Method for simultaneous detection and quantification of insulin, proinsulins, proinsulins metabolic intermediates, C-peptides and C-peptide variants by mass spectrometry

Abstract

A method for determining the amount of an analyte in a sample is described, wherein the analyte comprises insulin, proinsulin, a metabolic intermediate of proinsulin, a C-peptide, a mutant C-peptide, or a mixture of any two or more thereof. Provided herein are mass spectrometry methods for detecting and quantifying an analyte (e.g., insulin, proinsulins, metabolic intermediates of proinsulins, C-peptides, mutant C-peptides, or mixtures of any two or more thereof) in a biological sample using an enrichment and / or purification method in combination with mass spectrometry techniques.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 589,367, filed October 11, 2023, the entire disclosure of which is incorporated herein by reference in its entirety for any and all purposes. Background Technology

[0002] Insulin is a hormone essential for regulating carbohydrate and fat metabolism in the body. Abnormal insulin levels indicate a blood glucose disorder and / or insulin resistance syndrome, such as diabetes. Diabetes and its complications represent a significant public health problem.

[0003] Proinsulin is the precursor hormone of insulin. The most common method for measuring proinsulin is immunoassay, but this method has the problem of cross-reactivity with proinsulin intermediates (such as des31,32-proinsulin and des64,65-proinsulin).

[0004] Therefore, new measurement methods with improved reliability and accuracy are still needed. Summary of the Invention

[0005] On the one hand, this article provides a method for determining the amount of an analyte in a sample by mass spectrometry, the method comprising: The analyte is captured by immunization from the sample; The analyte, which is captured by the immune system, is subjected to an ionization source under conditions suitable for generating one or more ions that can be detected by mass spectrometry. The amount of one or more ions was determined by mass spectrometry; and The amount of the analyte in the sample is determined by the amount of the one or more ions; in The analytes include insulin, proinsulin, metabolic intermediates of proinsulin, C-peptide, mutant C-peptide, or any mixture of two or more thereof. The metabolic intermediate of proinsulin is des-31,32-proinsulin, des-64,65-proinsulin, or a mixture thereof; and The mutant C-peptide is a C-peptide (C-peptide+R) having an additional arginine residue at the C-terminus, a C-peptide (C-peptide+RR) having two additional arginine residues at the C-terminus, or a mixture thereof.

[0006] On the other hand, this article provides a method for determining the amount of proinsulin in a sample, the method comprising: Proinsulin was captured by immunization from the sample; The proinsulin captured by the immune system is subjected to an ionization source under conditions suitable for generating one or more proinsulin ions detectable by mass spectrometry. The amount of one or more proinsulin ions was determined by mass spectrometry; and The amount of proinsulin in the sample is determined by the amount of one or more proinsulin ions.

[0007] On the other hand, this article provides a method for determining the amount of metabolic intermediates of proinsulin in a sample, the method comprising: The metabolic intermediates were captured by immunization from the sample; The metabolic intermediates that are captured by the immune system are subjected to an ionization source under conditions suitable for generating one or more metabolic intermediate ions that can be detected by mass spectrometry. The amounts of one or more metabolic intermediate ions were determined by mass spectrometry; and The amount of the proinsulin metabolic intermediate in the sample is determined by the amount of one or more of the metabolic intermediate ions; in The metabolic intermediate of proinsulin is de31,32-proinsulin, de64,65-proinsulin, or a mixture thereof.

[0008] On the other hand, this article provides a method for determining the amount of mutant C-peptide in a sample, the method comprising: The mutant C-peptide was captured by immunization from the sample; The immune-captured mutant C-peptide is subjected to an ionization source under conditions suitable for generating one or more mutant C-peptide ions detectable by mass spectrometry. The amount of one or more mutant C-peptide ions was determined by mass spectrometry; and The amount of the mutant C-peptide in the sample is determined by the amount of the one or more mutant C-peptide ions; in The mutant C-peptide is a C-peptide (C-peptide+R) having an additional arginine residue at the C-terminus, a C-peptide (C-peptide+RR) having two additional arginine residues at the C-terminus, or a mixture thereof.

[0009] In some embodiments, the analyte includes proinsulin. In some embodiments, the one or more ions include one or more proinsulin ions. In some embodiments, the one or more proinsulin ions include proinsulin precursor ions with a mass-to-charge ratio (m / z) of 1342.20 ± 0.5. In some embodiments, the one or more proinsulin ions include one or more proinsulin fragment ions selected from the group consisting of ions with m / z of 183.30 ± 0.5, 120.20 ± 0.5, and 219.30 ± 0.5.

[0010] In some embodiments, the analyte comprises a metabolic intermediate of proinsulin. In some embodiments, the metabolic intermediate of proinsulin comprises des-31,32-proinsulin. In some embodiments, the one or more ions comprise one or more des-31,32-proinsulin ions. In some embodiments, the one or more des-31,32-proinsulin ions comprise a des-31,32-proinsulin precursor ion with an m / z of 1299.50 ± 0.5. In some embodiments, the one or more des-31,32-proinsulin ions comprise one or more des-31,32-proinsulin fragment ions selected from the group consisting of ions with m / z of 129.20 ± 0.5, 226.20 ± 0.5, and 183.25 ± 0.5.

[0011] In some embodiments, the metabolic intermediate of proinsulin includes des-64,65-proinsulin. In some embodiments, the one or more ions include one or more des-64,65-proinsulin ions. In some embodiments, the one or more des-64,65-proinsulin ions include a des-64,65-proinsulin precursor ion with an m / z of 1304.30 ± 0.5. In some embodiments, the one or more des-64,65-proinsulin ions include one or more des-64,65-proinsulin fragment ions selected from the group consisting of ions with m / z of 147.15 ± 0.5, 130.15 ± 0.5, and 183.20 ± 0.5.

[0012] In some embodiments, the analyte comprises a mutant C-peptide. In some embodiments, the mutant C-peptide comprises a C-peptide (C-peptide+R) having an additional arginine residue at its C-terminus. In some embodiments, the one or more ions comprise one or more C-peptide+R ions. In some embodiments, the one or more C-peptide+R ions comprise a precursor C-peptide+R ion with an m / z of 1059.20 ± 0.5. In some embodiments, the one or more C-peptide+R ions comprise one or more C-peptide+R fragment ions selected from the group consisting of ions with m / z of 966.40 ± 0.5, 260.00 ± 0.5, and 844.30 ± 0.5. In some embodiments, the mutant C-peptide comprises a C-peptide (C-peptide+RR) having two additional arginine residues at its C-terminus. In some embodiments, the one or more ions comprise one or more C-peptide+RR ions. In some embodiments, the one or more C-peptide+RR ions comprise a C-peptide+RR precursor ion with an m / z of 1111.50 ± 0.5. In some embodiments, the one or more C-peptide + RR ions include one or more C-peptide + RR fragment ions selected from the group consisting of ions with m / z of 896.50±0.5, 499.90±0.5, and 260.30±0.5.

[0013] In some embodiments, the analyte includes insulin. In some embodiments, the one or more ions include one or more insulin ions. In some embodiments, the one or more insulin ions include insulin precursor ions with an m / z of 1162.30 ± 0.5. In some embodiments, the one or more insulin ions include one or more insulin fragment ions selected from the group consisting of ions with m / z of 226.20 ± 0.5, 136.10 ± 0.5, and 345.20 ± 0.5.

[0014] In some embodiments, the analyte comprises a C-peptide. In some embodiments, the one or more ions comprise one or more C-peptide ions. In some embodiments, the one or more C-peptide ions comprise a C-peptide precursor ion with an m / z of 1007.70 ± 0.5. In some embodiments, the one or more C-peptide ions comprise one or more C-peptide fragment ions selected from the group consisting of ions with m / z of 927.50 ± 0.5, 646.40 ± 0.5, and 533.30 ± 0.5.

[0015] In some embodiments, the analyte is not insulin, C-peptide, or a mixture of insulin and C-peptide.

[0016] In some embodiments, the sample includes a plasma or serum sample.

[0017] In some embodiments, the ionization source is an electrospray (ESI) ionization source. In some embodiments, ionization is in positive ion mode.

[0018] In some embodiments, the sample is subjected to acidic conditions prior to mass spectrometry. In some embodiments, subjecting the sample to acidic conditions includes subjecting the sample to formic acid. In some embodiments, the sample is subjected to basic conditions prior to mass spectrometry. In some embodiments, subjecting the sample to basic conditions includes subjecting the sample to trizma and / or ethanol.

[0019] In some embodiments, the sample is defatted before quantification by mass spectrometry.

[0020] In some embodiments, the methods described herein further include purifying the sample prior to mass spectrometry. In some embodiments, the purification includes subjecting the sample to liquid chromatography. In some embodiments, the liquid chromatography includes high-performance liquid chromatography (HPLC) or high-turbulence liquid chromatography (HTLC). In some embodiments, the purification includes subjecting the sample to solid-phase extraction (SPE).

[0021] In some embodiments, the mass spectrometry method is tandem mass spectrometry, high-resolution mass spectrometry, or high-resolution / high-accuracy mass spectrometry.

[0022] In some embodiments, the immunocapture includes the use of antibodies (e.g., monoclonal antibodies). In some embodiments, the antibodies are immobilized on magnetic beads. In some embodiments, the method described herein further includes washing and eluting the material immunocaptured on the magnetic beads. Attached Figure Description

[0023] Figure 1 Example chromatograms of detectable analytes in multiplexed LC-MS assays as described in Example 1.

[0024] Figure 2 This study compares the proinsulin immunoassay and LC-MS methods. A strong correlation was observed between the methods (Spearman r = 0.75).

[0025] Figure 3 This diagram shows the primary and secondary processing pathways of proinsulin, which produce 65,66-splitproinsulin, de64,65-proinsulin, 32,33-splitproinsulin, de31,32-proinsulin, C-peptide, and / or insulin. Detailed Implementation

[0026] definition To facilitate understanding of this technology, certain terms are defined below. Further definitions of the following and other terms are provided throughout this specification. Publications and other references cited herein—which describe the background of the technology and provide additional details on its practice—are incorporated herein by reference.

[0027] As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, a reference to “a protein” includes multiple protein molecules.

[0028] As used herein, the terms “purification,” “purifying,” and “enrichment” do not refer to the removal of all material from a sample—except for one or more analytes of interest. Rather, these terms refer to a procedure for enriching one or more analytes of interest in a quantity relative to other components in the sample that may interfere with the detection of the analyte of interest. Purifying a sample by various means allows for a relative reduction of one or more interfering substances, such as substances that may or may not interfere with the detection of selected precursor or daughter ions by mass spectrometry. Relative reduction—when this term is used—does not require the complete removal by purification of any substances present in the material to be purified along with the analyte of interest.

[0029] As used herein, the terms "immunopurification" or "immunopurify" refer to a purification procedure that uses antibodies (including polyclonal or monoclonal antibodies) to enrich one or more analytes of interest. Immunopurification can be performed using any immunopurification method known in the art. Typically, immunopurification procedures utilize antibodies that are bound, conjugated, or otherwise attached to a solid support (e.g., a column, pore, tube, gel, capsule, particle, or the like). Immunopurification as used herein includes, but is not limited to, procedures commonly referred to in the art as immunoprecipitation, and procedures commonly referred to in the art as affinity chromatography or immunoaffinity chromatography.

[0030] As used herein, the term "immunoparticle" refers to a capsule, bead, gel particle, or the like having antibodies bound, conjugated, or otherwise attached to its surface (on and / or within the particle). In some preferred embodiments, the immune particles are agarose gel beads or agarose beads. In alternative preferred embodiments, the immune particles include glass beads, plastic beads, or silica beads, or silica gel.

[0031] As used herein, the term "anti-insulin antibody" refers to any polyclonal or monoclonal antibody that has an affinity for insulin, and the term "anti-C-peptide antibody" refers to any polyclonal or monoclonal antibody that has an affinity for C-peptides. In various embodiments, the specificity of the antibody (e.g., an insulin antibody or an anti-C-peptide antibody) to chemical species other than the target antigen (e.g., insulin or C-peptide) can vary. For example, in some embodiments, the antibody is specific to the target antigen and therefore has little affinity for chemical species other than the target antigen; while in other embodiments, the antibody is nonspecific and therefore binds to certain chemical species other than the target antigen.

[0032] As used herein, the term "sample" means any sample that may contain an analyte of interest. As used herein, the term "body fluid" means any fluid that can be separated from an individual's body. For example, "body fluid" may include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc. In a preferred embodiment, the sample includes a body fluid sample from a person; preferably plasma or serum.

[0033] As used herein, the term "solid-phase extraction" or "SPE" refers to the process by which a chemical mixture is separated into components due to the affinity of components dissolved or suspended in a solution (i.e., the mobile phase) for a solid (i.e., the solid phase) through which the solution passes or surrounds. In some cases, the solid phase may retain undesired components of the mobile phase as the mobile phase passes through or surrounds it, resulting in the purification of the analyte in the mobile phase. In other cases, the solid phase may retain the analyte, allowing undesired components of the mobile phase to pass through or surround it. In these cases, a second mobile phase is then used to elute the retained analyte from the solid phase for further processing or analysis. SPE (including TFLC) can be operated via single-mode or mixed-mode mechanisms. Mixed-mode mechanisms utilize ion exchange and hydrophobic retention within the same column; for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention, or it may exhibit strong cation exchange and hydrophobic retention.

[0034] Typically, the affinity of SPE column packing material for the analyte can be due to any of a variety of mechanisms, such as one or more chemical interactions or immunoaffinity interactions. In some embodiments, SPE of insulin is performed without the use of immunoaffinity column packing material. That is, in some embodiments, insulin is purified from the sample through an SPE column that is not an immunoaffinity column.

[0035] As used herein, the term “chromatography” refers to the process by which a chemical mixture carried by a liquid or gas is separated into its constituent components due to the differential distribution of the chemical entities as the mixture flows around or through a stationary liquid or solid phase.

[0036] As used herein, the term "liquid chromatography" or "LC" means the process of selectively blocking one or more components of a fluid solution when a fluid is uniformly permeated through a column of finely broken material or through a capillary channel. Blockage is caused by the distribution of components in the mixture between one or more stationary phases and the bulk fluid (i.e., the mobile phase) as this fluid moves relative to the stationary phase(s). Examples of "liquid chromatography" include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent liquid chromatography (TFLC) (sometimes referred to as high-turbulence liquid chromatography (HTLC) or high-throughput liquid chromatography).

[0037] As used in this article, the term "high performance liquid chromatography" or "HPLC" (sometimes referred to as "high pressure liquid chromatography") refers to liquid chromatography that increases resolution by forcing the mobile phase through a stationary phase (usually a densely packed column) under pressure.

[0038] As used herein, the term "turbulent liquid chromatography" or "TFLC" (sometimes referred to as high-turbulence liquid chromatography or high-throughput liquid chromatography) refers to a form of chromatography that utilizes the turbulence of the analyte passing through a column packing as the basis for separation. TFLC has been applied to the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, for example, Zimmer et al., J Chromatogr A 854:23-35 (1999); see also U.S. Patent Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further explain TFLC. Those skilled in the art understand “turbulence.” When a fluid flows slowly and smoothly, the flow is called “laminar.” For example, fluid moving at a low velocity through an HPLC column is laminar. In laminar flow, the motion of fluid particles is ordered, with the particles generally moving in a straight line. At higher velocities, the inertia of water overcomes fluid friction, and turbulence is generated. Fluid that does not come into contact with irregular boundaries “overtakes” fluid that is slowed by friction or deflected by uneven surfaces. When a fluid flows turbulently, it flows in the form of eddies and vortices (or eddies), having more “drag” than when the flow is laminar. Numerous references can be used to help determine when a fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction , PS Bernard & JM Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow , Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

[0039] As used herein, the term “gas chromatography” or “GC” refers to a chromatographic method in which a sample mixture is vaporized and injected into a stream of carrier gas (such as nitrogen or helium), moves through a column containing a stationary phase consisting of liquid or particulate solids, and is separated into its component compounds according to the affinity of the compounds for the stationary phase.

[0040] As used herein, the terms "large particle column" or "extraction / extraction column" refer to a chromatographic column containing an average particle size greater than about 50 μm. As used in this context, the term "about" means ±10%.

[0041] As used herein, the term "analytical column" refers to a chromatographic column with sufficient plates to achieve separation of materials in a sample, eluting from the column in a manner sufficient to allow determination of the presence or amount of an analyte. This type of column is generally distinguished from an "extraction column," whose general purpose is to separate from non-retained materials or to extract retained materials to obtain a purified sample for further analysis. As used in this context, the term "about" means ±10%. In a preferred embodiment, the analytical column contains particles with a diameter of about 5 μm.

[0042] As used herein, the terms "on-line" or "inline," such as in "online automated method" or "online extraction," refer to procedures that can be performed without operator intervention. In contrast, the term "off-line," as used herein, refers to procedures that require manual operator intervention. Therefore, if the sample undergoes precipitation and the supernatant is then manually loaded into the autosampler, the precipitation and loading steps are offline from subsequent steps. In various embodiments of the method, one or more steps can be performed in an online automated manner.

[0043] As used herein, the term "sample injection" refers to the introduction of an aliquot of a single sample into an analyzer (e.g., a mass spectrometer). This introduction can be performed directly or indirectly. Indirect sample injection can be achieved, for example, by injecting an aliquot into an HPLC column connected inline to the mass spectrometer.

[0044] As used in this article, the term "same sample injection" in relation to multiple analytes by mass spectrometry means that by measuring the ions of different analytes from the same (i.e., identical) sample injection, the ions of two or more different analytes are essentially determined simultaneously.

[0045] As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique for identifying compounds by their mass. MS refers to the method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m / z”. MS techniques generally involve (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating their mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ion generator, a mass analyzer, and an ion detector. Typically, one or more molecules of interest are ionized, and the ions are then introduced into the mass spectrometer, where they follow a spatial path that depends on their mass (“m”) and charge (“z”) due to a combination of magnetic and electric fields. See, for example, U.S. Patent No. 6,204,500, entitled "Mass Spectrometry From Surfaces"; U.S. Patent No. 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry"; U.S. Patent No. 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry"; U.S. Patent No. 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes"; Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

[0046] As used in this article, "high-resolution / high-accuracy mass spectrometry" refers to mass spectrometry performed using a mass analyzer capable of measuring the mass-to-charge ratio of charged species with sufficient precision and accuracy to identify unique chemical ions. Identifying a unique chemical ion is possible when the individual isotopic peaks from the ion are easily distinguishable. The specific resolution and mass accuracy required to identify unique chemical ions vary with the ion's mass and charge state.

[0047] As used herein, the term "resolution capability" or "resolution capability (FWHM)" (also referred to in the art as "m / Δm") is used. 50% "" refers to the width of the mass peak at 50% maximum height, which is the observed mass-to-charge ratio divided by the maximum height (full width half maximum, "FWHM").

[0048] As used in this article, a “unique chemical ion” in mass spectrometry refers to a single ion consisting of a single atom. A single ion can be single-charged or multi-charged.

[0049] As used in this article, the term "accuracy" (or "mass accuracy") in mass spectrometry refers to the potential deviation between the instrument response and the true m / z of the ion being studied. Accuracy is usually expressed in parts per million (ppm).

[0050] High-resolution / high-accuracy mass spectrometry methods can be implemented on instruments capable of mass analysis at FWHMs greater than 10,000, 15,000, 20,000, 25,000, 50,000, 100,000, or even higher. Similarly, the methods of this invention can be implemented on instruments capable of mass analysis at accuracies less than 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5 ppm, 3 ppm, or even lower. Instruments capable of possessing these performance characteristics may include certain orbital trap mass analyzers, time-of-flight (“TOF”) mass analyzers, or Fourier transform ion cyclotron resonance mass analyzers.

[0051] The term "orbit trap" describes an ion trap consisting of an external barrel-shaped electrode and a coaxial internal electrode. Ions are tangentially injected into the electric field between the electrodes, and as the ions orbit the coaxial internal electrode, they are trapped by centrifugal force balanced by the electrostatic interaction between the ions and the electrodes. During orbital orbit, the trajectory of the trapped ions oscillates along the axis of the central electrode at a resonant frequency relative to the ion's mass-to-charge ratio. Detecting the orbital oscillation frequency allows the orbit trap to be used as a mass analyzer with high accuracy (down to 1-2 ppm) and high resolution (FWHM) (up to about 200,000). Orbit trap-based mass analyzers are described in detail in U.S. Patent No. 6,995,364 (which is incorporated herein by reference in its entirety). The use of orbit trap analyzers for qualitative and quantitative analysis of a variety of analytes has been reported. See, for example, U.S. Patent Application Publication No. 2008 / 0118932 (filed November 9, 2007); Bredehöft, et al., Rapid Commun. Mass Spectrom., 2008, 22:477-485; Le Breton, et al., Rapid Commun. Mass Spectrom., 2008, 22:3130-36; Thevis, et al., Mass Spectrom.Reviews, 2008, 27:35-50; Thomas, et al., J. Mass Spectrom., 2008, 43: 908-15; Schenk, et al., BMC Medical Genomics, 2008, 1:41; and Olsen, et al., Nature Methods, 2007, 4:709-12.

[0052] As used herein, the term "operating in negative ion mode" refers to mass spectrometry methods that generate and detect negative ions. As used herein, the term "operating in positive ion mode" refers to mass spectrometry methods that generate and detect positive ions. In a preferred embodiment, the mass spectrometry is performed in positive ion mode.

[0053] As used herein, the term "ionization" or "ionizing" refers to the process of generating analyte ions with a net charge equal to or equal to one or more electron units. A negative ion is an ion with a net negative charge of one or more electron units, while a positive ion is an ion with a net positive charge of one or more electron units.

[0054] As used herein, the term "electron ionization" or "EI" refers to a method in which an analyte of interest in the gas or vapor phase interacts with a stream of electrons. Collisions between electrons and the analyte produce analyte ions, which can then be subjected to mass spectrometry techniques.

[0055] As used herein, the term “chemical ionization” or “CI” refers to a method in which a reagent gas (e.g., ammonia) is subjected to electron collisions and analyte ions are formed through the interaction of reagent gas ions and analyte molecules.

[0056] As used herein, the term "Fast Atom Bombardment" or "FAB" refers to a method in which a high-energy atomic beam (typically Xe or Ar) collides with a non-volatile sample, desorbing and ionizing the molecules contained within the sample. The test sample is dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6-crown ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine, and triethanolamine. Selecting a suitable matrix for the compound or sample is an empirical process.

[0057] As used herein, the term "matrix-assisted laser desorption / ionization" or "MALDI" refers to a method of exposing non-volatile samples to laser irradiation, which desorbs and ionizes the analytes in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. In MALDI, the sample is mixed with an energy-absorbing matrix that promotes the desorption of analyte molecules.

[0058] As used herein, the term "surface-enhanced laser desorption / ionization" or "SELDI" refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through a variety of ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more analytes of interest. As in MALDI, this process may also employ energy-absorbing materials to facilitate ionization.

[0059] As used herein, the term "electrospray ionization" or "ESI" refers to a method of passing a solution along a short length of a capillary—at which a high positive or negative potential is applied. The solution reaching the capillary end is vaporized (atomized) into a jet or spray of very small solution droplets in solvent vapor. This droplet mist flows through an evaporation chamber. As the droplets become smaller, the surface charge density increases until the natural repulsion between like charges causes the release of ions and neutral molecules.

[0060] As used herein, the terms "atmospheric pressure chemical ionization" or "APCI" refer to a mass spectrometry method similar to ESI; however, APCI generates ions through ion-molecule reactions occurring within a plasma at atmospheric pressure. The plasma is maintained by a discharge between a spray capillary and a counter electrode. The ions are then typically extracted into the mass analyzer using a set of differentially pumped skimmer stages. Countercurrent flow of dry, preheated N2 gas can be used to improve solvent removal. Gas-phase ionization in APCI can be used more efficiently than ESI for analyzing low-polarity species.

[0061] As used herein, the term "atmospheric pressure photoionization" or "APPI" refers to the mass spectrometric form of ionization of the molecule M via photon absorption and electron emission to form the molecular ion M+. Because the photon energy is typically just above the ionization potential, the molecular ion is less prone to dissociation. In many cases, it is possible to analyze samples without the need for chromatography, thus saving significant time and costs. In the presence of water vapor or proton solvents, the molecular ion can extract H to form MH+. This often occurs when M has a high proton affinity. Because the sum of M+ and MH+ is constant, this does not affect quantitative accuracy. Drug compounds in proton solvents are often observed as MH+, while nonpolar compounds (such as naphthalene or testosterone) typically form M+. See, for example, Robb et al., Anal. Chem 2000, 72(15): 3653-3659.

[0062] As used herein, the term “inductively coupled plasma” or “ICP” refers to a method in which a sample interacts with a partially ionized gas at a sufficiently high temperature such that most of the elements are atomized and ionized.

[0063] As used in this article, the term "field desorption" refers to a method in which a non-volatile test sample is placed on an ionization surface and a strong electric field is used to generate analyte ions.

[0064] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the transfer of the analyte into the gas phase. Laser desorption / thermal desorption is a technique in which a sample containing the analyte is thermally desorbed into the gas phase by a laser pulse. The laser strikes the back of a specially designed 96-well plate with a metallic substrate. The laser pulse heats the substrate, and the heat causes the sample to transfer into the gas phase. The gas phase sample is then drawn into a mass spectrometer.

[0065] As used in this article, the term “selective ion monitoring” is a detection mode of a mass spectrometer in which only ions within a relatively narrow mass range (typically about one mass unit) are detected.

[0066] As used in this article, “multi-reaction mode” (sometimes called “selective reaction monitoring”) is a detection mode of a mass spectrometer in which precursor ions and one or more fragment ions are selectively detected.

[0067] As used herein, the terms “lower limit of quantitation,” “lower limit of quantitation,” or “LLOQ” refer to the point at which a measurement becomes quantitatively meaningful. At this LOQ, the analyte response is identifiable, discrete, and reproducible, with a relative standard deviation (RSD%) of less than 20% and an accuracy of 85% to 115%.

[0068] As used herein, the term “limit of detection” or “LOD” is the point at which a measured value exceeds the uncertainty associated with it. LOD is the point at which a value exceeds the uncertainty associated with its measurement and is defined as 3 times the RSD of the mean at zero concentration.

[0069] As used herein, the “amount” of an analyte in a body fluid sample typically refers to the absolute value of the mass of the analyte detectable in the volume of the sample. However, amount also considers a relative amount compared to the amount of another analyte. For example, the amount of an analyte in a sample may be greater than the control or normal level of an analyte normally present in the sample.

[0070] As used in this document for quantitative measurements (excluding measurements of ion mass), the term "about" means the indicated value plus or minus 10%. Mass spectrometry instruments can vary slightly when determining the mass of a given analyte. The term "about" in the context of ion mass or ion mass / charge ratio means + / - 0.50 atomic mass units.

[0071] As used herein, the term “amino acid” refers to natural amino acids, non-natural amino acids, and amino acid analogs, which, unless otherwise stated, are D and L stereoisomers (if their structures allow for such stereoisomerism).

[0072] As used herein, the term "peptide" refers to a short polymer of amino acids linked together by peptide bonds. Compared to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are, for example, about 50 amino acids or fewer in length. Peptides can include natural amino acids, non-natural amino acids, amino acid analogs, and / or modified amino acids. Peptides can be subsequences of naturally occurring proteins or non-natural (synthetic) sequences.

[0073] As used herein, the term "mutant peptide" refers to a peptide having an amino acid sequence that differs from the most common variant found in nature (known as the "wild-type" sequence). A mutant peptide can be a subsequence of a mutated protein or polypeptide (e.g., a subsequence of a naturally occurring protein that is not the most common sequence in nature), or a peptide that is not a subsequence of a naturally occurring protein or polypeptide. For example, a "mutant C-peptide" or "C-peptide variant" can be a subsequence of a naturally occurring non-wild-type C-peptide, or a unique sequence not found in naturally occurring C-peptides.

[0074] The above overview is non-limiting, and other features and advantages of the present technology will become apparent from the following detailed description and claims.

[0075] method Insulin is a small peptide composed of 51 amino acids. It consists of two chains (α-chain and β-chain) linked by disulfide bonds between cysteine ​​residues. The α-chain has 21 amino acids and the β-chain has 30 amino acids.

[0076] Proinsulin is the precursor hormone of insulin. Metabolic intermediates of proinsulin can be formed during proinsulin processing, such as de31,32-proinsulin and de64,65-proinsulin. C-peptides are peptides that link the two polypeptide chains of insulin and are released from proinsulin during processing, subsequently co-secreted by pancreatic β-cells. Due to differences in half-life and hepatic clearance, peripheral blood levels of C-peptides and insulin are no longer equimolarly, but remain highly correlated. Mutant C-peptides (or C-peptide variants) can be subsequences of naturally occurring non-wild-type C-peptides or unique sequences not present in naturally occurring C-peptides. In some embodiments, a mutant C-peptide (or C-peptide variant) is a C-peptide with an additional arginine residue at the C-terminus (referred to as "C-peptide+R"). In some embodiments, a mutant C-peptide (or C-peptide variant) is a C-peptide with two additional arginine residues at the C-terminus (referred to as "C-peptide+RR").

[0077] The determination of serum insulin is primarily used to diagnose glycemic disorders in patients with diabetes and prediabetes during the assessment of insulin resistance syndrome. Therefore, the methods described herein can be used to diagnose glycemic disorders or insulin resistance syndrome in patients with diabetes and prediabetes. In some embodiments, the methods described herein can be used to diagnose diabetes. In some embodiments, the methods described herein can be used to assess insulin resistance syndrome. In some embodiments, the methods described herein can be used to differentiate insulin-secreting tumors from exogenous insulin administration as a cause of hypoglycemia. In some embodiments, the methods described herein can be used to differentiate type 1 diabetes from type 2 diabetes. In some embodiments, the methods described herein can be used to assess the risk of diabetes in patients with prediabetes.

[0078] On one hand, this document provides a method for measuring insulin levels in a patient by using mass spectrometry to determine the amount of an analyte in a sample. As described herein, the analyte may include insulin, proinsulin, proinsulin metabolic intermediates (e.g., de31,32-proinsulin, de64,65-proinsulin, or mixtures thereof), C-peptide, mutant C-peptide (e.g., C-peptide+R, C-peptide+RR, or mixtures thereof), or any mixture of two or more thereof. In some embodiments, the method provided herein includes multiplexing, which simultaneously measures the amount of insulin, proinsulin, proinsulin metabolic intermediates, C-peptide, mutant C-peptide, or any mixture of two or more thereof in a sample by mass spectrometry. In some embodiments, the amount of analyte in the sample is correlated with the amount of insulin in the patient. In some embodiments, the amounts of, for example, insulin and C-peptide in the sample are used to determine a corresponding ratio (e.g., the ratio of insulin to C-peptide) in the patient.

[0079] In some embodiments, the methods described herein include: subjecting a sample to an enrichment process to obtain a fraction rich in the analytes described herein; subjecting the enriched analytes to an ionization source under conditions suitable for generating one or more analyte ions detectable by mass spectrometry; and determining the amount of one or more analyte ions by mass spectrometry. In some embodiments, the determined amount of one or more analyte ions is used to determine the amount of analyte in the sample. In some embodiments, the amount of analyte in the sample is related to the amount of insulin in a patient.

[0080] In some embodiments, the methods described herein include subjecting a sample to an enrichment process to obtain a fraction rich in the analytes described herein. For example, the methods described herein may include immunocapturing the analyte from a sample. In some embodiments, the immunocapturing provided herein includes using an antibody (e.g., an anti-insulin antibody or an anti-C-peptide antibody). In some embodiments, the antibody provided herein is a monoclonal antibody. In some embodiments, the antibody provided herein is a mouse monoclonal antibody. In some embodiments, the antibody provided herein is a monoclonal IgG antibody. In some embodiments, the antibody provided herein is a polyclonal antibody. In some embodiments, the antibody (e.g., an anti-insulin antibody or an anti-C-peptide antibody) is immobilized on magnetic beads. In some embodiments, the analyte immunocaptured on the magnetic beads is washed and eluted.

[0081] In some embodiments, the serum is defatted before quantification by mass spectrometry. In some embodiments, one or more defatting agents are used to remove lipids from the sample. In some embodiments, the defatting agent is CLEANASCITE®.

[0082] In some embodiments, the methods provided herein include purifying the sample prior to mass spectrometry. In some embodiments, the methods include purifying the sample using liquid chromatography. In some embodiments, the liquid chromatography includes high-performance liquid chromatography (HPLC) or high-turbulence liquid chromatography (HTLC). In some embodiments, the methods include subjecting the sample to solid-phase extraction (SPE).

[0083] In some embodiments, the mass spectrometry method includes tandem mass spectrometry. In some embodiments, the mass spectrometry method is high-resolution mass spectrometry. In some embodiments, the mass spectrometry method is high-resolution / high-accuracy mass spectrometry.

[0084] In some embodiments, ionization is performed via electrospray ionization (ESI). In some embodiments, ionization is performed via atmospheric pressure chemical ionization (APCI). In some embodiments, the ionization is in a positive ion mode.

[0085] In some embodiments, the methods provided herein include adding an internal standard to the sample. In some embodiments, the internal standard for insulin is bovine insulin. In some embodiments, the internal standard for C-peptide is a C-peptide heavy internal standard. In some embodiments, the internal standard is labeled. In some embodiments, the internal standard is deuterated or isotopically labeled.

[0086] In some embodiments, the patient sample is a serum sample. In some embodiments, the patient sample is a plasma sample. In some embodiments, the patient sample is a blood, saliva, or urine sample.

[0087] In some embodiments, the sample is subjected to acidic conditions before ionization. In some embodiments, subjecting the sample to acidic conditions includes subjecting the enriched analyte (e.g., the analyte described herein) to formic acid. In some embodiments, the sample is subjected to alkaline conditions before ionization. In some embodiments, subjecting the sample to alkaline conditions includes subjecting the sample to trizma. In some embodiments, subjecting the sample to alkaline conditions includes subjecting the sample to trizma and ethanol.

[0088] In some embodiments, this document provides a method for determining the amount of an analyte (e.g., the analyte described herein) in a sample using mass spectrometry, comprising: (a) enriching the analyte in the sample by an extraction technique; (b) subjecting the purified analyte from step (a) to liquid chromatography to obtain an analyte-rich fraction from the sample; (c) subjecting the enriched analyte to an ionization source under conditions suitable for generating analyte precursor ions detectable by mass spectrometry; (d) generating one or more fragment ions of the precursor ions; and (e) determining the amount of one or more fragment ions by mass spectrometry. In some embodiments, the determined amount of one or more ions is used to determine the amount of the analyte in the sample.

[0089] In some embodiments, the collision energy is about -90 eV to about -20 eV (e.g., about -89 eV to about -29 eV). In some embodiments, the collision energy is about 20 eV to about 50 eV (e.g., about 27 eV to about 40 eV).

[0090] Therefore, according to some aspects, this disclosure provides a method for determining the amount of an analyte (e.g., the analyte described herein) in a sample by mass spectrometry, the method comprising one or more of the following: immunocapturing the analyte from the sample; subjecting the immunocaptured analyte to an ionization source under conditions suitable for generating one or more ions detectable by mass spectrometry; determining the amount of one or more ions by mass spectrometry; and determining the amount of the analyte in the sample by the amount of one or more ions.

[0091] In some embodiments, the analyte includes insulin, proinsulin, a metabolic intermediate of proinsulin, a C-peptide, a mutant C-peptide, or any two or more of the above. In some embodiments, the analyte includes proinsulin. In some embodiments, the analyte includes a metabolic intermediate of proinsulin (e.g., de31,32-proinsulin, de64,65-proinsulin, or a mixture thereof). In some embodiments, the analyte includes a mutant C-peptide (e.g., C-peptide+R, C-peptide+RR, or a mixture thereof). In some embodiments, the analyte is not insulin (e.g., the analyte may include insulin, but further includes at least one other type, such as those selected from proinsulin, proinsulin metabolic intermediates, and mutant C-peptides). In some embodiments, the analyte is not a C-peptide (e.g., the analyte may include a C-peptide, but further includes at least one other type, such as those selected from proinsulin, proinsulin metabolic intermediates, and mutant C-peptides). In some embodiments, it is not a mixture of insulin and C-peptide (e.g., the analyte may include a mixture of insulin and C-peptide, but further includes at least one other type, such as those selected from proinsulin, proinsulin metabolic intermediates and mutant C-peptides).

[0092] In some embodiments, this disclosure provides a method for determining the amount of an analyte (e.g., the analyte described herein) in a sample by mass spectrometry, the method comprising one or more of the following: immunocapturing the analyte from the sample; subjecting the immunocaptured analyte to an ionization source under conditions suitable for generating one or more ions detectable by mass spectrometry; determining the amount of one or more ions by mass spectrometry; and determining the amount of the analyte in the sample by the amount of one or more ions; wherein the analyte comprises insulin, proinsulin, a metabolic intermediate of proinsulin, a C-peptide, a mutant C-peptide, or any two or more of the above; the metabolic intermediate of proinsulin is de-31,32-proinsulin, de-64,65-proinsulin, or a mixture thereof; and the mutant C-peptide is C-peptide+R, C-peptide+RR, or a mixture thereof.

[0093] In some embodiments, this disclosure provides a method for determining the amount of an analyte (e.g., the analyte described herein) in a sample by mass spectrometry, the method comprising one or more of the following: immunocapturing the analyte from the sample; subjecting the immunocaptured analyte to an ionization source under conditions suitable for generating one or more ions detectable by mass spectrometry; determining the amount of one or more ions by mass spectrometry; and determining the amount of the analyte in the sample by the amount of one or more ions; wherein the analyte comprises insulin, proinsulin, a metabolic intermediate of proinsulin, a C-peptide, a mutant C-peptide, or any two or more of the above; the metabolic intermediate of proinsulin is de-31,32-proinsulin, de-64,65-proinsulin, or a mixture thereof; the mutant C-peptide is C-peptide+R, C-peptide+RR, or a mixture thereof; and the analyte is not insulin, C-peptide, and / or a mixture of insulin and C-peptide.

[0094] In some embodiments, the analyte described herein includes or is proinsulin. Therefore, this disclosure provides a method for determining the amount of proinsulin in a sample, the method comprising one or more of the following: immunocapturing proinsulin from the sample; subjecting the immunocaptured proinsulin to an ionization source under conditions suitable for generating one or more proinsulin ions detectable by mass spectrometry; determining the amount of one or more proinsulin ions by mass spectrometry; and determining the amount of proinsulin in the sample from the amount of one or more proinsulin ions.

[0095] In some embodiments, the analytes described herein include or are metabolic intermediates of proinsulin (e.g., de31,32-proinsulin, de64,65-proinsulin, or mixtures thereof). Therefore, this disclosure provides a method for determining the amount of metabolic intermediates of proinsulin in a sample, the method comprising one or more of the following: immunocapturing metabolic intermediates from a sample; subjecting the immunocaptured metabolic intermediates to an ionization source under conditions suitable for generating one or more metabolic intermediate ions detectable by mass spectrometry; determining the amount of one or more metabolic intermediate ions by mass spectrometry; and determining the amount of metabolic intermediates of proinsulin in the sample from the amount of one or more metabolic intermediate ions.

[0096] In some embodiments, the analytes described herein include or are mutant C-peptides (e.g., C-peptide + R, C-peptide + RR, or mixtures thereof). Therefore, this disclosure provides a method for determining the amount of mutant C-peptides in a sample, the method comprising one or more of the following: immunocapturing mutant C-peptides from a sample; subjecting the immunocaptured mutant C-peptides to an ionization source under conditions suitable for generating one or more mutant C-peptide ions detectable by mass spectrometry; determining the amount of one or more mutant C-peptide ions by mass spectrometry; and determining the amount of mutant C-peptides in the sample from the amount of one or more mutant C-peptide ions. Test samples suitable for the methods of the present invention include any test sample that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, cell culture, or organ culture. In some preferred embodiments, the sample is obtained from a mammal, such as a dog, cat, horse, etc. Particularly preferred mammals are primates, and most preferably, men or women. Preferred samples include bodily fluids, such as blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; preferably, plasma and serum. Such samples may be obtained, for example, from a patient; that is, a living person—male or female—in a clinical setting used for the diagnosis, prognosis, or treatment of a disease or condition. In embodiments where the sample includes a biological sample, when the sample is obtained from a biological source, the method can be used to determine the amount of analyte in the sample.

[0097] The present invention also contemplates kits for quantitative assays (e.g., for determining the amount of analytes described herein, such as insulin, proinsulin, proinsulin metabolic intermediates (e.g., de31,32-proinsulin, de64,65-proinsulin, or mixtures thereof), C-peptides, mutant C-peptides (e.g., C-peptide+R, C-peptide+RR, or mixtures thereof), or any mixture of two or more thereof). Kits for quantitative assays may include kits containing compositions provided herein. For example, a kit may include packaging material and an isotopically labeled internal standard in an amount sufficient to perform at least one assay. Typically, a kit also includes instructions for using the packaging reagents for quantitative assays, recorded in tangible form (e.g., contained on paper or electronic media).

[0098] The calibration and QC pool used in embodiments of the present invention is preferably prepared using a matrix similar to the intended sample matrix, provided that insulin is substantially absent.

[0099] Sample preparation for mass spectrometry analysis In the preparation of mass spectrometry analysis, analytes described herein can be enriched relative to one or more other components in a sample by a variety of methods known in the art, including, for example, immunocapture, liquid chromatography, filtration, centrifugation, thin-layer chromatography (TLC), electrophoresis (including capillary electrophoresis, affinity separation (including immunoaffinity separation, extraction methods (including ethyl acetate or methanol extraction))) and the use of a dissociative agent or any combination of the above methods, or similar methods.

[0100] One sample purification method that can be used prior to mass spectrometry involves applying the sample to a solid-phase extraction (SPE) column under conditions that reversibly retain the analyte of interest through the column packing material, without retaining one or more other materials. In this technique, a first mobile phase condition can be used to retain the analyte of interest through the column, and a second mobile phase condition can be used to remove the retained material from the column after washing away the non-retained material.

[0101] In some embodiments, the analytes in the sample can be reversibly retained on the SPE column using a packing material comprising alkyl-bonded surfaces. For example, in some embodiments, a C-8 online SPE column (such as the Oasis HLB online SPE column / body (2.1 mm x 20 mm) or equivalent from Phenomenex, Inc.) can be used to enrich insulin prior to mass spectrometry analysis. In some embodiments, the SPE column is used with HPLC-grade 0.2% aqueous formic acid as the washing buffer and with 0.2% formic acid in acetonitrile as the eluent.

[0102] In other embodiments, the method includes immunopurifying the analyte prior to mass spectrometry analysis. Any immunopurification method known in the art can be used for the immunopurification step. Typically, immunopurification procedures utilize antibodies that are bound, conjugated, immobilized, or otherwise attached to a solid support (e.g., a column, pore, tube, capsule, particle, or the like). Generally, immunopurification methods involve (1) incubating a sample containing the analyte of interest with an antibody to bind the analyte to the antibody, (2) performing one or more washing steps, and (3) eluting the analyte from the antibody.

[0103] In some embodiments, an incubation step for immunopurification is performed using antibodies free in solution, and the antibodies are subsequently bound or attached to a solid surface prior to a washing step. In some embodiments, this can be achieved using a primary antibody (e.g., an anti-insulin antibody and / or an anti-C-peptide antibody) and a secondary antibody attached to a solid surface with affinity for the primary anti-insulin antibody. In alternative embodiments, the primary antibody is bound to the solid surface prior to the incubation step.

[0104] Suitable solid supports include, without limitation, tubes, slides, columns, beads, capsules, granules, gels, etc. In some preferred embodiments, the solid support is a multi-well plate, such as, for example, a 96-well plate, a 384-well plate, or a similar plate. In some embodiments, the solid support is an agarose gel or agarose beads or gel. Numerous methods known in the art exist by which antibodies (e.g., insulin antibodies or secondary antibodies) can be bound, attached, immobilized, or coupled to a solid support, for example, by covalent or non-covalent adsorption, affinity binding, ionic bonding, etc. In some embodiments, CNBr is used to couple antibodies; for example, antibodies can be coupled to CNBr-activated agarose. In other embodiments, antibodies are attached to the solid support via antibody-binding proteins (such as protein A, protein G, protein A / G, or protein L).

[0105] The washing step in immunopurification generally involves washing the solid support so that insulin remains bound to the corresponding antibody on the solid support. The elution step in immunopurification generally involves adding a solution that disrupts the binding of the analyte to the corresponding antibody. Exemplary eluents include organic solutions, salt solutions, and high or low pH solutions.

[0106] Another sample purification method that can be used prior to mass spectrometry is liquid chromatography (LC). In LC, analytes are purified by applying a sample to a chromatographic column under mobile phase conditions—whereby the analyte of interest elutes at a rate different from one or more other materials. Such a procedure can enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

[0107] Some methods of liquid chromatography (including HPLC) rely on relatively slow laminar flow techniques. Traditional HPLC analysis relies on column packing, where laminar flow of the sample through the column is the basis for separating the analyte of interest from the sample. Those skilled in the art will understand that separation in such a column is a partitioning process and that suitable LCs for use with C-peptides can be selected, including HPLC, instruments, and columns. Chromatographic columns typically include a medium (i.e., packing material) that facilitates the separation (i.e., fractionation) of chemical fractions. The medium may include small particles. The particles typically include bound surfaces that interact with multiple chemical fractions to facilitate their separation. A suitable bound surface is a hydrophobic bound surface, such as an alkyl bound surface or a cyano bound surface. Alkyl bound surfaces may include C-4, C-8, C-12, or C-18 bound alkyl groups. In some embodiments, the chromatographic column is a monolithic C-18 column. The chromatographic column includes an inlet for receiving the sample and an outlet for discharging the effluent, including fractionated samples. The sample may be supplied directly to the inlet or from an SPE column (such as an inline SPE column or a TFLC column). In some implementations, an in-line filter may be used before the SPE column and / or HPLC column to remove particulates and phospholipids from the sample before it reaches the SPE and / or TFLC and / or HPLC column.

[0108] In one implementation, the sample may be applied to the LC column at the inlet, eluted with a solvent or solvent mixture, and discharged at the outlet. Different solvent modes can be selected for eluting one or more analytes of interest. For example, gradient mode, isocratic mode, or polymorphic (i.e., mixed) mode may be used to perform liquid chromatography. During chromatography, the separation of the materials is achieved through variables such as the selection of eluent (also known as the "mobile phase"), elution mode, gradient conditions, and temperature.

[0109] In some embodiments, analytes in the sample are enriched using HPLC. This HPLC can be performed using a monolithic C-18 column chromatography system (e.g., an Onyx monolithic C-18 column (50 x 2.0 mm) or equivalent from Phenomenex Inc.). In some embodiments, HPLC is performed using HPLC-grade 0.2% aqueous formic acid as solvent A and 0.2% formic acid in acetonitrile as solvent B.

[0110] By carefully selecting valves and connector plumbing, two or more chromatographic columns can be connected as needed, allowing material transfer from one column to the next without any manual steps. In a preferred embodiment, the selection of valves and plumbing is controlled by a computer pre-programmed to perform the necessary steps. Most preferably, the chromatographic system is also connected online to a detector system, such as an MS system. Thus, the operator can place a tray of samples in the autosampler and perform the remaining operations under computer control, resulting in the purification and analysis of all selected samples.

[0111] In some embodiments, TFLC can be used to purify analytes prior to mass spectrometry. In such embodiments, a TFLC column that captures the analyte can be used to extract the sample. The analyte is then eluted and transferred online to an analytical HPLC column. For example, sample extraction can be performed using a TFLC extraction column with a large particle size (50 μm) packing. The sample eluted from this column can then be transferred online to an HPLC analytical column for further purification, prior to mass spectrometry. Because the steps involved in these chromatographic procedures can be automated, the need for operator intervention during analyte purification is minimized. This feature results in time and cost savings and eliminates the chance of operator error.

[0112] In some embodiments, one or more of the above purification techniques can be used in parallel to purify analytes to enable simultaneous processing of multiple samples. In some embodiments, the purification techniques employed do not include immunopurification techniques, such as immunoaffinity chromatography.

[0113] Detection and quantification by mass spectrometry Mass spectrometry is performed using a mass spectrometer—which includes an ion source for ionizing and fractionating samples and generating charged molecules for further analysis. In various embodiments, the analyte can be ionized by any method known to those skilled in the art. For example, analytes can be ionized by electron ionization, chemical ionization, electrospray ionization (ESI), proton ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), laser diode thermal desorption (LDTD), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption / ionization (MALDI), field ionization, field desorption, thermal spray / plasma spray ionization, surface-enhanced laser desorption / ionization (SELDI), inductively coupled plasma (ICP), and particle beam ionization. Those skilled in the art will understand that the choice of ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the choice of positive versus negative mode, etc. The analyte can be ionized in either positive or negative mode. In a preferred embodiment, the analyte is ionized by ESI in positive ion mode.

[0114] In mass spectrometry, after a sample has been ionized, the resulting positively or negatively charged ions are analyzed to determine the mass-to-charge ratio (m / z). Various analyzers used to determine m / z include quadrupole analyzers, ion trap analyzers, time-of-flight analyzers, Fourier transform ion cyclotron resonance mass analyzers, and orbital trap analyzers. (In Bartolucci et al.,...) Rapid Commun. Mass Spectrom Some exemplary ion trap methods are described in 2000, 14:967-73.

[0115] Several detection modes can be used to detect ions. For example, selected ions can be detected (i.e., selective ion monitoring mode (SIM)), or alternatively, mass transitions caused by collision-induced dissociation or loss of neutrality can be monitored (e.g., multiple reaction monitoring (MRM) or selective reaction monitoring (SRM)). In some embodiments, a quadrupole analyzer is used to determine the mass-to-charge ratio. For example, in a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between the electrodes, the amplitude of the RF signal, and the mass / charge ratio. Selectable voltages and amplitudes allow only ions with a specific mass / charge ratio to travel the length of the quadrupole, while all other ions are deflected. Thus, a quadrupole instrument can simultaneously act as a "mass filter" and a "mass detector" for ions injected into the instrument.

[0116] Technicians can enhance the resolution of MS techniques using certain mass spectrometers through tandem mass spectrometry, or MS / MS. In this technique, precursor ions (also called parent ions) generated by the molecule of interest are filtered in the MS instrument, and then fragmented to produce one or more fragment ions (also called daughter ions or product ions), which are then analyzed in a second MS program. By carefully selecting the precursor ions, only ions generated by certain analytes are sent to the fragmentation chamber, where they collide with atoms of an inert gas to produce fragment ions. Because precursor and fragment ions are generated simultaneously in a reproducible manner under a given set of ionization / fragmentation conditions, MS / MS provides an extremely powerful analytical tool. For example, the combination of filtration / fragmentation can be used to eliminate interfering substances and is particularly useful in complex samples, such as biological samples. In some implementations, tandem mass spectrometry is performed using mass spectrometers with multiple quadrupole analyzers, such as triple quadrupole instruments.

[0117] In some implementations, such as MS / MS techniques, precursor ions are separated for further fragmentation, and collision-activated dissociation (CAD) is often used to generate fragment ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas and are subsequently fragmented through a process known as "unimolecular decomposition." Sufficient energy must be accumulated in the precursor ions so that some bonds within the ions can be broken due to the increased vibrational energy.

[0118] In some implementations, MS / MS is used to detect and / or quantify analytes in a sample as follows: Analytes in the sample are enriched by subjecting the sample to SPE followed by liquid chromatography (preferably HPLC); a liquid solvent stream from the chromatographic column enters a heated nebulizer port of the MS / MS analyzer; and the solvent / analyte mixture is vaporized in a heated, energized conduit at the port. During these processes, the analyte is ionized. Ions (e.g., precursor ions) pass through the instrument orifice and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that allow selection of ions based on their mass-to-charge ratio (m / z) (i.e., selection of “precursor” ions and “fragment” ions in Q1 and Q3, respectively). Quadrupole 2 (Q2) is a collision cell where ions are fragmented. The first quadrupole (Q1) of the mass spectrometer selects molecules with the m / z of the analyte ions. Precursor ions with the correct m / z are allowed to enter the collision chamber (Q2), while unwanted ions with any other m / z collide with the sides of the quadrupole and are eliminated. The precursor ions entering Q2 collide with neutral gas molecules (such as argon molecules) and fragment. The resulting fragment ions are then fed into quadrupole 3 (Q3), where fragment ions are selected for detection.

[0119] The method may involve MS / MS performed in positive or negative ion mode; preferably, positive ion mode. Using standard methods known in the art, those skilled in the art can identify one or more fragment ions of a specific precursor ion of the analyte described herein, which can be selected in quadrupole 3 (Q3). In some embodiments, the relative abundance of a single fragment ion from a single precursor ion may be measured. Alternatively, the relative abundance of two or more fragment ions from a single precursor ion may be measured. In these embodiments, the relative abundance of each fragment ion may be subjected to any known mathematical processing to quantitatively assess the initial analyte in the sample. In other embodiments, one or more fragment ions from two or more precursor ions may be measured as above and used to quantitatively assess the initial insulin in the sample.

[0120] When ions collide with the detector, they generate electronic pulses that are converted into digital signals. The acquired data is relayed to a computer, which plots the counts of collected ions versus time. The resulting mass chromatogram is similar to that generated in conventional HPLC methods. The area under the peak corresponding to a specific ion or the amplitude of such a peak is measured and correlated with the amount of the analyte of interest. In some embodiments, the area under the curve or the amplitude of the peak of fragment ions (one or more) and / or precursor ions is measured to determine the amount of one or more analytes detected. As described above, using a calibration standard curve, the relative abundance of a given ion or parent / fragment ion pair can be converted into the absolute amount of the original analyte (e.g., the analyte described herein) based on the peaks of one or more ions from internal molecular standards (e.g., internal molecular standards described herein).

[0121] The ionization of an analyte can result in multi-charged precursor ions (such as 4+, 5+, 6+, etc.). In some embodiments, the analyte may include insulin. Therefore, ionization conditions, particularly the pH of the buffer used in electrospray ionization, greatly influence the identity and quantity of the insulin precursor ions generated. For example, under acidic conditions, positive electrospray ionization may primarily generate 5+ and 6+ charged insulin precursor ions. However, under alkaline conditions, positive electrospray ionization may primarily generate 4+ and 5+ charged insulin precursor ions. The method can be performed under acidic or alkaline conditions; preferably, acidic conditions.

[0122] Optional modes of operation for the tandem mass spectrometer that may be used in some embodiments include product ion scanning and precursor ion scanning. For a description of these operating modes, see, for example, E. Michael Thurman, et al., Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (Amadeo R. Fernandez-Alba, ed., Elsevier 2005)(387).

[0123] In other embodiments, a high-resolution / high-accuracy mass spectrometer can be used to quantitatively analyze the analytes described herein according to the method of the present invention. In some embodiments, to achieve acceptable accuracy in the quantitative results, the mass spectrometer is required to exhibit a resolution of 10,000 or greater (FWHM) for the ions of interest, while maintaining an accuracy of about 50 ppm or less. In some embodiments, the mass spectrometer exhibits a resolution of 18,000 or better (FWHM) while maintaining an accuracy of about 5 ppm or less; such as 20,000 or better (FWHM) and an accuracy of about 3 ppm or less; such as 25,000 or better (FWHM) and an accuracy of about 3 ppm or less. Exemplary analyzers capable of exhibiting the required performance levels include orbital trap mass analyzers, certain TOF mass analyzers, and Fourier transform ion cyclotron resonance mass analyzers.

[0124] Elements found in biologically active molecules (such as carbon, oxygen, and nitrogen) exist naturally in a variety of different isotopic forms. For example, most carbon exists as... 12 It exists in the form of C, but about 1% of all naturally occurring carbon is in the form of C. 13 It exists in the form of C. Therefore, a certain proportion of naturally occurring molecules containing at least one carbon atom will contain at least one 13 C atoms. The inclusion of naturally occurring elemental isotopes in molecules results in multiple molecular isotopic forms. The mass difference between molecular isotopic forms is at least one atomic mass unit (amu). This is because elemental isotopes differ by at least one neutron (the mass of one neutron ≈ 1 amu). When molecular isotopic forms are ionized to multi-charged states, the mass difference between isotopic forms can become difficult to distinguish because mass spectrometry is based on mass-to-charge ratio (m / z). For example, two isotopic forms ionized to the 5+ state with a mass difference of 1 amu will show a difference of only 0.2 in their m / z. High-resolution / high-accuracy mass spectrometers can resolve isotopic forms of highly multi-charged ions (such as ions with ±2, ±3, ±4, ±5, or higher charges).

[0125] Due to naturally occurring elemental isotopes, each molecular ion typically exists in multiple isotopic forms (each of which can result in a detectable peak if analyzed using a sufficiently sensitive mass spectrometer). The m / z ratios and relative abundances of these multiple isotopic forms collectively constitute the isotopic characterization of the molecular ion. In some embodiments, the m / z ratios and relative abundances of two or more molecular isotopic forms can be used to identify the molecular ion under study. In some embodiments, mass spectral peaks from one or more isotopic forms are used to quantify the molecular ion. In some related embodiments, a single mass spectral peak from one isotopic form is used to quantify the molecular ion. In other related embodiments, multiple isotopic peaks are used to quantify the molecular ion. In these subsequent embodiments, the multiple isotopic peaks can undergo any suitable mathematical processing. Several mathematical processing methods are known in the art and include, but are not limited to, summing the areas under multiple peaks or averaging the responses from multiple peaks.

[0126] In some embodiments, a high-resolution / high-accuracy mass spectrometer is used to measure the relative abundance of one or more ions to quantitatively assess the amount of an analyte (e.g., the analyte described herein) in a sample. In some embodiments, the one or more ions measured by high-resolution / high-accuracy mass spectrometry are multiply charged analyte ions.

[0127] The use of high-resolution orbital trap analyzers for qualitative and quantitative analysis of a variety of analytes has been reported. See, for example, U.S. Patent Application Publication No. 2008 / 0118932 (filed November 9, 2007); Bredehoft, et al., Rapid Commun. Mass Spectrom., 2008, 22:477-485; Le Breton, et al., Rapid Commun. Mass Spectrom., 2008, 22:3130-36; Thevis, et al., Mass Spectrom. Reviews, 2008, 27:35-50; Thomas, et al., J. Mass Spectrom., 2008, 43:908-15; Schenk, et al., BMC Medical Genomics, 2008, 1:41; and Olsen, et al., Nature Methods, 2007, 4:709-12.

[0128] The results of analyte determinations can be correlated with the amount of analyte in the original sample using numerous methods known in the art. For example, with careful control of sampling and analytical parameters, the relative abundance of a given ion can be compared with a table converting that relative abundance into the absolute amount of the original molecule. Optionally, an external standard can be run with the sample, and a standard curve can be constructed based on the ions generated from those standards. Using such a standard curve, the relative abundance of a given ion can be converted into the absolute amount of the original molecule. In some preferred embodiments, an internal standard is used to generate a standard curve for calculating the amount of insulin. Methods for generating and using such a standard curve are well known in the art, and those skilled in the art can select an appropriate internal standard. For example, molecules labeled with one or more forms of isotopes can be used as internal standards. Numerous other methods for correlating the amount of ions with the amount of the original molecules will be well known to those skilled in the art.

[0129] As used in this article, when analyzed by mass spectrometry, "isotopic labeling" produces a mass shift in labeled molecules relative to unlabeled molecules. Examples of suitable labels include deuterium (…). 2 H) 13 C and 15 N. One or more isotopic labels may be incorporated at one or more positions in the molecule, and one or more isotopic labels may be used on molecules with the same isotopic label.

[0130] In other embodiments, insulin may be chemically treated prior to mass spectrometry analysis to generate the constituent chains of insulin. The β-chain of insulin can be separated by any chemical treatment known in the art to induce disulfide reduction. For example, insulin can be treated with TCEP (tris(2-carboxyethyl)phosphine) to reduce the disulfide bonds of insulin and separate the α-chain and β-chain.

[0131] The β-chain can then undergo any one or more of the purification steps described above for purifying insulin. In a preferred embodiment, the β-chain undergoes purification by HPLC prior to mass spectrometry analysis.

[0132] Once purified, the β-chain is then subjected to an ionization source. Similar to insulin, those skilled in the art will understand that the choice of ionization method can be determined based on the analyte being measured, the type of sample, the type of detector, and the choice of positive versus negative mode. The insulin β-chain can be ionized in either positive or negative mode. In a preferred embodiment, the insulin β-chain is ionized by ESI in positive mode.

[0133] Ionizing the insulin β-chain can generate multi-charged β-chain precursor ions (such as 3+, 4+, 5+, etc.). Similar to insulin, the identity and quantity of multi-charged precursor ions generated by ionizing the insulin β-chain are affected by the ionization conditions employed. In a preferred embodiment, the insulin β-chain is ionized under acidic conditions.

[0134] One or more steps of any of the methods described above may be performed using automated machines. In some embodiments, one or more purification steps are performed online, and more preferably, all purification and mass spectrometry steps can be performed online.

[0135] In some embodiments, the analyte includes or may include proinsulin. Ionization of proinsulin can produce multi-charged proinsulin precursor ions (such as 3+, 4+, 5+, etc.). For example, positive electrospray ionization of the proinsulin molecule can generate a 7+ charged proinsulin precursor ion with an m / z of approximately 1342.20 ± 0.5. Fragmentation of this proinsulin precursor ion can generate fragment ions with m / z of approximately 183.30 ± 0.5, 120.20 ± 0.5, and 219.30 ± 0.5.

[0136] In some embodiments, the analyte includes or is de31,32-proinsulin. Ionization of de31,32-proinsulin can generate multi-charged de31,32-proinsulin precursor ions (such as 3+, 4+, 5+, etc.). For example, positive electrospray ionization of the de31,32-proinsulin molecule can generate a 7+ charged de31,32-proinsulin precursor ion with an m / z of about 1299.50 ± 0.5. Fragmentation of this de31,32-proinsulin precursor ion can generate de31,32-proinsulin fragment ions with m / z of about 129.20 ± 0.5, 226.20 ± 0.5, and 183.25 ± 0.5.

[0137] In some embodiments, the analyte includes or is de64,65-proinsulin. Ionization of de64,65-proinsulin can generate multi-charged de64,65-proinsulin precursor ions (such as 3+, 4+, 5+, etc.). For example, positive electrospray ionization of the de64,65-proinsulin molecule can generate a 7+ charged de64,65-proinsulin precursor ion with an m / z of about 1304.30 ± 0.5. Fragmentation of this de64,65-proinsulin precursor ion can generate de64,65-proinsulin fragment ions with m / z of about 147.15 ± 0.5, 130.15 ± 0.5, and 183.20 ± 0.5.

[0138] In some embodiments, the analyte includes or is C-peptide+R. Ionization of C-peptide+R can generate multi-charged C-peptide+R precursor ions (such as 3+, 4+, 5+, etc.). For example, positive electrospray ionization of the C-peptide+R molecule can generate a 3+ charged C-peptide+R precursor ion with an m / z of about 1059.20 ± 0.5. Fragmentation of this C-peptide+R precursor ion can generate C-peptide+R fragment ions with m / z of about 966.40 ± 0.5, 260.00 ± 0.5, and 844.30 ± 0.5.

[0139] In some embodiments, the analyte includes or may include C-peptide+RR. Ionization of C-peptide+RR can generate multi-charged C-peptide+RR precursor ions (such as 3+, 4+, 5+, etc.). For example, positive electrospray ionization of the C-peptide+RR molecule can generate a 3+ charged C-peptide+RR precursor ion with an m / z of about 1111.50 ± 0.5. Fragmentation of this C-peptide+RR precursor ion can generate C-peptide+RR fragment ions with m / z of about 896.50 ± 0.5, 499.90 ± 0.5, and 260.30 ± 0.5.

[0140] In some embodiments, the analyte includes but is not insulin (e.g., the analyte further includes at least one other class, such as those selected from proinsulin, proinsulin metabolic intermediates, and mutant C-peptides). Ionization of insulin can generate multi-charged insulin precursor ions (such as 3+, 4+, 5+, etc.). For example, positive electrospray ionization of an insulin molecule can generate a 5+ charged insulin precursor ion with an m / z of about 1162.30 ± 0.5. Fragmentation of this insulin precursor ion can generate insulin fragment ions with m / z of about 226.20 ± 0.5, 136.10 ± 0.5, and 345.20 ± 0.5.

[0141] In some embodiments, the analyte includes but is not a C-peptide (e.g., the analyte further includes at least one other class, such as those selected from proinsulin, proinsulin metabolic intermediates, and mutant C-peptides). Ionization of the C-peptide can generate multi-charged C-peptide precursor ions (such as 3+, 4+, 5+, etc. precursor ions). For example, positive electrospray ionization of a C-peptide molecule can generate a 3+ charged C-peptide precursor ion with an m / z of about 1007.70 ± 0.5. Fragmentation of this C-peptide precursor ion can generate C-peptide fragment ions with m / z of about 927.50 ± 0.5, 646.40 ± 0.5, and 533.30 ± 0.5.

[0142] In some embodiments, the limit of quantitation (LOQ) of the methods described herein (e.g., methods for determining the amount of insulin) is less than or equal to about 3 uIU / mL. In some embodiments, the LQ of the methods described herein (e.g., methods for determining the amount of C-peptide or mutant C-peptide) is less than or equal to about 0.2 ng / mL (e.g., less than or equal to about 0.11 ng / mL). In some embodiments, the LQ of the methods described herein (e.g., methods for determining the amount of proinsulin or a metabolic intermediate of proinsulin) is less than or equal to about 3 pmol / L (e.g., less than or equal to about 2.9 pmol / L).

[0143] In some embodiments, amounts of two or more, or all of, of the following are determined in the same sample injection: insulin, proinsulin, proinsulin metabolic intermediates (e.g., de-31,32-proinsulin, de-64,65-proinsulin, or mixtures thereof), C-peptides, and mutant C-peptides (e.g., C-peptide+R, C-peptide+RR, or mixtures thereof). In some embodiments, amounts of insulin, proinsulin, de-31,32-proinsulin, de-64,65-proinsulin, C-peptides, C-peptide+R, and C-peptide+RR are determined in the same sample injection.

[0144] The invention thus generally described will be more readily understood by referring to the following embodiments, which are provided by way of example and are not intended to limit the invention.

[0145] Example Example 1 Insulin, C-peptide, proinsulin, des31,32-proinsulin, des64,65-proinsulin, C-peptide+R and / or C-peptide+RR in serum samples can be detected and quantified according to the methods outlined below.

[0146] In this assay, serum is first defatted, and then analytes (e.g., insulin, C-peptide, proinsulin, des31,32-proinsulin, des64,65-proinsulin, C-peptide + R, and / or C-peptide + RR) are immunoprecipitated using antibodies immobilized on magnetic beads. The beads are then subjected to a rigorous washing protocol to remove non-specifically bound material, followed by elution of the peptides from the beads with acidified acetonitrile in water. Aliquots of the sample are added with Trizma base to enhance the stability of the peptides in the elution plate.

[0147] The TECAN Evo liquid handling station automates the processes of calibrator preparation, internal standard addition, defatting, bead deposition, immune capture, washing, and peptide elution from the beads. Manual steps involve initial dilution of the calibrator stock, defatting transfer to a centrifuge, and bead preparation.

[0148] The eluent plate was transferred from the TECAN robot deck to the autosampler of the Shimadzu QX (Nexera XR) system and immediately run. The sample was injected onto a hydrophilic / lipophilic balanced (HLB) capture column, where the analyte was further enriched from background contaminants. After washing, a plug of transfer solvent was used to release the peptide from the extraction column and transfer it to a reversed-phase analytical column. An acetonitrile gradient chromatographically separated the analyte from the remaining background contaminants and from each other.

[0149] The solvent stream from the HPLC column is directed to the heated electrospray source of the Shimadzu 8060NX mass spectrometer. In the mass spectrometer, only ions with the desired mass-to-charge ratio are allowed to pass through the quadrupole 1 (Q1) region into the collision chamber (Q2). The accelerated ions then collide with neutral argon molecules, breaking into small fragments. Finally, in Q3, only selected ions are selected to reach the detector (see Table 1 below). The signal intensity at the detector is proportional to the number of molecules entering the mass spectrometer. The peak area ratio is then calculated against a known set of calibrators, and a calibration curve is established. The concentration of the analyte in the sample (e.g., insulin, C-peptide, proinsulin, des31,32-proinsulin, des64,65-proinsulin, C-peptide+R, and / or C-peptide+RR) can then be determined using the calibration equation.

[0150] Table 1. Paragraph 2. The method in Paragraph 1, wherein the analyte includes proinsulin.

[0151] Paragraph 3. The method of paragraph 1 or 2, wherein one or more ions include one or more proinsulin ions.

[0152] Paragraph 4. The method of any one of paragraphs 1-3, wherein one or more proinsulin ions include a proinsulin precursor ion with a mass-to-charge ratio (m / z) of 1342.20 ± 0.5.

[0153] Paragraph 5. The method of paragraph 3, wherein one or more proinsulin ions comprise one or more proinsulin fragment ions selected from the group consisting of ions with m / z of 183.30±0.5, 120.20±0.5 and 219.30±0.5.

[0154] Paragraph 6. The method of any one of paragraphs 1-5, wherein the analyte includes a metabolic intermediate of proinsulin.

[0155] Paragraph 7. The method in paragraph 6, wherein the metabolic intermediate of proinsulin includes des31,32-proinsulin.

[0156] Paragraph 8. The method in paragraph 7, wherein one or more ions include one or more de31,32-proinsulin ions.

[0157] Paragraph 9. The method of paragraph 8, wherein one or more de31,32-proinsulin ions include de31,32-proinsulin precursor ions with an m / z of 1299.50 ± 0.5.

[0158] Paragraph 10. The method of paragraph 8, wherein one or more de31,32-proinsulin ions comprise one or more de31,32-proinsulin fragment ions selected from the group consisting of ions with m / z of 129.20±0.5, 226.20±0.5 and 183.25±0.5.

[0159] Paragraph 11. The method in paragraph 6, wherein the metabolic intermediate of proinsulin includes de64,65-proinsulin.

[0160] Paragraph 12. The method of paragraph 11, wherein one or more ions include one or more de64,65-proinsulin ions.

[0161] Paragraph 13. The method of paragraph 12, wherein one or more de64,65-proinsulin ions include a de64,65-proinsulin precursor ion with an m / z of 1304.30 ± 0.5.

[0162] Paragraph 14. The method of paragraph 12, wherein one or more de64,65-proinsulin ions comprise one or more de64,65-proinsulin fragment ions selected from the group consisting of ions with m / z of 147.15±0.5, 130.15±0.5 and 183.20±0.5.

[0163] Paragraph 15. The method of any one of paragraphs 1-14, wherein the analyte includes a mutant C-peptide.

[0164] Paragraph 16. The method of paragraph 15, wherein the mutant C-peptide includes a C-peptide having an additional arginine residue at the C-terminus (C-peptide+R).

[0165] Paragraph 17. The method of paragraph 16, wherein one or more ions include one or more C-peptide + R ions.

[0166] Paragraph 18. The method of paragraph 17, wherein one or more C-peptide+R ions include a precursor C-peptide+R ion with an m / z of 1059.20 ± 0.5.

[0167] Paragraph 19. The method of paragraph 17, wherein one or more C-peptide+R ions comprise one or more C-peptide+R fragment ions selected from the group consisting of ions with m / z of 966.40±0.5, 260.00±0.5 and 844.30±0.5.

[0168] Paragraph 20. The method of paragraph 15, wherein the mutant C-peptide includes a C-peptide having two additional arginine residues at the C-terminus (C-peptide+RR).

[0169] Paragraph 21. The method of paragraph 20, wherein one or more ions include one or more C-peptide + RR ions.

[0170] Paragraph 22. The method of paragraph 21, wherein one or more C-peptide+RR ions include C-peptide+RR precursor ions with an m / z of 1111.50 ± 0.5.

[0171] Paragraph 23. The method of paragraph 21, wherein one or more C-peptide + RR ions comprise one or more C-peptide + RR fragment ions selected from the group consisting of ions with m / z of 896.50±0.5, 499.90±0.5 and 260.30±0.5.

[0172] Paragraph 24. The method of any one of paragraphs 1-21, wherein the analyte includes insulin.

[0173] Paragraph 25. The method in paragraph 24, wherein one or more ions include one or more insulin ions.

[0174] Paragraph 26. The method of paragraph 25, wherein one or more insulin ions include an insulin precursor ion with an m / z of 1162.30 ± 0.5.

[0175] Paragraph 27. The method of paragraph 25, wherein one or more insulin ions comprise one or more insulin fragment ions selected from the group consisting of ions with m / z of 226.20±0.5, 136.10±0.5 and 345.20±0.5.

[0176] Paragraph 28. The method of any one of paragraphs 1-27, wherein the analyte includes a C-peptide.

[0177] Paragraph 29. The method of paragraph 28, wherein one or more ions include one or more C-peptide ions.

[0178] Paragraph 30. The method of paragraph 29, wherein one or more C-peptide ions include C-peptide precursor ions with an m / z of 1007.70 ± 0.5.

[0179] Paragraph 31. The method of paragraph 29, wherein one or more C-peptide ions comprise one or more C-peptide fragment ions selected from the group consisting of ions with m / z of 927.50±0.5, 646.40±0.5 and 533.30±0.5.

[0180] Paragraph 32. The method of any one of paragraphs 1-31, wherein the analyte is not insulin, C-peptide, or a mixture of insulin and C-peptide.

[0181] Paragraph 33. A method for determining the amount of proinsulin in a sample, the method comprising: immunocapturing proinsulin from the sample; subjecting the immunocaptured proinsulin to an ionization source under conditions suitable for generating one or more proinsulin ions detectable by mass spectrometry; determining the amount of one or more proinsulin ions by mass spectrometry; and determining the amount of proinsulin in the sample from the amount of one or more proinsulin ions.

[0182] Paragraph 34. Method 33 of the paragraph, wherein one or more proinsulin ions include proinsulin precursor ions with a mass-to-charge ratio (m / z) of 1342.20 ± 0.5.

[0183] Paragraph 35. The method of paragraph 33 or 34, wherein one or more proinsulin ions comprise one or more proinsulin fragment ions selected from the group consisting of ions with m / z of 183.30±0.5, 120.20±0.5 and 219.30±0.5.

[0184] Paragraph 36. A method for determining the amount of a metabolic intermediate of proinsulin in a sample, the method comprising: immunocapturing the metabolic intermediate from the sample; subjecting the immunocaptured metabolic intermediate to an ionization source under conditions suitable for generating one or more metabolic intermediate ions detectable by mass spectrometry; determining the amount of one or more metabolic intermediate ions by mass spectrometry; and determining the amount of a metabolic intermediate of proinsulin in the sample from the amount of one or more metabolic intermediate ions; wherein the metabolic intermediate of proinsulin is de31,32-proinsulin, de64,65-proinsulin, or a mixture thereof.

[0185] Paragraph 37. The method in paragraph 36, wherein the metabolic intermediate of proinsulin includes des31,32-proinsulin.

[0186] Paragraph 38. The method described in paragraphs 36 or 37, wherein one or more metabolic intermediate ions include one or more de31,32-proinsulin ions.

[0187] Paragraph 39. The method of paragraph 38, wherein one or more de31,32-proinsulin ions include a de31,32-proinsulin precursor ion with an m / z of 1299.50 ± 0.5.

[0188] Paragraph 40. The method of paragraph 38, wherein one or more de31,32-proinsulin ions comprise one or more de31,32-proinsulin fragment ions selected from the group consisting of ions with m / z of 129.20±0.5, 226.20±0.5 and 183.25±0.5.

[0189] Paragraph 41. Any of the methods in paragraphs 36-40, wherein the metabolic intermediate of proinsulin includes de64,65-proinsulin.

[0190] Paragraph 42. A method of any one of paragraphs 36-41, wherein one or more metabolic intermediate ions include one or more de64,65-proinsulin ions.

[0191] Paragraph 43. The method of paragraph 42, wherein one or more de64,65-proinsulin ions include a de64,65-proinsulin precursor ion with an m / z of 1304.30 ± 0.5.

[0192] Paragraph 44. The method of paragraph 42, wherein one or more de64,65-proinsulin ions comprise one or more de64,65-proinsulin fragment ions selected from the group consisting of ions with m / z of 147.15±0.5, 130.15±0.5 and 183.20±0.5.

[0193] Paragraph 45. A method for determining the amount of a mutant C-peptide in a sample, the method comprising: immunocapturing the mutant C-peptide from the sample; subjecting the immunocaptured mutant C-peptide to an ionization source under conditions suitable for generating one or more mutant C-peptide ions detectable by mass spectrometry; determining the amount of one or more mutant C-peptide ions by mass spectrometry; and determining the amount of the mutant C-peptide in the sample by the amount of one or more mutant C-peptide ions; wherein the mutant C-peptide is a C-peptide having an additional arginine residue at the C-terminus (C-peptide+R), a C-peptide having two additional arginine residues at the C-terminus (C-peptide+RR), or a mixture thereof.

[0194] Paragraph 46. The method of paragraph 45, wherein the mutant C-peptide includes a C-peptide having an additional arginine residue at the C-terminus (C-peptide+R).

[0195] Paragraph 47. The method in paragraphs 45 or 46, wherein one or more mutant C-peptide ions include one or more C-peptide+R ions.

[0196] Paragraph 48. The method of paragraph 47, wherein one or more C-peptide+R ions include a precursor C-peptide+R ion with an m / z of 1059.20 ± 0.5.

[0197] Paragraph 49. The method of paragraph 47, wherein one or more C-peptide+R ions comprise one or more C-peptide+R fragment ions selected from the group consisting of ions with m / z of 966.40±0.5, 260.00±0.5 and 844.30±0.5.

[0198] Paragraph 50. The method of any one of paragraphs 45-49, wherein the mutant C-peptide includes a C-peptide having two additional arginine residues at the C-terminus (C-peptide+RR).

[0199] Paragraph 51. The method of any one of paragraphs 45-50, wherein one or more mutant C-peptide ions include one or more C-peptide+RR ions.

[0200] Paragraph 52. The method of paragraph 51, wherein one or more C-peptide+RR ions include C-peptide+RR precursor ions with an m / z of 1111.50 ± 0.5.

[0201] Paragraph 53. The method of paragraph 51, wherein one or more C-peptide + RR ions comprise one or more C-peptide + RR fragment ions selected from the group consisting of ions with m / z of 896.50±0.5, 499.90±0.5 and 260.30±0.5.

[0202] Paragraph 54. The method of any of the preceding paragraphs, wherein the sample includes a plasma or serum sample.

[0203] Paragraph 55. The method of any of the preceding paragraphs, wherein the ionization source is an electrospray (ESI) ionization source.

[0204] Paragraph 56. Any of the methods in the preceding paragraphs, wherein ionization is in a positive ion mode.

[0205] Paragraph 57. The method of any of the preceding paragraphs, wherein the sample is subjected to acidic conditions prior to mass spectrometry.

[0206] Paragraph 58. The method in paragraph 57, wherein subjecting the sample to acidic conditions includes subjecting the sample to formic acid.

[0207] Paragraph 59. The method of any of the preceding paragraphs, wherein the sample is subjected to alkaline conditions prior to mass spectrometry.

[0208] Paragraph 60. The method of paragraph 59, wherein subjecting the sample to alkaline conditions includes subjecting the sample to trizma and / or ethanol.

[0209] Paragraph 61. The method of any of the preceding paragraphs, wherein the sample is degreased prior to quantification by mass spectrometry.

[0210] Paragraph 62. The method of any of the preceding paragraphs further includes purifying the sample prior to mass spectrometry.

[0211] Paragraph 63. The method of paragraph 62, wherein the purification includes subjecting the sample to liquid chromatography.

[0212] Paragraph 64. The methods in paragraph 63, wherein liquid chromatography includes high performance liquid chromatography (HPLC) or high turbulence liquid chromatography (HTLC).

[0213] Paragraph 65. The method of paragraph 62, wherein the purification includes subjecting the sample to solid-phase extraction (SPE).

[0214] Paragraph 66. Any of the methods in the preceding paragraphs, wherein the mass spectrometry method is tandem mass spectrometry, high-resolution mass spectrometry, or high-resolution / high-accuracy mass spectrometry.

[0215] Paragraph 67. Any of the methods in the preceding paragraphs, wherein immune capture includes the use of antibodies.

[0216] Paragraph 68. The method in paragraph 67, wherein the antibody is a monoclonal antibody.

[0217] Paragraph 69. The method described in paragraphs 67 or 68, in which antibodies are immobilized on magnetic beads.

[0218] Paragraph 70. The method of any of the preceding paragraphs further includes washing and eluting material captured on the magnetic beads by the immune system.

[0219] While certain embodiments have been shown and described, it should be understood that changes and modifications can be made to them by those skilled in the art without departing from the art in the broader aspects of the art as defined in the appended claims.

[0220] This disclosure is not limited to the specific embodiments described herein. It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. In addition to those listed herein, functionally equivalent methods and compositions within the scope of this disclosure will become apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. This disclosure is limited only by the terminology of the appended claims and the full scope of their equivalents. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, which can certainly be modified. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and not for limitation.

[0221] Furthermore, when describing features or aspects of this disclosure in accordance with the Markush Group, those skilled in the art will recognize that this disclosure is also described in accordance with any individual member or subgroup member of the Markush Group.

[0222] As those skilled in the art will understand, for any and all purposes, particularly in providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any enumerated scope can be readily considered adequately descriptive and the same scope can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily broken down into lower thirds, middle thirds, and upper thirds, etc. As those skilled in the art will also understand, all terms (such as “up to,” “at least,” “greater than,” “less than,” etc.) include the stated quantity and refer to scopes that can subsequently be broken down into subscopes as discussed above. Finally, as those skilled in the art will understand, a scope includes each individual member.

[0223] All publications, patent applications, granted patents, and other documents mentioned in this specification are incorporated herein by reference as if each individual publication, patent application, granted patent, or other document were specifically and individually indicated as incorporated herein by reference in its entirety. Any definition contained in the text incorporated by reference that conflicts with a definition in this disclosure is excluded.

[0224] Other embodiments are set forth in the appended claims.

Claims

1. A method for determining the amount of an analyte in a sample by mass spectrometry, the method comprising: The analyte is captured by immunization from the sample; The analyte, which is captured by the immune system, is subjected to an ionization source under conditions suitable for generating one or more ions that can be detected by mass spectrometry. The amount of one or more ions was determined by mass spectrometry; and The amount of the analyte in the sample is determined by the amount of the one or more ions; in The analytes include insulin, proinsulin, metabolic intermediates of proinsulin, C-peptide, mutant C-peptide, or any mixture of two or more thereof. The metabolic intermediate of proinsulin is de31,32-proinsulin, de64,65-proinsulin, or a mixture thereof; and The mutant C-peptide is a C-peptide (C-peptide+R) having an additional arginine residue at the C-terminus, a C-peptide (C-peptide+RR) having two additional arginine residues at the C-terminus, or a mixture thereof.

2. The method of claim 1, wherein the analyte comprises proinsulin.

3. The method according to claim 2, wherein the one or more ions include one or more proinsulin ions.

4. The method according to claim 3, wherein the one or more proinsulin ions comprise proinsulin precursor ions with a mass-to-charge ratio (m / z) of 1342.20 ± 0.

5.

5. The method according to claim 3, wherein the one or more proinsulin ions comprise one or more proinsulin fragment ions selected from the group consisting of ions with m / z of 183.30±0.5, 120.20±0.5, and 219.30±0.

5.

6. The method of claim 1, wherein the analyte comprises a metabolic intermediate of proinsulin.

7. The method of claim 6, wherein the metabolic intermediate of proinsulin comprises de-31,32-proinsulin.

8. The method of claim 7, wherein the one or more ions comprise one or more de31,32-proinsulin ions.

9. The method of claim 8, wherein the one or more de31,32-proinsulin ions comprise de31,32-proinsulin precursor ions with an m / z of 1299.50 ± 0.

5.

10. The method of claim 8, wherein the one or more de-31,32-proinsulin ions comprise one or more de-31,32-proinsulin fragment ions selected from the group consisting of ions with m / z of 129.20±0.5, 226.20±0.5, and 183.25±0.

5.

11. The method of claim 6, wherein the metabolic intermediate of proinsulin comprises de64,65-proinsulin.

12. The method of claim 11, wherein the one or more ions comprise one or more de64,65-proinsulin ions.

13. The method of claim 12, wherein the one or more de64,65-proinsulin ions comprise de64,65-proinsulin precursor ions with an m / z of 1304.30 ± 0.

5.

14. The method of claim 12, wherein the one or more de64,65-proinsulin ions comprise one or more de64,65-proinsulin fragment ions selected from the group consisting of ions with m / z of 147.15±0.5, 130.15±0.5, and 183.20±0.

5.

15. The method of claim 1, wherein the analyte comprises a mutant C-peptide.

16. The method of claim 15, wherein the mutant C-peptide comprises a C-peptide having an additional arginine residue at the C-terminus (C-peptide+R).

17. The method of claim 16, wherein the one or more ions comprise one or more C-peptide + R ions.

18. The method of claim 17, wherein the one or more C-peptide+R ions comprise precursor C-peptide+R ions with an m / z of 1059.20 ± 0.

5.

19. The method of claim 17, wherein the one or more C-peptide+R ions comprise one or more C-peptide+R fragment ions selected from the group consisting of ions with m / z of 966.40±0.5, 260.00±0.5, and 844.30±0.

5.

20. The method of claim 15, wherein the mutant C-peptide comprises a C-peptide (C-peptide+RR) having two additional arginine residues at the C-terminus.

21. The method of claim 20, wherein the one or more ions comprise one or more C-peptide + RR ions.

22. The method of claim 21, wherein the one or more C-peptide+RR ions comprise C-peptide+RR precursor ions with an m / z of 1111.50 ± 0.

5.

23. The method of claim 21, wherein the one or more C-peptide + RR ions comprise one or more C-peptide + RR fragment ions selected from the group consisting of ions with m / z of 896.50±0.5, 499.90±0.5, and 260.30±0.

5.

24. The method of claim 1, wherein the analyte comprises insulin.

25. The method of claim 24, wherein the one or more ions comprise one or more insulin ions.

26. The method of claim 25, wherein the one or more insulin ions comprise insulin precursor ions with an m / z of 1162.30 ± 0.

5.

27. The method of claim 25, wherein the one or more insulin ions comprise one or more insulin fragment ions selected from the group consisting of ions with m / z of 226.20±0.5, 136.10±0.5, and 345.20±0.

5.

28. The method of claim 1, wherein the analyte comprises a C-peptide.

29. The method of claim 28, wherein the one or more ions comprise one or more C-peptide ions.

30. The method of claim 29, wherein the one or more C-peptide ions comprise C-peptide precursor ions with an m / z of 1007.70 ± 0.

5.

31. The method of claim 29, wherein the one or more C-peptide ions comprise one or more C-peptide fragment ions selected from the group consisting of ions with m / z of 927.50±0.5, 646.40±0.5, and 533.30±0.

5.

32. The method of claim 1, wherein the analyte is not insulin, C-peptide, or a mixture of insulin and C-peptide.

33. A method for determining the amount of proinsulin in a sample, the method comprising: Proinsulin was captured by immunization from the sample; The proinsulin captured by the immune system is subjected to an ionization source under conditions suitable for generating one or more proinsulin ions detectable by mass spectrometry. The amount of one or more proinsulin ions was determined by mass spectrometry; and The amount of proinsulin in the sample is determined by the amount of one or more proinsulin ions.

34. The method of claim 33, wherein the one or more proinsulin ions comprise proinsulin precursor ions with a mass-to-charge ratio (m / z) of 1342.20 ± 0.

5.

35. The method of claim 33, wherein the one or more proinsulin ions comprise one or more proinsulin fragment ions selected from the group consisting of ions with m / z of 183.30±0.5, 120.20±0.5, and 219.30±0.

5.

36. A method for determining the amount of a metabolic intermediate of proinsulin in a sample, the method comprising: The metabolic intermediates were captured by immunization from the sample; The metabolic intermediates that are captured by the immune system are subjected to an ionization source under conditions suitable for generating one or more metabolic intermediate ions that can be detected by mass spectrometry. The amount of one or more metabolic intermediate ions was determined by mass spectrometry; and The amount of the proinsulin metabolic intermediate in the sample is determined by the amount of one or more of the metabolic intermediate ions; in The metabolic intermediate of proinsulin is de31,32-proinsulin, de64,65-proinsulin, or a mixture thereof.

37. The method of claim 36, wherein the metabolic intermediate of proinsulin comprises des31,32-proinsulin.

38. The method of claim 36, wherein the one or more metabolic intermediate ions comprise one or more des31,32-proinsulin ions.

39. The method of claim 38, wherein the one or more de31,32-proinsulin ions comprise de31,32-proinsulin precursor ions with an m / z of 1299.50 ± 0.

5.

40. The method of claim 38, wherein the one or more de-31,32-proinsulin ions comprise one or more de-31,32-proinsulin fragment ions selected from the group consisting of ions with m / z of 129.20±0.5, 226.20±0.5, and 183.25±0.

5.

41. The method of claim 36, wherein the metabolic intermediate of proinsulin comprises de64,65-proinsulin.

42. The method of claim 36, wherein the one or more metabolic intermediate ions comprise one or more de64,65-proinsulin ions.

43. The method of claim 42, wherein the one or more de64,65-proinsulin ions comprise de64,65-proinsulin precursor ions with an m / z of 1304.30 ± 0.

5.

44. The method of claim 42, wherein the one or more de64,65-proinsulin ions comprise one or more de64,65-proinsulin fragment ions selected from the group consisting of ions with m / z of 147.15±0.5, 130.15±0.5, and 183.20±0.

5.

45. A method for determining the amount of a mutant C-peptide in a sample, the method comprising: The mutant C-peptide was captured by immunization from the sample; The immune-captured mutant C-peptide is subjected to an ionization source under conditions suitable for generating one or more mutant C-peptide ions detectable by mass spectrometry. The amount of one or more mutant C-peptide ions was determined by mass spectrometry; and The amount of the mutant C-peptide in the sample is determined by the amount of the one or more mutant C-peptide ions; in The mutant C-peptide is a C-peptide (C-peptide+R) having an additional arginine residue at the C-terminus, a C-peptide (C-peptide+RR) having two additional arginine residues at the C-terminus, or a mixture thereof.

46. ​​The method of claim 45, wherein the mutant C-peptide comprises a C-peptide having an additional arginine residue at the C-terminus (C-peptide+R).

47. The method of claim 45, wherein the one or more mutant C-peptide ions comprise one or more C-peptide+R ions.

48. The method of claim 47, wherein the one or more C-peptide+R ions comprise precursor C-peptide+R ions with an m / z of 1059.20 ± 0.

5.

49. The method of claim 47, wherein the one or more C-peptide+R ions comprise one or more C-peptide+R fragment ions selected from the group consisting of ions with m / z of 966.40±0.5, 260.00±0.5, and 844.30±0.

5.

50. The method of claim 45, wherein the mutant C-peptide comprises a C-peptide (C-peptide+RR) having two additional arginine residues at the C-terminus.

51. The method of claim 45, wherein the one or more mutant C-peptide ions comprise one or more C-peptide+RR ions.

52. The method according to claim 51, wherein the one or more C-peptide+RR ions comprise C-peptide+RR precursor ions with an m / z of 1111.50 ± 0.

5.

53. The method of claim 51, wherein the one or more C-peptide + RR ions comprise one or more C-peptide + RR fragment ions selected from the group consisting of ions with m / z of 896.50±0.5, 499.90±0.5, and 260.30±0.

5.

54. The method according to any one of the preceding claims, wherein the sample comprises a plasma or serum sample.

55. The method according to any one of the preceding claims, wherein the ionization source is an electrospray (ESI) ionization source.

56. The method according to any one of the preceding claims, wherein ionization is in a positive ion mode.

57. The method according to any one of the preceding claims, wherein the sample is subjected to acidic conditions prior to mass spectrometry.

58. The method of claim 57, wherein subjecting the sample to acidic conditions comprises subjecting the sample to formic acid.

59. The method according to any one of the preceding claims, wherein the sample is subjected to alkaline conditions prior to mass spectrometry.

60. The method of claim 59, wherein subjecting the sample to alkaline conditions comprises subjecting the sample to trizma and / or ethanol.

61. The method according to any one of the preceding claims, wherein the sample is defatted before quantification by mass spectrometry.

62. The method according to any one of the preceding claims, further comprising purifying the sample prior to mass spectrometry.

63. The method of claim 62, wherein the purification comprises subjecting the sample to liquid chromatography.

64. The method of claim 63, wherein the liquid chromatography method includes high performance liquid chromatography (HPLC) or high turbulence liquid chromatography (HTLC).

65. The method of claim 62, wherein the purification comprises subjecting the sample to solid-phase extraction (SPE).

66. The method according to any one of the preceding claims, wherein the mass spectrometry is tandem mass spectrometry, high-resolution mass spectrometry, or high-resolution / high-accuracy mass spectrometry.

67. The method according to any one of the preceding claims, wherein the immune capture includes the use of an antibody.

68. The method of claim 67, wherein the antibody is a monoclonal antibody.

69. The method according to claim 67 or 68, wherein the antibody is immobilized on a magnetic bead.

70. The method according to any one of the preceding claims, further comprising washing and eluting the material captured on the magnetic beads by the immune system.

Images