internal standard is isotopically labeled.

21. The internal standard is C １３ N １５ 20. The method of claim 19, comprising a labeled amino acid.

22. 10. The method of claim 1, wherein the internal standard is labeled at leucine (L) or lysine (K).

9. The method according to claim 9.

23. The internal standard has the sequence EGSANRRPEDQELESL*SAIEAELEK*VA HQL (SEQ ID NO: 5), wherein L* and K* are each C １３ N １５ Labeled amino 20. The method of claim 19, wherein the acid is an acid.

23. An internal standard precursor ion having a mass-to-charge ratio of 734.6±0.5, or 839.5± The amount of product ions with a mass-to-charge ratio of 0.5 or 997.6±0.5 was measured.

20. The method of claim 19, comprising determining

24. 10. The method of claim 1, wherein the limit of quantitation of the method is less than or equal to 50 ng / mL.

25. 10. The method of claim 1, wherein the detection limit of the method is less than or equal to 35.5 ng / mL. 。

26. The claimed method has linearity of quantitation over a range of 50 ng / mL to 50,000 ng / mL. The method according to claim 1.

27. 10. The method of claim 1, having inter- and intra-assay reproducibility of CV≦15%.

28. The mass spectrometry is a selective reaction monitoring (SRM) mass spectrometry. The method of claim 1, wherein the tree is a tree.

29. 10. The method of claim 1, which is fully automated.

30. The method of claim 1 , which does not include an antibody.

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