Detection of insulin-like growth factor-2 variants by mass spectrometry

Abstract

Methods are provided for detecting and / or quantifying insulin-like growth factor-2 (IGF-2) variant(s) in a sample. The methods provided herein are further directed to determining the presence of IGF2 variant(s) in a sample using one or more detected ions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 328,649, filed April 7, 2022, the contents of which are incorporated by reference herein in their entirety.

[0002] The present invention relates to the identification and quantification of insulin-like growth factor 2 variants using mass spectrometry. [Background technology]

[0003] Insulin-like growth factor 2 (IGF-II or IGF-2) plays an essential role in prenatal growth and development. Research suggests that IGF-2 promotes cell growth and division in many different tissues. The IGF-2 gene is highly active during fetal development but is much less active after birth.

[0004] Although high-resolution LC-MS-based approaches are effective for quantification of peptides in samples from most patients, amino acid substitutions due to polymorphisms can result in mass changes that essentially "hide" the polymorphic variant. When monitoring larger polypeptides with more amino acid residues, the likelihood of harboring polymorphisms increases.

[0005] Protein quantification is based on peak intensity at a particular mass-to-charge ratio (m / z) of the wild-type (WT) protein, so quantification of IGF-2 includes only the WT and does not include polymorphisms. Described herein are methods for detecting novel IGF-2 variants. Summary of the Invention

[0006] The methods provided herein include detecting and quantitating one or more novel IGF-2 variants by mass spectrometry.

[0007] In some embodiments, the method comprises detecting the following mass / charge (m / z) ratios for each of the variants of interest:

[0008] In some embodiments, a method provided herein is a method of detecting insulin-like growth factor 2 (IGF-2) variant(s) in a sample, the method comprising: ionizing IGF-2 in the sample to produce one or more ions detectable by mass spectrometry; detecting one or more of the ions by mass spectrometry, including an ion having a mass-to-charge ratio of 1089.8±0.5; and determining the presence of IGF2 variant(s) in the sample using the one or more detected ions.

[0009] In some embodiments, the method includes detecting one or more of the ions by mass spectrometry, including an ion having a mass-to-charge ratio of 1089.66±0.5.

[0010] In some embodiments, the method includes detecting one or more of the ions by mass spectrometry, including an ion having a mass-to-charge ratio of 1090.23±0.5.

[0011] In some embodiments, the methods provided herein detect variants having an extra arginine at position 68.

[0012] In some embodiments, the methods provided herein include purifying the protein by high performance liquid chromatography (HPLC) prior to ionization.

[0013] In some embodiments, the sample comprises plasma or serum.

[0014] In some embodiments, samples may be purified by high performance liquid chromatography (HPLC) prior to ionization. In a related embodiment, IGF-2 and its variants are extracted and resolved using a multiplexed high performance liquid chromatography (HPLC) system with on-line extraction (Aria TLX-4).

[0015] In some embodiments, the sample may be purified by solid phase extraction (SPE) prior to ionization.

[0016] In some embodiments, the mass spectrometer comprises a Q Exactive Focus Hybrid Quadrupole-Orbitrap instrument.

[0017] In some embodiments, the high resolution / high accuracy mass analysis is performed with a resolution (FWHM) of about 10,000 or more, such as about 15,000 or more, such as about 20,000 or more, such as about 25,000 or more. In some embodiments, the high resolution / high accuracy mass analysis is performed with an accuracy of about 50 ppm or less, such as about 20 ppm or less, such as about 10 ppm or less, such as about 5 ppm or less, such as about 3 ppm or less. In some embodiments, the high resolution / high accuracy mass analysis is performed with a resolution (FWHM) of about 10,000 or more and an accuracy of about 50 ppm or less. In some embodiments, the resolution is greater than about 15,000 and the accuracy is about 20 ppm or less. In some embodiments, the resolution is about 20,000 or more and the accuracy is about 10 ppm or less. Preferably, the resolution is about 25,000 or more and the accuracy is about 5 ppm or less, such as about 3 ppm or less.

[0018] In some embodiments, high resolution / high accuracy mass analysis may be performed using an Orbitrap mass spectrometer, a Time of Flight (TOF) mass spectrometer, or a Fourier Transform Ion Cyclotron Resonance mass spectrometer (also known as a Fourier Transform mass spectrometer). In some embodiments, the sample may comprise a biological sample, preferably plasma or serum.

[0019] In some embodiments, relating the amount of one or more IGF-2 ions detected by mass spectrometry to the amount of IGF-2 protein in the sample includes comparison to an internal standard, such as a human or non-human IGF-2 protein (e.g., recombinant mouse IGF-2). The internal standard may optionally be isotopically labeled.

[0020] The features of the above listed embodiments may be combined for use in the methods of the present invention, including but not limited to:

[0021] In certain preferred embodiments, mass spectrometry is performed in positive ion mode. Alternatively, mass spectrometry is performed in negative ion mode. A preferred ionization technique used in the methods described herein is electrospray ionization (ESI). Electrospray ionization can be performed, for example, using a heated ionization source. In some embodiments, the methods provided herein include heated electrospray ionization (HESI). In some embodiments, the methods provided herein include heated electrospray ionization (HESI) in positive ion mode.

[0022] In a preferred embodiment, one or more separately detectable internal standards are provided in the sample, and the amount thereof is also determined in the sample. In these embodiments, all or a portion of both the analyte of interest and one or more internal standards are ionized to generate a plurality of ions detectable by a mass spectrometer, and one or more ions generated from each are detected by mass spectrometry. The internal standard may be selected from the group consisting of non-human IGF-2 (e.g., recombinant mouse IGF-2, either isotopically labeled or unlabeled), isotopically labeled IGF-2 protein.

[0023] In other embodiments, the amount of IGF-2 variant protein in a sample may be determined by comparison to one or more external reference standards. Exemplary external reference standards include blank plasma or serum spiked with isotopically labeled or unlabeled human or non-human IGF-2 (e.g., isotopically labeled or unlabeled recombinant mouse IGF-2).

[0024] In some embodiments, an isotopic signature comprising mass spectrometry peaks from two or more molecular isotopic forms of an analyte may be used to confirm the identity of the analyte under study. In other embodiments, mass spectrometry peaks from one or more isotopic forms may be used to quantify the analyte of interest. In some related embodiments, a single mass spectrometry peak from one isotopic form may be used to quantify the analyte of interest. In other related embodiments, multiple isotopic peaks may be used to quantify the analyte. The multiple peaks may be subjected to any suitable mathematical treatment. Some mathematical treatments are known in the art, including, but not limited to, summing the areas under multiple peaks or averaging the responses from multiple peaks.

[0025] As used herein, the singular forms "a," "an," and "the" include plural references unless specifically stated otherwise. Thus, for example, reference to a "protein" includes a plurality of protein molecules.

[0026] As used herein, the term "IGF-2 protein" refers to full-length IGF-2 polypeptides or fragments thereof, as well as full-length IGF-2 variant polypeptides or fragments thereof. Exemplary chemical modifications may include reduction of one or more disulfide bridges or alkylation of one or more cystines. These exemplary chemical modifications result in an increase in the mass of the IGF-2 variant polypeptide compared to the mass of the corresponding unmodified IGF-2 polypeptide. The reduction of one or more disulfide bridges results in a relatively small change in the mass of the molecule, and the resulting mass-to-charge ratio is within the range of mass-to-charge ratios described herein. Other chemical modifications that result in a mass deviation from the unmodified IGF-2 polypeptide are also encompassed within the meaning of IGF-2 protein. Those skilled in the art will understand that the addition of atoms to the IGF-2 protein by chemical modification results in an observed increase in the mass-to-charge ratio during mass spectrometry. Thus, variants of IGF-2 protein resulting from chemical modifications are encompassed within the meaning of IGF-2 protein and are detectable according to the methods of the present invention.

[0027] As used herein, the term "intact" to describe a polypeptide refers to a full-length (i.e., non-fragmented) polypeptide. Non-intact forms (i.e., fragments) of IGF-2 can also be detected by the methods described herein. For example, fragments of IGF-2 protein having molecular weights of about 1,000 daltons or more, such as about 1500 daltons or more, such as about 2000 daltons or more, such as about 2500 daltons or more, such as about 3000 daltons or more, such as about 4000 daltons or more, such as about 5000 daltons or more, such as about 6000 daltons or more, such as about 7000 daltons or more can be detected by the methods described herein.

[0028] The term "purification" or "purifying" refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in a sample that may interfere with the detection of the analytes of interest. Although not required, "purification" may completely remove all interfering components, or even all materials other than the analytes of interest. Purification of a sample by various means may allow for the relative reduction of one or more interfering substances, for example, one or more substances that may or may not interfere with the detection of a selected parent or child ion by mass spectrometry. The relative reduction used in this term does not require that any substances present with the analytes of interest in the material being purified are completely removed by purification.

[0029] The term "sample" refers to any sample that may contain an analyte of interest. As used herein, the term "body fluid" refers to any fluid that may be isolated from the body of an individual. For example, "body fluid" may include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc. In a preferred embodiment, the sample comprises a body fluid sample, preferably plasma or serum.

[0030] The term "solid phase extraction" or "SPE" refers to a process in which a chemical mixture is separated into components as a result of the affinity of the components dissolved or suspended in a solution (i.e., mobile phase) for the solids (i.e., solid phase) on or around which the solution passes. In some examples, as the mobile phase passes through or around the solid phase, undesired components of the mobile phase may be retained by the solid phase, resulting in purification of the analytes in the mobile phase. In other examples, the analytes may be retained by the solid phase, allowing the undesired components of the mobile phase to pass through or around the solid phase. In these examples, a second mobile phase is then used to elute the retained analytes from the solid phase for further processing or analysis. SPE may operate via a single mode mechanism or a mixed mode mechanism. As used herein, SPE may be performed using an extraction column or cartridge, such as, for example, a turbulent flow liquid chromatography (TFLC) column. A mixed mode mechanism utilizes ion exchange and hydrophobic retention in the same column. For example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention, or may exhibit strong cation exchange and hydrophobic retention.

[0031] The term "chromatography" refers to a process by which chemical mixtures carried by a liquid or gas are separated into components as a result of the differential distribution of chemicals as they flow through a stationary solid phase.

[0032] The term "liquid chromatography" or "LC" refers to the process of selective retardation of one or more components of a fluid solution as the fluid percolates uniformly through a column of finely divided material or through a capillary passage. Retardation results from the distribution of components of the mixture between one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Examples of separation techniques that employ "liquid chromatography" include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent flow liquid chromatography (TFLC) (also known as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography). In some embodiments, an SPE column may be used in combination with an LC column. For example, a sample may be purified in a TFLC extraction column followed by further purification in an HPLC analytical column.

[0033] The term "high performance liquid chromatography" or "HPLC" (also known as "high pressure liquid chromatography") refers to liquid chromatography in which the degree of separation is increased by forcing a mobile phase under pressure through a stationary phase, typically a densely packed column.

[0034] The term "turbulent liquid chromatography" or "TFLC" (also known as high turbulence liquid chromatography or high throughput liquid chromatography) refers to a form of chromatography that utilizes the turbulent flow of the material to be assayed through a column packing as the basis for performing separations. TFLC has been applied to the preparation of a sample containing two unnamed drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et al., J Chromatogr A 854:23-35 (1999). See also U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further describe TFLC. Those skilled in the art understand "turbulent flow." When a fluid flows slowly and smoothly, the flow is called "laminar flow." For example, a fluid moving through an HPLC column at a low flow rate is laminar. In laminar flow, the motion of the particles of the fluid is orderly, and the particles generally move in straight lines. At higher velocities, the inertia of the water overcomes the forces of fluid friction and results in turbulence. Fluids that are not in contact with irregular boundaries will "overtake" fluids that are slowed down by friction or deflected by uneven surfaces. When a fluid is turbulent, it flows in eddies and whirls (or vortices), which creates more "drag" than when the flow is laminar. Numerous references are available to help determine if a fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction, PS Bernard & J.M. Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

[0035] The term "gas chromatography" or "GC" refers to chromatography in which a sample mixture is vaporized, injected into a stream of carrier gas (such as nitrogen or helium), travels through a column containing a stationary phase consisting of a liquid or particulate solid, and is separated into its component compounds according to the compounds' affinity for the stationary phase.

[0036] The terms "online" and "in-line", for example when used in "online automation mode" or "online extraction", refer to a procedure that is performed without the need for operator intervention. In contrast, the term "offline" as used herein refers to a procedure that requires manual operator intervention. Thus, if a sample undergoes precipitation and then the supernatant is manually loaded into an autosampler, the precipitation and loading steps are offline from the subsequent steps. In various embodiments of the method, one or more steps may be performed in an online automation mode.

[0037] The term "mass spectrometry" or "MS" refers to an analytical technique for identifying compounds by their mass. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio or "m / z". MS techniques generally involve (1) ionizing a compound to form a charged compound, and (2) detecting the molecular weight of the charged compound and calculating the mass-to-charge ratio. Compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized and then introduced into a mass spectrometry instrument where a combination of magnetic and electric fields causes the ions to follow paths in space that depend on their mass ("m") and charge ("z"). See, e.g., 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," and 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.

[0038] As used herein, "high resolution / accuracy mass analysis" refers to mass analysis performed in a mass analyzer capable of measuring the mass-to-charge ratio of charged species with sufficient accuracy and precision to identify unique chemical ions. Unique chemical ion identification is possible for an ion when the individual isotopic peaks from the ion are readily distinguishable. The particular resolution and mass accuracy required to identify unique chemical ions will vary depending on the mass and charge state of the ion.

[0039] As used herein, the term "resolution" or "resolution (FWHM)" (known in the art as "m / Δm 50% ") refers to the observed mass-to-charge ratio divided by the width of the mass peak at 50% maximum height (Full Width Half Maximum, "FWHM"). At this highest resolution, individual isotope peaks contain less than 1% contribution from the baseline.

[0040] As used herein, with respect to mass spectrometry, a "unique chemical ion" refers to a single ion having a single atomic structure. A single ion may be singly or multiply charged.

[0041] As used herein, the term "precision" (or "mass accuracy") in reference to mass spectrometry refers to the potential deviation of the instrument response from the true m / z of the ion being investigated. Precision is typically expressed in parts per million (ppm).

[0042] The high resolution / precision mass spectrometry method of the present invention may be performed on an instrument capable of performing mass analysis with a FWHM of 10,000, 15,000, 20,000, 25,000, 50,000, 100,000, or even higher. Similarly, the method of the present invention may be performed on an instrument capable of performing mass analysis with an accuracy of 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5 ppm, 3 ppm, or even lower. Instruments capable of these performance characteristics may incorporate certain Orbitrap mass analyzers, time-of-flight ("TOF") mass analyzers, or Fourier transform ion cyclotron resonance mass analyzers. In a preferred embodiment, the method is performed using an instrument that includes an Orbitrap mass analyzer or a TOF mass analyzer.

[0043] The term "Orbitrap" describes an ion trap consisting of an outer barrel-shaped electrode and a coaxial inner electrode. Ions are injected tangentially into the electric field between the electrodes and are trapped as the ions orbit the coaxial inner electrode because the electrostatic interaction between the ion and the electrode is balanced by centrifugal forces. As the ions orbit the coaxial inner electrode, the orbital path of the trapped ions oscillates along the axis of the central electrode at a harmonic frequency relative to the mass-to-charge ratio of the ion. Detection of the orbital oscillation frequency allows the Orbitrap to be used as a mass analyzer with high accuracy (down to 1-2 ppm) and high resolution (FWHM) (up to about 200,000). Mass analyzers based on the Orbitrap are described in detail in U.S. Pat. No. 6,995,364, which is incorporated herein by reference in its entirety. Use of the Orbitrap analyzer has been reported for qualitative and quantitative analysis of various analytes. See, e.g., 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.

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

[0045] The terms "ionization" or "ionizing" refer to the process of producing ions with a net charge equal to one or more electron units. A negative ion is one that has a net negative charge of one or more electron units, and a positive ion is one that has a net positive charge of one or more electron units.

[0046] The term "selective ion monitoring" refers to a detection mode of a mass spectrometry instrument in which only ions within a relatively narrow mass range, typically about one mass unit or less, are detected.

[0047] "Multiple reaction mode", also known as "selected reaction monitoring", is a detection mode of a mass spectrometer instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0048] The term "lower limit of quantification", "lower limit of quantitation", or "LLOQ" refers to the point at which a measurement becomes quantitatively meaningful. The analyte response at this LLOQ is identifiable, discrete, and reproducible with a relative standard deviation (RSD%) of less than 20% and an accuracy of 85%-115%.

[0049] The term "limit of detection" or "LOD" is the point at which a measured value is greater than the uncertainty associated with that measurement. The LOD is defined as four times the RSD of the mean at the zero concentration.

[0050] The term "simultaneous" as applied to simultaneously detecting the amount of two or more analytes from a sample means obtaining data reflecting the amount of two or more analytes in the sample from the same sample injection. Data for each analyte may be obtained sequentially or in parallel, depending on the instrument technology employed. For example, a single sample containing two analytes, such as IGF-1 and IGF-2 proteins, may be injected into an HPLC column, which may then elute each analyte in turn, resulting in the introduction of the analytes into the mass spectrometer in sequence. Determining the amount of each of these two analytes is done simultaneously for purposes herein, since both analytes result from the injection of the same sample into the HPLC.

[0051] The "amount" of an analyte in a body fluid sample generally refers to an absolute value reflecting the mass of the analyte detectable in a volume of the sample. However, the amount also contemplates a relative amount compared to the amount of another analyte. For example, the amount of an analyte in a sample can be an amount that is greater than a control level or normal level of the analyte that is normally present in the sample.

[0052] The term "about" as used herein in connection with quantitative measurements that do not include measurement of the mass of an ion refers to the indicated value plus or minus 10%. Mass spectrometry instruments may vary slightly in determining the mass of a given analyte. The term "about" in the context of the mass of an ion or the mass-to-charge ratio of an ion refers to + / - 0.50 atomic mass units.

[0053] The above summary of the invention is non-limiting; other features and advantages of the present invention will be apparent from the following detailed description of the invention and the claims. [Brief description of the drawings]

[0054] [Figure 1] 1 shows mass spectral detection of a previously unknown variant of IGF-2 (IGF-2+68R). [Diagram 2] 1 shows the mass spectral differences between an unknown variant of IGF-2 and IGF-1 wild type (WT). [Diagram 3]Improved mass spectrum of an unknown variant of IGF-2 (IGF-2+68R) is shown. [Figure 4] The results of a Basic Local Alignment Search Tool (BLAST) search for an unknown variant of IGF-2 (IGF-2+68R) are shown. [Diagram 5] 1 shows the mass spectrum of a novel variant of IGF-2 (IGF-2+68R). [Figure 6] A diagram of the IGF-2 protein domains is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Suitable test samples for use in the methods of the invention include any test sample that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample, i.e., a sample obtained from any biological source, such as an animal, cell culture, organ culture, etc. In certain preferred embodiments, the sample is obtained from a mammal, such as a dog, cat, horse, etc. Particularly preferred mammals are primates, most preferably humans, male or female. Preferred samples include 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, i.e., a living male or female individual presenting to a clinical setting for diagnosis, prognosis, or treatment of a disease or condition.

[0056] Quality control (QC) pools of known concentrations for use in embodiments of the present invention are preferably prepared using a matrix similar to the intended sample matrix.

[0057] Sample preparation for mass spectrometry In preparation for mass spectrometry, the IGF-2 protein may be enriched relative to one or more other components (e.g., other proteins) in the sample by a variety of methods known in the art, including, for example, solid phase extraction (SPE), LC, 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 chaotropic agents, or any combination of the above. In some embodiments, liquid chromatography, and / or SPE, and / or protein precipitation may be used in combination.

[0058] Protein precipitation is one method of preparing test samples, particularly biological samples such as serum or plasma. Protein purification methods are well known in the art, for example, Polson et al., Journal of Chromatography B 2003,785:263-275, describes protein precipitation techniques suitable for use in the methods of the present invention. Protein precipitation can be used to remove most of the proteins from the sample, leaving IGF-I and / or IGF-II proteins in the supernatant. The sample can be centrifuged to separate the liquid supernatant from the precipitated proteins, or alternatively, the sample can be filtered to remove the precipitated proteins. The resulting supernatant or filtrate can then be applied directly to mass spectrometry, or alternatively to solid phase extraction and / or liquid chromatography, and subsequent mass spectrometry. In certain embodiments, the use of protein precipitation (e.g., acidic ethanol protein precipitation) may eliminate the need for TFLC, SPE, or other online extraction before mass spectrometry or HPLC and mass spectrometry.

[0059] In preferred embodiments, liquid-liquid extraction methods (such as acid ethanol extraction) are used to extract the native IGF-2 variants from the sample. In these embodiments, 10 μl to 500 μl, for example 25 μl to 250 μl, for example about 100 μl of sample is added to a portion of the extraction solvent. The amount of extraction solvent is commensurate with the sample volume and may vary depending on the extraction solvent used, but is preferably about 50 μl to 1000 μl. The sample / solvent mixture is mixed and centrifuged, and a portion of the supernatant or organic phase (depending on the solvent used) is removed for further analysis. The solvent may be removed from the removed portion, for example under a nitrogen stream, and the residue is reconstituted in a solvent different from that used for the liquid-liquid extraction. At least a portion of the resulting solution may then undergo additional processing steps, such as SPE and / or LC, prior to mass spectrometry.

[0060] Another method of sample purification that may be used prior to mass spectrometry is liquid chromatography (LC). Certain methods of liquid chromatography, including HPLC, rely on relatively slow laminar flow techniques. Traditional HPLC analysis relies on column packings, where laminar flow of the sample through the column is the basis for separating the analytes of interest from the sample. Those skilled in the art will understand that separation in such columns is a diffusion process and may select LC, including HPLC, instruments, and columns suitable for use with IGF-2. Chromatography columns typically include a medium (i.e., packing) to facilitate separation (i.e., fractionation) of chemical moieties. The medium may include particulates or may include monolithic materials with porous channels. The surface of the medium typically includes a binding surface that interacts with various chemical moieties to facilitate separation of the chemical moieties. One suitable binding surface is a hydrophobic binding surface, such as an alkyl- or cyano-binding surface. The alkyl-binding surface may include C-4, C-8, C-12, or C-18-bound alkyl groups. In a preferred embodiment, the column is a C-18 alkyl bonded column (such as a Phenomenex Onyx monolithic C-18 column). The chromatography column includes an inlet port for receiving the sample and an outlet port for discharging the eluate containing the fractionated sample. The sample can be delivered directly to the inlet port or from an SPE column, such as an online SPE guard cartridge or a TFLC column.

[0061] In one embodiment, a sample may be applied to an LC column at an inlet port, eluted with a solvent or solvent mixture, and discharged at an outlet port. Different solvent modes may be selected to elute the analyte(s) of interest. For example, liquid chromatography may be performed using gradient, isocratic, or polymorphic (i.e., mixed) modes. During chromatography, separation of materials is affected by variables such as the choice of eluent (also known as the "mobile phase"), elution mode, gradient conditions, and temperature.

[0062] In certain embodiments, analytes may be purified by applying a sample to a column under conditions in which the analytes of interest are reversibly retained by the column packing while one or more other materials are not retained. In these embodiments, first mobile phase conditions may be employed in which the analytes of interest are retained by the column, and then second mobile phase conditions may be employed to wash away the unretained materials and remove the retained materials from the column. Alternatively, analytes may be purified by applying a sample to a column under mobile phase conditions in which the analytes of interest elute at a differential rate compared to one or more other materials. Such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

[0063] In some embodiments, HPLC is performed using an alkyl bond analytical column chromatography system. In certain embodiments, a C-18 analytical column (e.g., Phenomenex Onyx monolithic C18, or equivalent) is used. In certain embodiments, HPLC and / or TFLC is performed using HPLC grade 0.2% formic acid in water as mobile phase A, and 0.2% formic acid in acetonitrile as mobile phase B.

[0064] By careful selection of valves and connector tubing, two or more chromatography columns can be connected so that materials pass from one to the other as needed without the need for manual steps. In a preferred embodiment, the selection of valves and tubing is controlled by a computer preprogrammed to perform the necessary steps. Most preferably, the chromatography system is also connected to a detector system (e.g., an MS system) in such an on-line manner. Thus, an operator can place a tray of samples into the autosampler and the remaining operations are performed under computer control, resulting in the purification and analysis of all selected samples.

[0065] In some embodiments, TFLC can be used for purification of IGF-2 proteins or fragments prior to mass spectrometry. In such embodiments, samples can be extracted using a TFLC column to capture the analyte, then eluted and chromatographed on a second TFLC column or an analytical HPLC column prior to ionization. For example, sample extraction with a TFLC extraction column can be accomplished with a large particle size (50 μm) packed column. The sample eluted from this column can then be transferred to an analytical HPLC column for further purification prior to mass spectrometry. The steps involved in these chromatographic procedures can be linked in an automated fashion, thus minimizing the need for operator involvement during the purification of the analyte. This feature can save time and costs and eliminate the possibility of operator error.

[0066] In some embodiments, protein precipitation is achieved by acidic ethanol extraction from the serum, and the resulting solution is subjected to SPE, preferably performed on-line using a C-18 extraction column (e.g., a Phenomenex Onyx C-18 guard cartridge or equivalent). The eluate from the SPE column can then be applied to an analytical LC column, such as an HPLC column, in an on-line fashion prior to mass spectrometry.

[0067] Detection and quantification by mass spectrometry Mass analysis is performed using a mass spectrometer, which includes an ion source for ionizing the sample and generating charged molecules for further analysis. In various embodiments, the IGF-2 protein can be ionized by any suitable method known to those skilled in the art. For example, the ionization of the sample can be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray ionization / 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 selection of the ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the selection of positive mode vs. negative mode, and the like. Depending on the particular ionization method and conditions employed, IGF-2 protein can be ionized into several different charge states. The ionization source can be selected to minimize the dispersion of the generated charge states. In some embodiments, ESI (optionally heated) is used as the ionization source, and the ionization conditions are optimized to minimize the dispersion of the observed multiply charged IGF-2 protein ions.

[0068] The IGF-2 proteins may be ionized in positive or negative mode. In a preferred embodiment, one or more IGF-2 proteins are ionized in positive mode. The majority of the multiply charged ions generated within these ranges may be within narrower sub-ranges, such as the indicated m / z ±1.

[0069] In mass spectrometry techniques, generally, after a sample is ionized, the positively or negatively charged ions generated thereby can be analyzed to determine the mass-to-charge ratio (m / z). Various analyzers for determining m / z include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers, as well as orbitrap analyzers. According to the method of the present invention, high resolution / high accuracy mass spectrometry is used for quantitative analysis of IGF-2 protein. That is, mass spectrometry is performed using a mass spectrometer that can exhibit a resolution (FWHM) of at least 10,000 with an accuracy of about 50 ppm or less for the ions of interest. Preferably, the mass spectrometer exhibits a resolution (FWHM) of 20,000 or more and an accuracy of about 20 ppm or less, for example, a resolution (FWHM) of 25,000 or more and an accuracy of about 5 ppm or less, for example, a resolution (FWHM) of 25,000 or more and an accuracy of about 3 ppm or less. Three exemplary mass spectrometers capable of demonstrating the required level of performance for IGF-2 protein ions include an Orbitrap mass analyzer, certain TOF mass analyzers, or a Fourier transform ion cyclotron resonance mass analyzer.

[0070] Elements found in biologically active molecules, such as carbon, oxygen, and nitrogen, exist naturally in several different isotopic forms. For example, most carbon is 12 C, about 1% of all naturally occurring carbon is 13 C. Thus, some naturally occurring carbon-containing molecules have at least one 13C atoms. The inclusion of naturally occurring elemental isotopes in molecules results in multiple molecular isotopic forms. The difference in mass of molecular isotopic forms is at least 1 atomic mass unit (amu). This is because elemental isotopes differ by at least one neutron (mass of one neutron ≈ 1 amu). When molecular isotopic forms are ionized to multiply charged states, mass discrimination between isotopic forms can be difficult to distinguish because mass spectrometry detection is based on mass-to-charge ratio (m / z). For example, two isotopic forms that differ in mass by 1 amu, both ionized to the 5+ state, show a difference in their m / z of only 0.2 (1 amu difference / 5 charge states). High resolution / high accuracy mass spectrometers can distinguish isotopic forms of multiply charged ions (such as ions with charges of ±5, ±6, ±7, ±8, ±9, or more).

[0071] For naturally occurring elemental isotopes, there are typically multiple isotopic forms for every molecular ion (each of which may produce a separately detectable spectroscopic peak when analyzed with a sufficiently sensitive mass spectrometry instrument). The m / z ratios and relative abundances of the multiple isotopic forms collectively comprise the isotopic signature of the molecular ion. In some embodiments, the m / z ratios and relative abundances for two or more molecular isotopic forms may be utilized to confirm the identity of the molecular ion under investigation. In some embodiments, mass spectrometry peaks from one or more isotopic forms are used to quantify the molecular ion. In some related embodiments, a single mass spectrometry 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 latter embodiments, the multiple isotopic peaks may be subjected to any suitable mathematical treatment. Some mathematical treatments 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.

[0072] In mass spectrometry techniques, ions can generally be detected using several detection modes. For example, selected ions can be detected, i.e., using selected ion monitoring mode (SIM), or alternatively, ions can be detected using scan mode. When operating in scan mode, the mass spectrometer typically provides the user with an ion scan, i.e., the relative abundance of each ion having a particular mass / charge over a given range (e.g., 100-1000 amu). Furthermore, when using instruments capable of multiple mass spectrometry events, such as certain ion trap or triple quadrupole instruments, mass transitions resulting from collision-induced dissociation or neutral losses can be monitored (e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM)).

[0073] The results of the assay of an analyte, i.e., mass spectrum, can be related to the amount of the analyte in the original sample by a number of methods known in the art. For example, given that the sampling and analysis parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts the relative abundance to the absolute amount of the original molecule. Alternatively, an internal or external standard can be run with the sample and a standard curve constructed based on the ions generated from those standards. Using such a standard curve, the relative abundance of a given ion can be converted to the absolute amount of the original molecule. In certain preferred embodiments, one or more standards are used to generate a standard curve for calculating the amount of IGF-2 protein. Methods for generating and using such standard curves are well known in the art, and the skilled artisan can select an appropriate internal standard. For example, in a preferred embodiment, isotopically labeled or unlabeled non-human IGF-2 (e.g., recombinant mouse IGF-2), or isotopically labeled human IGF-2 can be used as a standard. Many other methods for relating the amount of an ion to the amount of the original molecule will be known to those skilled in the art.

[0074] As used herein, an "isotopic label" produces a mass shift in the labeled molecule compared to the unlabeled molecule when analyzed by mass spectrometry techniques. Examples of suitable labels include deuterium ( 2 H), 13 C, and 15 N. Isotopic labels can be incorporated at one or more positions within a molecule, and more than one type of isotopic label can be used on the same isotopically labeled molecule.

[0075] One or more steps of the method may be performed using automated machinery. In certain embodiments, one or more purification steps may be performed online, and more preferably, all of the purification and mass spectrometry steps may be performed online.

[0076] In some embodiments, IGF-2 variants in a sample are detected and / or quantified using MS as follows: The sample is subjected to liquid chromatography, preferably HPLC, and the liquid solvent flow from the chromatography column enters the heated nebulizer interface of the ESI ionization source, and the solvent / analyte mixture is converted to vapor in the heated charged tube of the interface. The analyte (e.g., IGF-2 variant) contained in the solvent is ionized by applying a large voltage to the solvent / analyte mixture. As the analyte exits the charged tube of the interface, the solvent / analyte mixture is nebulized, the solvent evaporates, and the analyte ions are in various charge states. Quantitative data is then collected for the intensity of one or more of the ions. Quantitative data of the signal intensity of one or more ions is then collected and related to the amount of IGF-2 variant in the sample.

[0077] For IGF-2 variants, ions of various charge states can be observed with m / z, and at this level of precision, the observed mass for any ion can vary slightly (e.g., ±0.1) due to instrumental differences.

[0078] In some embodiments, the use of a high resolution / high accuracy mass spectrometer may allow the signal intensity of a peak from a single isotopic form of a single ion to be selected for data acquisition. Alternatively, quantitative data of the signal intensity from one or more isotopic forms of a single ion, or across a narrow m / z range, may be collected and related to the amount of IGF-2 variant in the sample.

[0079] In some embodiments, quantitative data of signal intensity is collected for one or more IGF-2 ions from at least two different charge states.The intensities of these ions can then be used to quantitatively evaluate the IGF-2 variants in a sample.For example, IGF-2 variants can be quantified using the signal intensity from one or more ions of 8+ charge state or 7+ charge state.In embodiments where quantitative data of the signal intensity of two or more ions is collected, the intensities can be combined by any mathematical method known in the art (e.g., summation, or averaging of the area under the curve) to quantitatively evaluate the IGF-2 variants in a sample.

[0080] When an ion collides with the detector, a pulse of electrons is generated, which is converted into a digital signal. The acquired data is relayed to a computer, which plots the counts of collected ions against time. The resulting mass chromatogram is similar to that generated by conventional HPLC-MS methods. The area under the peak corresponding to a particular ion or the amplitude of such peak can be measured and correlated to the amount of the analyte of interest. In certain embodiments, the area under the curve or the amplitude of the peak is measured to determine the amount of IGF-I and / or IGF-II protein or fragment. As described above, the relative abundance of a given ion can be converted to the absolute amount of the original analyte using a calibration standard curve based on one or more ion peaks of an internal molecular standard.

[0081] In some embodiments, IGF-1 and IGF-2 are quantified simultaneously. In these embodiments, each of IGF-1 and IGF-2 may be quantified by any of the methods provided above.

[0082] In certain preferred embodiments, the lower limit of quantification (LLOQ) of the IGF-2 variant is within the range of about 30.0 ng / mL to 200 ng / dL (including the boundary value), preferably within the range of about 30.0 ng / dL to 100 ng / mL (including the boundary value), preferably within the range of about 30.0 ng / mL to 50 ng / mL (including the boundary value), preferably within the range of about 30.0 ng / mL to 25 ng / mL (including the boundary value), preferably within the range of about 30.0 ng / mL to 15 ng / mL (including the boundary value), preferably within the range of about 30.0 ng / mL to 10 ng / mL (including the boundary value), preferably about 30.0 ng / mL.

[0083] In certain preferred embodiments, the limit of detection (LOD) of the IGF-2 variant is within the range of about 8.2 ng / mL to 200 ng / mL (inclusive), preferably within the range of about 8.2 ng / mL to 100 ng / mL (inclusive), preferably within the range of about 8.2 ng / mL to 50 ng / mL (inclusive), preferably within the range of about 8.2 ng / mL to 25 ng / mL (inclusive), preferably within the range of about 8.2 ng / mL to 20 ng / mL, preferably about 8.2 ng / mL.

[0084] The following examples serve to illustrate the present invention. These examples are not intended to limit the scope of the method in any way. In particular, the following examples show the quantification of IGF-2 protein or fragments by mass spectrometry using a specific internal standard. The use of the internal standard shown is not intended to be limiting in any way. Any suitable chemical species can be used as an internal standard, as easily determined by a person skilled in the art. EXAMPLES

[0085] Example 1: Identification of IGF-2 variants by mass spectrometry During the analysis of IGF-1 variants, an unusual variant was detected in VG1. Detected in IP1, its relative retention time (rRT) was -0.19 min, which did not match any previously identified variants. MS / MS detected a PAK sequence from the C-terminus. The unusually high y ion mass, 214 Da heavier than expected, could be explained by either a heavy chain modification of the last two amino acids or a heavy chain modification of the C-terminus. However, no such candidate was found in the post-translational modification (PTM) list. Figure 1.

[0086] In addition to the unknowns, the total mass of the protein was 30.9 daltons lighter than that of WT IGF-1. This mystery remained unsolved until another specimen with the same modifications was discovered. Figure 2.

[0087] Another sample was sequenced by MS / MS, this time producing a better spectrum. Two additional C-terminal amino acids were identified making the sequence ATPAK. In addition, we attempted to sequence the N-terminus where the ETL sequence was detected. All these new findings only added to the mystery. All y-ions were 214 Da heavier than predicted, y6 and y7 did not match the WT IGF-1 sequence, the N-terminal ion, ETL, was 420 Da heavier than predicted and the total protein was 30 Da lighter than WT IGF-1. Figure 3.

[0088] The identified C-terminal sequence, ATPAK, was searched using BLAST. One of the potential candidates was the structurally similar protein, IGF-2.

[0089] The assumptions were changed and the hypothesis was that this was an IGF-2 variant. The N-terminus of IGF-2 starts with the sequence AYRPSETL, which contains the ETL fragment, but with a much heavier m / z due to the presence of residual amino acids (the IGF-1 sequence is simply GPETL). However, the C-terminus of the IGF-2 variant still had an additional 156 Da to be accounted for (here measured relative to the IGF-2 sequence). This mass difference corresponds to a C-terminal arginine (R) extension to the mature IGF-2 sequence (1-67). R68 immediately follows the C-terminus of the mature IGF-2 polypeptide sequence and is the first amino acid of the E domain of the prohormone. When this possibility is considered, all discrepancies are resolved. All b and y ions match the correct m / z ratios and the total m / z of the protein matches exactly the theoretical value within 0.6 ppm. Conclusion: this is the case for an IGF-2 protein (ATPAKSER) with an R extension at the C-terminus. Figure 5.

[0090] The m / z used to detect the IGF-2 variant was 1089.8092, detected in IP1.

[0091] In the IGF-2 prohormone, the E domain (68-156), beginning with R, is cleaved upon protein maturation, leaving only the WT IGF-2 sequence. In the present case, it seems likely that R was retained along with the IGF-2 sequence. To our knowledge, IGF-2(1-68) has not been observed before. It may have some relationship to the so-called "big IGF-2", described as an IGF-2 protein in which erroneous cleavage results in an extra amino acid from the E domain of the pro-IGF-2 sequence. Both big IGF-2 polypeptides 1-87 and 1-104 described in the literature are biomarkers for non-islet cell tumor hypoglycemia (NICTH). Figure 6.

[0092] Conclusions: Our workflow for routine monitoring of IGF-1 variants resulted in the detection of an abnormal IGF-2 species (1-68) indicative of unexpected prohormone processing with unknown clinical significance and unknown frequency of occurrence.

[0093] The contents of the publications, patents, and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such publications, patents, patent applications, or other physical and electronic documents.

[0094] The method illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing" and the like should be read broadly and without limitation. In addition, the terms and expressions used herein are used as terms of description, not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention as claimed. Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention disclosed and embodied herein may be utilized by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

[0095] The present invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the method. This includes the generic description of the method with a provisos or negative limitation removing any subject matter from that genus, regardless of whether the removed material is specifically recited herein.

[0096] Other embodiments are within the scope of the following claims. Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual members or subgroups of members of the Markush group.

Claims

1. 1. A method for detecting insulin-like growth factor-2 (IGF-2) variant(s) in a sample, comprising: a. ionizing the IGF-2 variant(s) in said sample to produce one or more ions detectable by mass spectrometry; b. detecting one or more of said ions by mass spectrometry, including an ion having a mass-to-charge ratio of 1089.8±0.5; c. using said detected ion or ions to determine the presence of IGF-2 variant(s) in said sample.

2. 2. The method of claim 1, wherein the method detects variants having an extra arginine at position 68.

3. 10. The method of claim 1, further comprising purifying the protein by high performance liquid chromatography (HPLC) prior to ionization.

4. The method of claim 1 , wherein the sample comprises plasma or serum.

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