Identification and quantification of insulin-like growth factor I variants by mass spectrometry

Abstract

Methods are provided for detecting insulin-like growth factor I (IGF-I) variants in a sample. The methods provided herein are further directed to determining the presence of an IGF1 variant in the sample using the detected ions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 163,502, filed March 19, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the identification and quantification of insulin-like growth factor 1 variants using mass spectrometry. [Background technology]

[0003] IGF-I is a hormone with a molecular structure similar to insulin. It is a peptide produced by the liver, containing 70 amino acids in a single chain and with three intramolecular disulfide bridges. IGF-I has a molecular weight of approximately 7,649 Da and is highly protein bound in serum. Production is stimulated by growth hormone and can be delayed by nutritional deficiencies, growth hormone insensitivity, lack of growth hormone receptors, or defective downstream signaling pathways. IGF-I plays an important role in childhood growth and continues to exert anabolic effects in adulthood.

[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-1 includes only WT and does not include polymorphisms. For example, the presence of IGF-1 variants may indicate that in heterozygous individuals, WT concentrations represent only half of the circulating IGF-1, or the absence of wild-type IGF-1 may be explained by individuals being homozygous for a variant or heterozygous for two different variants. Some variants have been reported to be pathogenic, while most others are of unknown clinical significance. This information is important for clinicians when interpreting IGF-1 test results for patients. Furthermore, knowing what variants are present in patients with a particular phenotype helps expand our understanding of genotype-phenotype relationships and their clinical significance.

[0006] Effective methods for identifying IGF-1 variants are needed. Summary of the Invention

[0007] Methods provided herein include isotopic peak indexing (ISI), which allows for the simultaneous monitoring of 15 IGF-1 variants by using 4 m / z ratios. Also provided herein is a relative retention time (RRT) parameter that allows for the differentiation of previously unresolved variants. Also provided herein is a method utilizing tandem mass spectrometry (MS / MS) to differentiate the most common variant pair: isobaric A67T and A70T.

[0008] The methods described herein include enhanced automation, avoidance of detailed manual calculations prone to human error, and the ability to monitor more and discover new IGF-1 variants. In this approach, six variants were identified from the ExAC database: P66A, A67S, S34N, A38V, A67T, and A70T, two of which were previously reported variants V44M and A67V, and six were unreported variants: Y31H, S33P, R50Q, R56K, T41I, and A62T. The methods provided herein include a profile of the IGF-1 status of a patient and can be used to explore genotype-phenotype relationships in IGF-1 variants.

[0009] The method described herein is for the simultaneous monitoring of 15 IGF-1 variants in groups using 4 mass / charge (m / z) ratios without compromising quantification of the WT. Most variants within each group are distinguished by their isotopic distribution and relative retention time, but reanalysis by MS / MS distinguishes A67T from A70T. Calculation and interpretation of LC-MS data is automated using commercial instrument software, requiring little operator intervention, thus protecting the workflow from human error in the interpretation of results.

[0010] In some embodiments, the method comprises detecting the following mass / charge (m / z) ratios for each of the variants of interest: [Table 1]

[0011] In some embodiments, a method provided herein is a method for detecting an insulin-like growth factor I (IGF-I) variant in a sample, the method comprising: ionizing IGF-I in the sample to produce one or more ions detectable by mass spectrometry; detecting by mass spectrometry one or more of the ions including ions having a mass-to-charge ratio selected from the group consisting of 1093.5±0.5, 1089.8±0.5, 1095±0.5, 1097.09±0.5, 1098.09±0.5; and using the detected ions to determine the presence of an IGF1 variant in the sample.

[0012] In some embodiments, detection of m / z 1093.5±0.5 determines the presence of WT4 or P66T1.

[0013] In some embodiments, detection of m / z 1089.8±0.5 determines the presence of P66A4, R55K6, or R36Q6.

[0014] In some embodiments, detection of m / z 1095.8±0.5 determines the presence of A67S4 or T29I8.

[0015] In some embodiments, detection of m / z 1097.09±0.5 is S34N2, A70P3, A38V1, M59R4, or A67S 13 Determine the existence of

[0016] In some embodiments, detection of m / z 1098.09±0.5 is S34N9, A70P 10 , A38V8, M59R 11 , V17 / 44M4, R50W6, T4M6, or A67 / 70T6.

[0017] In some embodiments, the methods provided herein include purifying the protein by high performance liquid chromatography (HPLC) prior to ionization.

[0018] In some embodiments, the sample comprises plasma or serum.

[0019] In some embodiments, samples may be purified by high performance liquid chromatography (HPLC) prior to ionization. In a related embodiment, IGF-1 and its variants are extracted and resolved using a multiplexed high performance liquid chromatography (HPLC) system (Aria TLX-4) equipped with on-line extraction.

[0020] In some embodiments, the sample may be purified by solid phase extraction (SPE) prior to ionization.

[0021] In some embodiments, the mass spectrometer comprises a Q Exactive Focus Hybrid quadrupole-orbitrap instrument.

[0022] 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.

[0023] 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.

[0024] In some embodiments, relating the amount of one or more IGF-I ions detected by mass spectrometry to the amount of IGF-I protein in the sample includes comparison to an internal standard, such as a human or non-human IGF-I protein (e.g., intact recombinant mouse IGF-I), which may optionally be isotopically labeled.

[0025] The features of the embodiments listed above can be combined for use in the methods of the present invention, including but not limited to:

[0026] 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 may 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.

[0027] In preferred embodiments, 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 part 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 intact non-human IGF-I (e.g., intact recombinant mouse IGF-I, either isotopically labeled or unlabeled), intact human IGF-I protein, isotopically labeled.

[0028] In other embodiments, the amount of intact IGF-I protein in a sample can 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 intact human or non-human IGF-I (e.g., isotopically labeled or unlabeled intact recombinant mouse IGF-I).

[0029] In some embodiments, an isotopic signature comprising mass spectrometry peaks from two or more molecular isotopic forms of an analyte can be used to confirm the identity of the analyte under study. In other embodiments, mass spectrometry peaks from one or more isotopic forms can be used to quantify the analyte of interest. In some related embodiments, a single mass spectrometry peak from one isotopic form can be used to quantify the analyte of interest. In other related embodiments, multiple isotopic peaks can be used to quantify the analyte. The multiple peaks can be subjected to any suitable mathematical treatment. Several 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.

[0030] 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.

[0031] As used herein, the term "IGF-I protein" refers to full-length IGF-I polypeptides or fragments thereof, as well as full-length IGF-I variant polypeptides or fragments thereof. IGF-I variants include, for example, long R3 IGF-I, which is an 83 amino acid analog of IGF-I that contains the complete human IGF-I sequence, with a substitution of Arg(R) for Glu(E) at position 3 (hence R3) and a 13 amino acid extension peptide at the N-terminus. This analog of IGF-I has been produced with the aim of increasing the biological activity of the IGF-I peptide. The mass of this analog is about 9111.4 daltons, and thus multiple charged long R3 IGF-I ions can be observed with m / z ratios of about 1014.1±1, 1140.7±1, 1303.5±1, and 1520.6±1. Other IGF-I variant polypeptides, including, for example, chemically modified full-length IGF-I polypeptides or fragments thereof, are readily recognized by those skilled in the art. 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-I variant polypeptide compared to the mass of the corresponding unmodified IGF-I 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-I polypeptide are also encompassed within the meaning of IGF-I protein. Those skilled in the art will understand that the addition of atoms to the IGF-I protein by chemical modification results in an observed increase in the mass-to-charge ratio during mass spectrometry. Thus, variants of IGF-I protein resulting from chemical modifications are encompassed within the meaning of IGF-I protein and are detectable according to the methods of the present invention.

[0032] As used herein, the term "intact" to describe a polypeptide refers to a full-length (i.e., non-fragmented) polypeptide. Intact IGF-I is, for example, a polypeptide containing 70 amino acid residues, and intact long R3 IGF-I is an 83 amino acid analog of IGF-I that contains the complete human IGF-I sequence, with a substitution of Arg(R) for GLu(E) at position 3 (hence R3) and a 13 amino acid extension peptide at the N-terminus. Non-intact forms of IGF-I (i.e., fragments) can also be detected by the methods described herein. For example, fragments of IGF-I protein having a molecular weight 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.

[0033] 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" can completely remove all interfering components, or all materials other than the analytes of interest. Purification of a sample by various means can 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.

[0034] 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.

[0035] 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 can operate via a single mode mechanism or a mixed mode mechanism. As used herein, SPE can be performed in 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.

[0036] 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.

[0037] 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 passageway. Retardation results from the distribution of the components of the mixture between one or more stationary phases and a bulk fluid (i.e., mobile phase), which moves relative to the stationary phase. Examples of separation techniques that use "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 can be used in combination with an LC column. For example, a sample may be purified on a TFLC extraction column, followed by further purification on an HPLC analytical column.

[0038] 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.

[0039] 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. Patent 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 "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 & JM Allace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

[0040] 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.

[0041] The terms "online" and "in-line" refer to a procedure that is performed without the need for operator intervention, for example as used in "online automated method" or "online extraction". In contrast, the term "offline" as used herein refers to a procedure that requires manual intervention by an operator. Thus, if a sample is subjected to 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 automated manner.

[0042] 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 include (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 can 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 a path in space that depends 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.

[0043] 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.

[0044] As used herein, the term "resolution" or "resolution (FWHM)" (known in the art as "m / Δm 50% The effect of differences in resolution is illustrated in Figures 1A-C, which show theoretical mass spectra of an ion with m / z of about 1093. Figure 1A shows a theoretical mass spectrum from a mass analyzer with a resolving power of about 3000 (typical operating conditions for a conventional quadrupole mass analyzer). As can be seen in Figure 1A, the individual isotope peaks are not distinguishable. In comparison, Figure 1B shows a theoretical mass spectrum from a mass analyzer with a resolving power of about 10,000, with clearly distinguishable individual isotope peaks. Figure 1C shows a theoretical mass spectrum from a mass analyzer with a resolving power of about 12,000. At this highest resolution, the individual isotope peaks contain less than 1% contribution from the baseline.

[0045] 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.

[0046] As used herein, the term "precision" (or "mass accuracy") in relation to mass spectrometry refers to the potential deviation of the instrument response from the true m / z of the ions being investigated. Precision is typically expressed in parts per million (ppm). The effect of differences in mass precision is illustrated in Figures 2A-D, which show the boundaries of the potential difference between the detected m / z and the actual m / z for a theoretical peak at m / z of 1093.52094. Figure 2A shows the potential range of m / z detected with an accuracy of 120 ppm. In contrast, Figure 2B shows the potential range of m / z detected with an accuracy of 50 ppm. Figures 2C and 2D show the narrower potential range of m / z detected with an accuracy of 20 ppm and 10 ppm.

[0047] 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 less than 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.

[0048] 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 ions and the electrodes 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 ions. 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); Bredehoeft, 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.

[0049] 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.

[0050] 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.

[0051] The term "selective ion monitoring" refers to a detection mode in a mass spectrometer in which only ions within a relatively narrow mass range, typically about one mass unit or less, are detected.

[0052] "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.

[0053] 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%.

[0054] 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.

[0055] 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 a sample from the same sample injection. Data for each analyte can be obtained sequentially or in parallel, depending on the instrument technology used. For example, a single sample containing two analytes, such as intact IGF-I and IGF-II proteins, may be injected into an HPLC column, and then each analyte may be eluted in turn, resulting in the analytes being introduced sequentially into the mass spectrometer. 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.

[0056] 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.

[0057] 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.

[0058] 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]

[0059] [Figure 1] We demonstrate the principle of the IPi concept, where the isotopic peak index is linked to the isotopic peak relative intensity. IGF-1 WT and all variants, whatever their charge state, follow this index intensity distribution. The IPrI is calculated relative to the intensity of the IP4 peak, as used for quantification of WT IGF-1. [Figure 2-1] LC-MS data containing the VG4 peak for samples from two different patients are shown. A: EIC of WT4 from patient 1, B: EIC of VG4 from patient 1, C: variant isotope envelope from patient 1, rectangle indicates IP6 was detected in VG4. [Figure 2-2] LC-MS data containing the VG4 peak for samples from two different patients are shown. D: EIC of WT4 from patient 2, E: EIC of VG4 from patient 2, F: variant isotope envelope from patient 2, rectangle indicates IP4 was detected in VG4. [Figure 3-1] Automated IGF-1 variant detection is shown. Reports are generated in TraceFinder 5.0 Clinical. The variant report only displays samples suspected of containing IGF-1 variants. The IGF-1 quantification report flags suspected homozygous variants. [Figure 3-2] Automated IGF-1 variant detection is shown. Reports are generated in TraceFinder 5.0 Clinical. The variant report only displays samples suspected of containing IGF-1 variants. The IGF-1 quantification report flags suspected homozygous variants. [Figure 4] The LC gradient for the extraction and analytical pumps is shown: Mobile phase A is water with 0.1% formic acid, and mobile phase B is 20% isopropyl alcohol, 80% acetonitrile with 0.1% formic acid. [Diagram 5] Comparison of SIM scans with in-source CID (SID) and HCD at various energies is shown. The red rectangle indicates the highest sensitivity. [Figure 6] An unknown variant from VG2 is shown. The m / z peak of VG2 corresponds to either IP10 or IP11 and did not match any known variants from that group (either IP4 or IP8). DNA sequencing revealed the variant S33P, which matches the calculated m / z of S33P10 of 1095.8130 (different from the m / z of VG2 by 6 ppm). [Figure 7] FIG. 1 shows extracted ion chromatograms of selected y ions after tandem MS / MS indicating the presence of WT and variant A67T in patient samples A and WT, and variant A70T in patient sample B. [Figure 8-1] Simultaneous IGF-1 quantification and detection of IGF-1 variants is shown. The MS experiment is designed as a 2-cycle wide isolation window PRM scan with low NCE (10) and high NCE (30). A low energy scan can be used to detect and quantitate IGF-1, while a high energy scan can be used to extract recycled y ions to distinguish the A67T and A70T variants. The number of scans per quantitative peak is 13 in this case. This can be further increased with faster instruments or lower detector resolution. [Figure 8-2] Simultaneous IGF-1 quantification and detection of IGF-1 variants is shown. The MS experiment is designed as a 2-cycle wide isolation window PRM scan with low NCE (10) and high NCE (30). A low energy scan can be used to detect and quantitate IGF-1, while a high energy scan can be used to extract recycled y ions to distinguish the A67T and A70T variants. The number of scans per quantitative peak is 13 in this case. This can be further increased with faster instruments or lower detector resolution. [Figure 9] The quantification and detection limits of the IGF-1 assay are shown. Concentration (x-axis) and SD (y-axis) values ​​are in ng / mL. [Figure 10] The quantification and detection limits of the IGF-2 assay are shown. Concentration (x-axis) and SD (y-axis) values ​​are in ng / mL. [Figure 11] FIG. 13 shows a comparison of IGF-1 assay aliquots for LC-MS versus RIA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] 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 (e.g., 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 can be obtained, for example, from a patient, i.e., a living person, male or female, in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition.

[0061] 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.

[0062] Sample preparation for mass spectrometry In preparation for mass spectrometry, the IGF-I 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, a combination of liquid chromatography, and / or SPE, and / or protein precipitation may be used.

[0063] 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 protein 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 directly applied to mass spectrometry, or alternatively solid phase extraction and / or liquid chromatography, followed by 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.

[0064] In preferred embodiments, liquid-liquid extraction methods (such as acid ethanol extraction) are used to extract native intact IGF-I and / or IGF-II from the sample. In these embodiments, 10 μl to 500 μl (e.g., 25 μl to 250 μl, e.g., 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 can 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 can 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 can then be subjected to additional processing steps, such as SPE and / or LC, prior to mass spectrometry.

[0065] 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 can select LC, including HPLC, instruments, and columns suitable for use with IGF-I and / or IGF-II. 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 a sample and an outlet port for discharging an eluate containing the fractionated sample. The sample may be fed into the inlet port directly or from an SPE column (e.g., an on-line SPE guard cartridge or a TFLC column).

[0066] 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 analytes 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.

[0067] In certain embodiments, analytes can be purified by applying a sample to a column under conditions in which the analytes of interest are reversibly retained by the column packing material while one or more other materials are not retained. In these embodiments, first mobile phase conditions can be employed in which the analytes of interest are retained by the column, and then second mobile phase conditions can be employed to wash away the unretained materials and remove the retained materials from the column. Alternatively, analytes can 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 can enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

[0068] 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.

[0069] By careful selection of valves and connector tubing, two or more chromatography columns can be connected, as needed, so that materials can pass from one to the other 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.

[0070] In some embodiments, TFLC may be used to purify IGF-I proteins or fragments prior to mass spectrometry. In such embodiments, samples may 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 may be accomplished with a large particle size (50 μm) packed column. The sample eluted from this column may then be transferred to an analytical HPLC column for further purification prior to mass spectrometry. The steps involved in these chromatographic procedures may be linked in an automated fashion, thus minimizing the need for operator involvement during the purification of the analyte. This feature may save time and costs, and eliminate the possibility of operator error.

[0071] 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 may then be applied in an on-line manner to an analytical LC column, such as an HPLC column, prior to mass spectrometry.

[0072] 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-I and / or IGF-II proteins 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 used, IGF-I and IGF-II proteins 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-I and / or IGF-II protein ions.

[0073] IGF-I and / or IGF-II proteins may be ionized in positive or negative mode. In preferred embodiments, one or more IGF-I and / or IGF-II proteins are ionized in positive mode. In some embodiments, intact, multiply charged IGF-I ions are generated with m / z ratios within the ranges of about 850.8±2, 957.1±2, 1093.7±2, and 1275.8±2. In some embodiments, intact, multiply charged IGF-II ions are generated with m / z ratios within the ranges of about 934.69±2, 1068.07±2, 1245.92±2, and 1494.89±2. The majority of the generated multiply charged ions within these ranges may be within narrower sub-ranges, such as the indicated m / z±1.

[0074] In mass spectrometry techniques, generally, a sample is ionized, and 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-I and / or IGF-II proteins. 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 analyzers capable of demonstrating the required level of performance for IGF-I and / or IGF-II protein ions include an Orbitrap mass analyzer, a specific TOF mass analyzer, or a Fourier transform ion cyclotron resonance mass analyzer.

[0075] Elements found in biologically active molecules, such as carbon, oxygen, and nitrogen, exist naturally in several different isotopic forms. For example, most carbon is 12C, about 1% of all naturally occurring carbon 13 C. Thus, some naturally occurring carbon-containing molecules have at least one 13 C 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 into multiple charge states, it can be difficult to distinguish mass distinctions between isotopic forms 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 of which are ionized to the 5+ state, show a difference in their m / z of only 0.2 (1 amu difference per 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).

[0076] Due to 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 can 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 can be subjected to any suitable mathematical treatment. Several 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.

[0077] 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 with 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 loss can be monitored (e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM)).

[0078] The results of an assay (i.e., mass spectrum) of an analyte can be related to the amount of the analyte in the original sample by a number of methods known in the art. For example, if 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-I and / or IGF-II protein. Methods for generating and using such standard curves are well known in the art, and one of skill in the art can select an appropriate internal standard. For example, in a preferred embodiment, isotopically labeled or unlabeled intact non-human IGF-I and / or IGF-II (e.g., recombinant mouse IGF-I and / or IGF-II), or isotopically labeled intact human IGF-I and / or IGF-II can be used as a standard. Many other methods for relating the amount of ion to the amount of the original molecule will be known to those skilled in the art.

[0079] 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.

[0080] 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.

[0081] In some embodiments, intact IGF-I and / or IGF-II in a sample is 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., intact IGF-I and / or IGF-II) 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 intact IGF-I and / or IGF-II in the sample.

[0082] For intact IGF-I, ions of various charge states may be observed at m / z within the range of about 850.8±2 (9+), 957.1±2 (8+), 1093.7±2 (7+), and 1275.8±2 (6+). In some embodiments, data from one or more IGF-I ions having m / z within the range of about 1093.7±2 are collected and used for quantitation. Exemplary ions within this m / z range include IGF-I ions having m / z of about 1091.9±0.1, 1092.8±0.1, 1092.9±0.1, 1093.1±0.1, 1093.2±0.1, 1093.4±0.1, 1093.5±0.1, 1093.7±0.1, 1093.8±0.1, 1093.9±0.1, 1094.1±0.1, 1094.2±0.1, 1094.4±0.1, 1094.5±0.1, 1094.7±0.1, and 1095.4±0.1. This list is not intended to be limiting. Many other ions may be suitable for use in the methods of the present invention, as shown in the spectrum shown in FIG. 3 (which shows detection of a group of isotopic ions at m / z of approximately 828.0509±2, 850.7373±2, 871.8730±2, 920.5544±2, 939.6930±2, 956.9532±2, 975.4576±2, 1034.8128±2, 1051.6337±2, 1073.9335±2, 1093.6597±2, 1114.5219±2, 1207.9401±2, 1226.5705±2, 1252.5871±2, and 1275.6019±2). However, as noted above, ions of individual isotopes within these ranges will primarily fall within the indicated m / z range of ±1. Also, at this level of precision, the observed mass for any ion may vary slightly (e.g., ±0.1) due to instrumental variations.

[0083] For intact IGF-II, ions of various charge states may be observed at m / z within the range of about 934.69±2 (8+), 1068.07±2 (7+), 1245.92±2 (6+), and 1494.89±2 (5+). In some embodiments, data from one or more IGF-II ions having m / z within the range of about 1068.07±2 are collected and used for quantitation. Exemplary ions within this m / z range include IGF-II ions having m / z of about 1067.36±0.1, 1067.51±0.1, 1067.65±0.1, 1067.80±0.1, 1067.94±0.1, 1068.08±0.1, 1068.23±0.1, 1068.37±0.1, 1068.51±0.1, 1068.65±0.1, 1068.80±0.1, 1068.94±0.1, and 1069.08±0.1. In some embodiments, the one or more IGF-II ions are selected from the group consisting of IGF-II ions having m / z of about 1067.94±0.1 and 1068.08±0.1. This list is not intended to be limiting, and other ions may be suitable for use in the present methods.

[0084] 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 (e.g., the single IGF-I ion peak at m / z of about 1093.66 as shown in FIG. 4, or the single IGF-II peak at m / z of about 1067.80 as shown in FIG. 12) to be selected for data acquisition. Alternatively, quantitative data of signal intensities from one or more isotopic forms of a single ion (e.g., one or more isotopic forms of IGF-I or IGF-II as shown in FIGS. 4 and 12) or signal intensities across a narrow m / z range (e.g., all IGF-I signal intensities for an m / z range of about 1093.7±1, or all IGF-II signal intensities for an m / z range of about 1068.2±1) can be collected and related to the amount of intact IGF-I and / or IGF-II in the sample.

[0085] In some embodiments, quantitative data of signal intensity is collected for one or more IGF-I ions and / or IGF-II ions from at least two different charge states. The intensities of these ions can then be used to quantitatively assess intact IGF-I and / or IGF-II in a sample. For example, IGF-I can be quantified using the signal intensity from one or more IGF-I ions in 8+ charge state (i.e., IGF-I ions within an m / z range of about 957.1±2) and one or more IGF-I ions in 7+ charge state (i.e., IGF-I ions within an m / z range of 1093.7±2). In embodiments in which quantitative data of 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 areas under the curve) for quantitatively assessing intact IGF-I and / or IGF-II in a sample.

[0086] 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 the peak of one or more ions of an internal molecular standard.

[0087] In some embodiments, IGF-I and IGF-II are quantified simultaneously. In these embodiments, each of IGF-I and IGF-II may be quantified by any of the methods provided above.

[0088] In certain preferred embodiments, the lower limit of quantification (LLOQ) of IGF-I is within the range of about 15.0 ng / mL to 200 ng / dL (including the boundary value), preferably within the range of about 15.0 ng / dL to 100 ng / mL (including the boundary value), preferably within the range of about 15.0 ng / mL to 50 ng / mL (including the boundary value), preferably within the range of about 15.0 ng / mL to 25 ng / mL (including the boundary value), preferably within the range of about 15.0 ng / mL to 15 ng / mL (including the boundary value), preferably within the range of about 15.0 ng / mL to 10 ng / mL (including the boundary value), preferably about 15.0 ng / mL.

[0089] In certain preferred embodiments, the lower limit of quantification (LLOQ) of IGF-II 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.

[0090] In certain preferred embodiments, the limit of detection (LOD) of IGF-I is within the range of about 4.9 ng / mL to 200 ng / mL (including the boundary value), preferably within the range of about 4.9 ng / mL to 100 ng / mL (including the boundary value), preferably within the range of about 4.9 ng / mL to 50 ng / mL (including the boundary value), preferably within the range of about 4.9 ng / mL to 25 ng / mL (including the boundary value), preferably within the range of about 4.9 ng / mL to 20 ng / mL (including the boundary value), preferably about 4.9 ng / mL.

[0091] In certain preferred embodiments, the limit of detection (LOD) of IGF-II is within the range of about 8.2 ng / mL to 200 ng / mL (including the boundary value), preferably within the range of about 8.2 ng / mL to 100 ng / mL (including the boundary value), preferably within the range of about 8.2 ng / mL to 50 ng / mL (including the boundary value), preferably within the range of about 8.2 ng / mL to 25 ng / mL (including the boundary value), preferably within the range of about 8.2 ng / mL to 20 ng / mL, preferably about 8.2 ng / mL.

[0092] 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-I and IGF-II proteins or fragments by mass spectrometry using specific internal standards. The use of the internal standards 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 one skilled in the art. EXAMPLES

[0093] Example 1: Identification of IGF-1 variants by mass spectrometry Specimen Source Specimens were originally submitted to Quest Diagnostics for quantification of intact IGF-1 by LC-MS and de-identified prior to further analysis. IRB sponsor protocol number is BR13-002, IRB protocol number is 20121940.

[0094] Variant Database To find IGF-1 variants in patient samples, we used the Exome Aggregation Consortium database, ExAC (14) (accessed 2 / 6 / 2019, in Supplementary Table 2), which contains all currently known variants, including 13 variants of unknown clinical significance: T4M, V17M, T29I, S34N, A38V, R55K, M59R, P66A, P66T, A67S, A67T, A70P, A70T, and three previously reported pathogenic variants: V44M, R50W, and R36Q.

[0095] Sample preparation and LC-MS Patient samples were prepared using a modification of the method previously described by Bystrom et al. (3). Briefly, 100 μL of patient serum, quality controls (Bio-Rad), and calibrators (Ajinomoto) were thawed, vortexed, and transferred to a deep-well plate (Thermo Scientific). Then, 10 μL of internal standard ( 15 A mixture of 1000μL of N-labeled IGF-1, ProSpec) and 400μL of acidified ethanol solution (87.5% EtOH, 12.5% ​​1N HCl) was added, the sample was mixed vigorously and incubated at room temperature for 30 minutes. The sample was then centrifuged (5,500RCF for 10 minutes at 10°C) and 350μL of the supernatant was mixed with 60μL of 1.5M Tris base. The sample was cooled at -20°C for 60 minutes to generate further protein precipitation, followed by centrifugation at the previous settings.

[0096] After centrifugation, IGF-1 and its variants were separated using a multiplexed high-performance liquid chromatography (HPLC) system (Aria TLX-4) with online extraction (Thermo Scientific, San Jose, CA) using a gradient of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) (Supplementary Figure 1). The extraction column was a Phenomenex Monolithic Onyx C18 guard cartridge, and the analytical column was a Phenomenex Kinetex C18 50 × 4.6 mm, 100 Å pore size. Mass spectrometry was performed using a Q Exactive Focus Hybrid quadrupole-orbitrap instrument (Thermo Scientific) equipped with a heated electrospray ionization (HESI) source and operated in positive total ion fragmentation (AIF) mode with approximately twice the sensitivity compared to the SIM scan mode (Supplementary Figure 2 and Supplementary Table 1). The following MS parameters were used: Resolution 70,000, scan range 1,060-1,110 Da, normalized collision energy: 10, automatic gain control (AGC): 2e4, Max injection time (IT): automatic. For the determination of the A67T / A70T pair, the mass spectrometer was configured with a resolution of 35,000, isolation window 2.0 m / z, NCE 30, fixed first mass 150 Da, AGC: 2e4, MaxIT: 250 ms and 1 microscan.

[0097] MS Data Analysis and Visualization LC-MS data were analyzed using Thermo TraceFinder 5.0 Clinical and visualized using the Qual Browser in Thermo Xcalibur v.4.2.

[0098] Confirmation by DNA sequencing DNA extraction from de-identified serum specimen waste was performed using the Apostle MiniMax™ High Efficiency Isolation Kit (Beckman Coulter life science, Indianapolis, IN). The extracted DNA was used to sequence the exon 4 region (Chr12:102419509-102419690 in Build GRCh38) and exon 3 region (Chr12:102475582-102475855 in Build GRCh38) of the IGF-1 gene. Primer design excluded common SNP positions (>1% population MAF). Sequencing was performed using the ABI Prism BigDye Terminator v3.1 cycle sequencing kit and an ABI3500 genetic analyzer (Applied Biosystems) according to the manufacturer's instructions.

[0099] Results and Discussion Using the concepts of isotopic peak index (IPi) and relative retention time (rRT), we developed and automated a process for the detection of IGF-1 variants using tandem mass spectrometry to distinguish the most common variant pair, A67T and A70T.

[0100] Isotope Peak Index (IPi) The natural non-reduced form (C 331 H 513 N 94 O 101 Based on the elemental composition of WT IGF-1 in S7) and its known variants, the peaks in the isotopic envelope for each of the observed charge states of the polypeptide can be accurately predicted in terms of their exact m / z and distribution of peak intensities.

[0101] In a previous study, we briefly described the nomenclature of peaks within the isotopic envelope. (12) Herein, we further develop the concepts of "isotopic peak index (IPi)" and "isotopic peak relative intensity (IPrI)" (Figure 1). The isotopic envelopes of IGF-1 and variants follow this index intensity distribution, regardless of their monoisotopic m / z and charge states.

[0102] IPi is the numerical index of a peak in the isotopic envelope, indicated in subscript and starting from zero. The monoisotopic peak is designated as IP0 (read as "isotopic peak zero" or "IP zero"), followed by the heavier isotopic peaks, IP1 through IP 13 This nomenclature applies to the isotopic envelope of the WT and all variants. For example, IP0 of the V44M variant is designated as V44M0, representing the monoisotopic peak within the isotopic envelope of the V44M variant. During routine detection and quantification of WT IGF-1, IP4 is used under the label WT4.

[0103] IPrI is the distribution of the intensities of the peaks within the isotopic envelope. The specific pattern depends only on the elemental composition of the molecule, which is very similar in IGF-1 variants, and therefore the IPrI should be similar for WT and all variants. The distribution of peak relative intensities in Figure 1 is calculated relative to the intensity of the IP4 peak averaged from actual patient samples and used to quantify WT IGF-1. The use of the relative intensities of "qualifier" isotopes adjacent to a "quantifier" isotope to confirm IGF-1 identity is a practical example of the use of IPrI in the clinical field (7).

[0104] Monitoring all IGF-1 variants is challenging and has limitations. The mass resolution of the Orbitrap mass analyzer in the Q Exactive Focus instrument (70,000 at 200 m / z) is sufficient to recognize isotopic peaks within the isotopic envelope (MH7 +7(0.14 m / z difference for P66T and P70T). A limitation of any MS approach is the inability to distinguish between isotopic variants A67T and A70T, as well as V17M and V44M. Furthermore, current instrument resolution is unable to resolve variants with very close monoisotopic m / z (±10 ppm): R55K and R36Q; R50W, T4M, and A67 / 70T. Finally, all P66T isotopic peaks have interference from the WT IGF-1 isotopic envelope, so the isotopic envelope of the P66T variants overlaps with the WT IGF-1 and cannot be detected by routine analysis.

[0105] Based on the theoretical isotopic envelope of all variants, peaks that could not be resolved were identified by calculating the exact m / z shifts relative to WT IGF based on the difference in elemental composition of the variants as follows: The exact masses of the monoisotopic peaks were first calculated by subtracting the m / z shifts of MH7 from the m / z shifts of MH7 in all variants with unreduced disulfide bonds. +7 was calculated for each group (Supplementary Table 3). The exact m / z for the following 13 variant isotopes were then entered into a table and regrouped by sorting all m / z values ​​from smallest to largest (Isotope Peak Master Table, Supplementary Table 4). Adjacent m / z differences of <10 ppm were identified and grouped as peaks that could not be resolved from each other but could be resolved from peaks in other groups. In total, 5 m / z values ​​(orange cells, Supplementary Table 4) were selected to monitor the WT and 4 variant groups (VG, Table 1).

[0106] The isotopic peaks of some variants belong to two variant groups (VG3 and VG4), and their IPrIs provide further confidence for their identification (Table 1). For example, the extracted ion chromatogram (EIC) of S34N2 (VG3) is expected to be 57% of that of S34N4 according to its relative intensity in the isotopic envelope of IGF-1 (see Figure 1). However, in VG4, S34N9 is expected to be only 25% of WT and about half the area of ​​S34N2. This difference in EIC can be used to distinguish S34N from other variants. By switching from VG4 to VG3, the detection sensitivity of S34N can be increased by two times. The same is true for other variants from VG3, except for A38V, whose sensitivity is higher in VG4.

[0107] In our method, all variants are monitored at only 4 m / z ratios (Table 1). The presence of a peak at any of these m / z ratios indicates the presence of a variant belonging to a given VG. For example, Figure 2 shows LC-MS data including a VG4 peak for samples from two different patients. In the sample from patient 1, IP6 was detected as the m / z of VG4, and thus IP i The concept predicts that patient 1 must have the R50W, T4M, A67T, or A70T variant (Table 1). Similarly, patient 2's sample should have either the V17M or V44M variant, since IP4 was detected as an m / z of VG4. DNA sequencing confirmed that patient 1 has A70T and patient 2 has V44M, thus providing the validity of IPi as an approach to distinguish variants. Most variants can be distinguished using only the IPi approach, i.e., based solely on the isotopic distribution of peaks in their mass spectra.

[0108] Although this approach is targeted, it may also lead to the discovery of new variants. Close inspection of the LC-MS data from patient samples in which members of VG2 were detected revealed that IPi did not match either of the two variants of VG2: A67S (IP4) or T29I (IP8). Instead, the peak was not related to IP 10 or IP 11 (Supplementary Figure 3). DNA sequencing revealed that S33P 10 Our results revealed a variant, S33P, that matches the calculated m / z of 1095.8130 for VG1 (different by 6 ppm from the m / z of VG2, Supplementary Table 4) but has no match in the ExAC database, thus validating IPi as an approach for the discovery of new variants.

[0109] Relative retention time (rRT) Our preliminary studies show that some IGF-1 variants do not co-elute with WT. This difference between the RT of the WT and the RT of the variant is called the relative retention time (rRT) and can be used to distinguish between variants. By using the IPi concept to predict and assign variants to different VGs, comparing their observed rRTs, and confirming their identity by DNA sequencing, we have validated rRT as a tool for variant discrimination and discovery (Table 2).

[0110] For example, the EICs of WT4 and VG4 show subtle differences in the rRT of known IGF-1 variants from the two samples, thus distinguishing variants A70T (Fig. 2B), which co-elutes with WT (Fig. 2A), and V44M (Fig. 2E), which elutes later than WT (Fig. 2D).

[0111] Notably, in some VGs, IPi predicted a single variant, whereas rRT revealed the presence of novel IGF-1 variants (Table 2). For example, there was only one known variant (A38V) that could have IP1 at m / z in VG3, which should co-elute with the WT. However, we found a specimen in which the IP1 variant eluted slightly later than the WT. DNA sequencing confirmed that the co-eluting variant was A38V. However, the later-eluting variant was A67V, a variant not found in the ExAC database (i.e., a variant with the same amino acid substitution, but at a different position) (Table 2). Similarly, for IP4 in VG1, we found an unreported Y31H variant in a patient specimen, which elutes slightly earlier than the WT, with only a 1.5 ppm difference in calculated m / z compared to P66A (Supplementary Table 2) (Table 2). Also, in VG1, rRT showed the presence of new variants R50Q or R56K identified by DNA sequencing. Both of these new variants had the same amino acid substitutions as the known variants, but at different positions (Table 2). These examples provide validation that rRT is a tool for variant identification and new variant discovery.

[0112] Tandem Mass Spectrometry In the ExAC database, A67T and A70T have the highest occurrence (total 93%, Supplementary Table 1, generated from ExAC browser). They can be distinguished from each other by the difference in m / z of the y3 fragment ion and from WT by the difference in m / z of the y5 fragment ion upon reanalysis by MS / MS (Supplementary Table 5). In our workflow, we use parallel reaction monitoring (PRM) with an inclusion list of m / z 1093.5215 (WT4) and 1098.0969 (VG4).

[0113] We validated this approach using samples with IP6 detection of VG4, indicating the presence of A67T, A70T, T4M, or R50W variants. For example, when two patient samples were reanalyzed, MS / MS spectra revealed y ions, confirming that the patients were heterozygous for WT and A67T variants (patient A) and WT and A70T variants (patient B), respectively (Supplementary Figure 4). In total, we confirmed that MS / MS was concordant with DNA sequencing results in 28 samples tested (9 samples containing the A67T variant and 19 samples containing the A70T variant).

[0114] By alternating MS and MS / MS scans, it may be possible to detect A67T and A70T in a single run (Supplementary Figure 5), but this workflow requires proof that quantification of IGF-1 is not compromised due to the smaller number of MS scans across the chromatographic peak. Furthermore, this workflow may be difficult to justify given that the most abundant polymorphism, A67 / 70T, is present in only 0.2% of patient samples (Supplementary Table 2).

[0115] Automatic variant detection Detection of IGF-1 variants was automated and incorporated into the routine IGF-1 quantification workflow. All spectra were analyzed with Thermo TraceFinder 5.0 Clinical with mass extraction tolerance set to 20 ppm. A TraceFinder Master method was set up to search analyte spectra for the following compounds: WT4 (with WT3 and WT5 added as confirmatory ions), internal standard (ISTD5), and m / z corresponding to the four variant groups designated as internal standards. The software generated two reports:

[0116] For variant reports, the TraceFinder report type was configured as batch, with the following filter string: AND(quanresults.sampleType=“Specimen”,quanresults.totalArea<>“N / F”,quanresults.peakArea>300000,compound.compoundname<>“IGF-1”,Compound.CompoundName<>“ISTD”) This generates a list of samples suspected of containing IGF-1 variants (Figure 3, Panel A). In the example shown, the samples are suspected of containing the VG4 variant. Information regarding the presence of the variants is then reported to the physician via standard comments in the Laboratory Information System (LIS).

[0117] Based on the mass spectrometry data, a rare heterozygous IGF-1 variant was detected in this specimen. The levels of this variant are not included in the reported IGF-1 results above, explaining that they may be lower than expected. The levels of the variant are expected to be the same as those reported for the wild-type IGF-1 protein. The clinical significance of this IGF-1 variant remains unknown. Additional DNA sequencing is recommended to confirm the presence of the variant.

[0118] Using the concepts of IPi and rRT, the identity of the variants can be predicted and then confirmed by DNA sequencing or tandem mass spectrometry, in the case of the A67T / A70T pair.

[0119] The IGF-1 quantification report was designed to show information relevant to the detection and quantification of WT (Figure 3, Panel B). TraceFinder report types include a complex custom formula<compound.compoundType=“eTargetCompound”> In addition, the report is set to the following cell formula: IF(AND(sample.sampletype=“Specimen”,quanresults.peaksfound=FALSE),“POSSIBLE HOMO VAR”,IF(AND(sample.sampletype=“Specimen”,quanresults.peaksfound=TRUE),IF(quanresults.calcamount-compound.loq<0,“POSSIBLE HOMO VAR”,“”),“”)) Specimens suspected of containing homozygous IGF-1 variants are labeled. In this case, the results are manually inspected and the levels of the variants are estimated based on the WT IGF-1 calibration curve and reported to the physician via standard comments in the LIS system (quantitative values ​​are used as an example).

[0120] Based on the mass spectrometry data, a rare homozygous IGF-1 variant was detected in this specimen. The level of the variant was not included in the reported IGF-1 results above. This explains the reported IGF-1 concentration, as all IGF-1 is produced in the form of the variant protein. The estimated concentration of the variant is 75 ng / mL. The clinical significance of this variant remains unknown. Additional DNA sequencing is recommended to confirm the presence of the variant.

[0121] Clinical Applications The present inventors have developed a robust, high-throughput IGF-1 variant detection and discovery method using high-resolution LC-MS in a clinical laboratory. i , rRT, and MS / MS were validated by DNA sequencing. Our approach to simultaneously monitor and identify IGF-1 variants represents a significant improvement compared to other methods in clinical laboratories. Automated interpretation of LC-MS data by commercially available instrument software reduces the risk of human error.

[0122] During the development and validation of the approach, we identified six variants P66A, A67S, S34N, A38V, A67T, A70T, and two previously reported variants V44M and A67V from the ExAC database. In addition, the approach discovered six previously unreported variants Y31H, S33P, R50Q, R56K, T41I, and A62T. Given the large number of new discoveries, it becomes clear that our current knowledge of IGF-1 variants is limited and that the universe of IGF-1 variants is much larger.

[0123] We anticipate that our methodology may be more generally applicable to high-resolution LC-MS approaches for automated quantification of polypeptides and proteins and efficient detection of their polymorphic variants in clinical laboratories. In the case of IGF-1, the method offers the opportunity to provide clinicians with a greater profile of their patients' IGF-1 status, as well as to further explore genotype-phenotype relationships.

[0124] In the future, the list of monitored IGF-1 variants will include newly reported ones. It is also possible to develop MS / MS strategies to confirm the P66T, A67S, and A67V variants without DNA sequencing. Given the current improvements in mass spectrometers and their scan speeds, parallel MS quantification of WT IGF-1 and MS / MS validation of some of its variants may become feasible in clinical laboratories.

[0125] By applying this approach to data from a large number of samples, future studies aim to compare the frequency of different IGF-1 variants between general and clinical populations. Discrepancies in frequency could imply that a variant is pathogenic or likely pathogenic, thus informing diagnosis and patient care. [Table 2] [Table 3]

[0126] Within each variant group, the IPi concept was applied to each variant detected. After IPi prediction, rRTs were identified and compared within the unresolved variants. rRTs led to the separation of R50Q from R56K variants and A38V from A67V. It also led to the discovery of Y31H, S33P, and A62T variants.

[0127] Summary of SIM scan comparison with in-source CID (SID) and HCD of various energies as shown in Table 3. Calibrators and QCs. Results show a significant increase in response to the first calibrator and internal standard (cells highlighted in yellow) applying HCD at 10 NCE. [Table 4] [Table 5] [Table 6]

[0128] Theoretical accurate m / z of all IGF-1 variants in charge state +7 and non-reduced disulfide bonds and their isotopic peaks. [Table 7]

[0129] The theoretical m / z values ​​of all variants were sorted from minimum to maximum. Adjacent m / z differences of <10 ppm (pink cells) were then identified and grouped as peaks that could not be resolved from each other but could be resolved from peaks in other groups. In total, 5 m / z values ​​(orange cells) were selected to monitor the WT and the four variant groups.

Table 8

[0130] Example 2: Sensitivity Quantification of IGF-1 and IGF-2 by Mass Spectrometry Method. Samples are prepared by adding an acidified ethanol solution containing an isotopically labeled internal standard and placing them in a freezer for 60 minutes. After centrifugation, the supernatant is collected, the analytes are extracted, and separated by a multiplex high-performance liquid chromatography (HPLC) system with online extraction. Detection is performed by an ion trap Orbitrap mass spectrometer equipped with an ESI source and operating in positive all-ion fragmentation (AIF) mode. The ratio of the analyte response to the internal standard is used to construct a calibration curve and quantify IGF-1 or IGF-2 in patient samples.

Table 9

[0131] Precision Study. Definition: Reproducibility of Samples within an Assay Prior Art: Acceptance Criteria: SD < TEa / 3; Sigma for each process must be > 3.0 based on SD and CV (%).

[0132] Advanced Technology: The acceptance criteria are determined by the medical and scientific directors overseeing the measurements. The acceptance criteria must be reasonable and supported by data.

[0133] Accuracy within Run. Acceptance Criteria: SD must be less than the acceptable SD (wr) or ≦ TEa / 4.

[0134] The within-assay accuracy is defined as the reproducibility of the quality control pool within the measurement. All levels of QC were analyzed to determine the mean, SD, and coefficient of variation (%CV). Each level was measured 5 times per run over 5 runs, for a total of 25 replicates. The summary data for the analytes are shown below. The original data are listed in the table below.

Table 10

[0135] The results show that the acceptance criteria for the detection of IGF-1 and IGF-2 in human serum were met.

[0136] Overall precision. Acceptance criteria: If the overall SD ≥ 1 / 2TEa it is unacceptable or the overall SD must be less than the defined maximum SD or CV.

[0137] Inter-assay precision is defined as the reproducibility of samples between assays. All levels of QC were analyzed to determine the mean, SD, and coefficient of variation (%CV). Each level was measured twice per day for 20 days with a total of 40 replicates. Summary data from the analyses is shown below. The raw data is listed in the table below. [Table 12] [Table 13]

[0138] The results show that the acceptance criteria for the detection of IGF-1 and IGF-2 in human serum were met.

[0139] Limit blank (LOB). LOB was determined as the mean + 2SD of the zero calibrator. The LOB for IGF-1 and IGF-2 are 1.621 ng / mL and 10.162 ng / mL, respectively.

[0140] Limits of detection (LOD). LOD was determined as the mean + 4 SD of the zero calibrator. The LODs for IGF-1 and IGF-2 are 2.446 ng / mL and 12.276 ng / mL, respectively.

[0141] Limit of Quantitation (LOQ). Acceptance Criteria: The lowest concentration at which SD < TEa / 3 Three pools prepared using the standard calibrator material in methanol added to treated urine were tested to determine the LOQ. Each pool was measured five times per run over five runs. The LOQ was determined by the lowest concentration at which SD < TEa / 3. The summary data for the limit of detection are shown below. The LOQs for IGF-1 and IGF-2 are 7.794 ng / mL and 31.983 ng / mL, respectively.

Table 14

Table 15

[0142] Verification of Calibration. Acceptance Criteria: The mean of the observed values must not deviate from the expected range by more than TEa / 4.

[0143] The linearity of the assay was determined by analyzing the results of two different calibrator lots. The calibrator lots were freshly prepared and tested as unknowns against the currently used calibrator (previous preparation). The difference between the targeted and observed concentrations was analyzed. The statistics performed on the data indicate acceptable performance.

[0144] The analytical measurement ranges (AMRs) for IGF-1 and IGF-2 are 7.8 - 2,000 ng / mL and 31.3 - 2,000 ng / mL, respectively.

[0145] Reportable Range (RR). The clinical reportable ranges (CRRs) for IGF-1 and IGF-2 are 7.8 - 2,000 ng / mL and 31.3 - 2,000 ng / mL, respectively.

[0146] Six patient samples with known concentrations of IGF-1 were diluted in treated serum 2, 4, 8, and 16 times. Recoveries at the 2, 4, and 8 dilution levels were within 100±20% of recovery error.

[0147] Six patient samples with known concentrations of IGF-2 were diluted 2, 4, 8, and 16-fold in treated serum. All recoveries at each dilution level were within 100±20% of recovery error.

[0148] Recovery Testing. Acceptance Criteria: The error due to lack of complete recovery (amount recovered minus amount added) must be ≦TEa for each individual measurement.

[0149] A spike recovery study was performed to assess any matrix effects within the assay. IGF-1 base recovery samples (six total) were prepared by combining 200uL of treated serum with 400uL of each sample. Spiked samples were prepared by adding 200uL of a 2,000ng / mL stock solution of IGF-1 to 400uL of each sample. Results are within the acceptable precision range.

[0150] IGF-2 base recovery samples (6 total) were prepared by combining 100uL of treated serum with 100uL of each sample. Spiked samples were prepared by adding 100uL of a 2,000ng / mL stock solution of IGF-2 to 100uL of each sample. Results are within acceptable precision.

[0151] Split sample comparison study. Acceptance criteria: The absolute value of the mean difference must be less than TEa / 4.

[0152] RIA vs. Current Agilent TOF LC-MS. A split sample comparison study was conducted comparing radioimmunoassay (RIA) with current Agilent TOF LC-MS technology for quantification of IGF-1. Samples from 100 patients were split and analyzed using the LC-MS method on an Aria TX-4 system. Samples were assayed simultaneously using the RIA method at Esoterix Laboratory (Calabasas, CA). (Test code #500282, RIA block after acid:alcohol extraction). Data were analyzed by Deming regression (n=100, m=1.039±0.01572, b=-11.55±5.673). The LC-MS method was found to be in close agreement with the RIA.

[0153] HPLC-MS Method Comparison. A split-sample comparison of IGF-1 by LC-MS was performed between the current Agilent TOF and the proposed Q Exactive Focus HPLC-MS method. 197 patient samples (n=197) were tested. The linear regression was 1.0266x+0.57, r 2 =0.9861. The results indicate that the acceptance criteria for the quantification of IGF-1 in human serum were met.

[0154] A split-sample comparison of IGF-2 by LC-MS was performed between the current and proposed HPLC-MS methods. 214 patient samples (n=144) were tested. The linear regression was 0.9327x+38.17, r 2 =0.9132.

[0155] Measurement Uncertainty. Definition: The degree to which the result of a particular measurement is certain. Expressed as the 95th percentile confidence interval around a given number determined by the mean of multiple determinations + / - 1.96*SD.

[0156] For modified or unmodified FDA-cleared or approved tests, measurement uncertainty is determined by measuring each QC level five times per day for five separate days. If MU testing needs to be completed over a period shorter than five days, each set of five QC values ​​should be run separately with the shortened time frame recorded in the final precision test.

[0157] MU confidence intervals are calculated using the precision sheet of the assay validation calculator template. Data are analyzed by calculating the mean and standard deviation, then MU is calculated by multiplying the SD by 1.96 and adding or subtracting this from the mean.

[0158] The confidence intervals for each QC level of IGF-1 and IGF-2 in human serum are shown in the table below. [Table 16] [Table 17]

[0159] Specimen stability. Acceptance criteria: A sample is considered stable as long as the % difference between the baseline value and the time / temperature sample value is ≦TEa for that analyte. Serum pools were obtained from the clinical correlation department, pre-aliquoted and ready for set-up.

[0160] To test the recovery of IGF-1 and IGF-2 at various temperatures, 10 samples of each were evaluated. Aliquots from each pool were incubated at various temperatures for the times indicated in Tables 7-11. When the stability test pools reached the appropriate incubation times, they were placed in a -90°C to -60°C freezer until the assay date when all test pools were assayed together.

[0161] Freeze / thaw stability. Six samples were evaluated for freeze / thaw recovery of IGF-1 material in human serum. All pools were then aliquoted and stored at -90°C to -60°C. Six aliquots from each pool were subjected to different numbers of freeze / thaw cycles (0 to 5). The data in Table 7a show that for all pools tested, acceptable levels of activity of IGF-1 in human serum were retained even with five additional freeze / thaw cycles from the first freeze / thaw (cycle "0"). The first freeze / thaw cycle (cycle "0") represents the freeze / thaw that all specimens undergo during the recovery process. Recovery of IGF-2 in patient samples was performed in a similar manner over up to five freeze / thaw cycles.

[0162] IGF-1 patient samples are stable to at least 6 freeze / thaw cycles. IGF-2 patient samples are stable to at least 5 freeze / thaw cycles.

[0163] Refrigerated stability (2.0°C to 8.0°C). Data indicate that human serum retained acceptable levels of IGF-1 and IGF-2 activity at refrigerated temperatures for at least 7 days.

[0164] Ambient stability (18.0°C to 25.0°C). Data indicate that human serum retained acceptable levels of IGF-1 and IGF-2 activity at room temperature for at least 2 days and at least 7 days, respectively.

[0165] Freezing stability (-30.0℃ to -10.0℃). Data indicate that human serum retained acceptable levels of IGF-1 and IGF-2 activity at freezing temperatures for at least 2 months and at least 21 days, respectively.

[0166] Freezing stability: Ultra-low temperature (-90.0℃~-60.0℃). Data indicate that human serum retained acceptable levels of IGF-1 and IGF-2 activity at room temperature for at least 3 months and at least 34 days, respectively.

[0167] Interference Testing. Acceptability Criteria: The mean difference due to potential interfering substances must be ≦TEa / 4 to be considered acceptable.

[0168] Hemolysis interference. The effect of hemolysis on the assay was assessed by spiking low, medium, and high concentrations of hemolyzed RBCs into three patient samples (100 uL) to represent mild, moderate, and gross hemolysis.

[0169] For IGF-1, the mean recoveries were 82.4%, 72.4%, and 63.9% for mild, moderate, and severe hemolysis, respectively. Mild hemolysis does not interfere with IGF-1 in serum assays, whereas moderate and severe hemolysis samples are unacceptable.

[0170] The mean recoveries of IGF-2 were 111.9%, 119.6%, and 130.1% for mild, moderate, and severe hemolysis, respectively. The mean recoveries for mild and moderate hemolysis are within the acceptable range, but the individual values ​​are not. Hemolysis at all levels interferes with IGF-2 in serum assays. Samples with any degree of hemolysis are also unacceptable.

[0171] Lipidemia Interference. The effect of lipemia in the assay was evaluated by spiking low, medium, and high concentrations of Intralipid (20%, emulsion) to triplicate patient samples (100 uL), representing mild, moderate, and severe lipemia.

[0172] For IGF-1, the mean recoveries were 102.8%, 97.0%, and 96.8% for mild, moderate, and severe lipemia, respectively. Lipemia does not interfere with IGF-1 in serum assays. Samples with any degree of lipemia are acceptable.

[0173] For IGF-2, the mean recoveries were 105.1%, 108.6%, and 122.5% for mild, moderate, and severe lipemia, respectively. Lipemia does not interfere with IGF-2 in serum assays at mild and moderate levels. Samples with severe lipemia are not accepted.

[0174] Bilirubin Interference. The effect of jaundice on the assay was evaluated by spiking triplicate patient samples (100 uL) with low, medium, and high concentrations of bilirubin powder, representing mild, moderate, and severe jaundice.

[0175] For IGF-1, the mean recoveries were 102.8%, 99.0%, and 98.9% for mild, moderate, and severe jaundice, respectively. Jaundice does not interfere with IGF-1 in serum assays. Samples with any degree of jaundice are acceptable.

[0176] The mean recoveries of IGF-2 were 99.7%, 102.0%, and 106.0% for mild, moderate, and severe jaundice, respectively. Jaundice does not interfere with IGF-2 in serum assays. Samples with any degree of jaundice are acceptable.

[0177] Analytical specificity. IGF-1 binding protein interference was also tested using a mixture containing the following compounds: IGF binding protein 1 (IGFBP1), IGF binding protein 2 (IGFBP2) and IGF binding protein 3 (IGFBP3).

[0178] Six patient samples were collected and run to establish a baseline. Equivalent amounts (200 uL) of each sample and binding proteins (IGFBP1 at 800 ng / mL, IGFBP2 at 2,000 ng / mL, and IGFBP3 at 10,000 ng / mL) were then combined and tested to calculate the recovery of IGF-1. Following these, six solutions of the mixture of binding proteins (at the same concentrations as before) were run. The results show that the recovery was within 100±20%, fulfilling the acceptance criteria for the quantification of IGF-1 in human serum. Sample recoveries showed that no interferences were present.

[0179] One patient sample was taken and run to establish a baseline. Equivalent amounts (200 uL) of each sample and binding proteins (IGFBP1 at 800 ng / mL, IGFBP2 at 2,000 ng / mL, and IGFBP3 at 10,000 ng / mL) were then combined and tested to calculate the recovery of IGF-2. After these, a solution of the mixture of binding proteins (same concentrations as before) was run. The results show that the recovery was within 100 ± 20%, fulfilling the acceptance criteria for the quantification of IGF-2 in human serum. Sample recovery showed that no interferences were present. Note: The low signal of IGF-2 in the binding protein solution can be explained by the low amount of IGF-2 present in the treated serum used as the matrix.

[0180] Ion suppression study. The objective of this study was to determine whether ion suppression or ion enhancement effects were observed during the gradient or upon injection of matrix components. To assess this, isotopically labeled internal standards (N 15 A post-column "T" injection of labeled IGF-1 was performed. In this experiment, the total ion count (TIC) showed less than a 20% change during the elution time of the analyte of interest. Testing indicates that the assay passed the test and no ion suppression was observed for both IGF-1 and IGF-2.

[0181] Carryover. Twelve replicates of blank serum were tested to assess carryover on each LC column (corresponding to each of the four channel Aria LC systems) immediately followed by eight replicates of the high concentration standard (2,000 ng / mL). The low calibrator replicates showed no carryover for both IGF-1 and IGF-2 in human serum.

[0182] Reference Interval (RI) [Table 18] [Table 19] [Table 20] [Table 21] [Table 22]

[0183] IGF-2 Reference Range Adults, men and women: 267-616ng / mL Children, males and females (2-18 years) 260-630ng / mL Specimen Type / Alternative Specimen Types. The IGF-1 assay is validated on human serum collected in red top (no gel) tubes. A comparison of IGF-1 from SST tubes to red top tubes was completed. SST tubes met acceptance criteria.

[0184] The IGF-2 assay is validated for human serum collected in red-top tubes. Forty subjects were recruited with paired red-top and SST samples and analyzed for IGF-2. SST serum was compared to red-top serum. No significant differences were observed between sample tube types. The SST tubes met the acceptance criteria.

Table 23

[0185] Plasma samples have previously been noted to exhibit viscosities that are not compatible with automated pipetting systems. For this reason, plasma samples are not acceptable for IGF-1 and IGF-2 assays.

[0186] Clinical utility. The measurement of IGF-1 and IGF-2 is useful in the diagnosis of growth disorders. Dwarfism is associated with decreased serum IGF-1 and IGF-2 levels, while acromegaly is associated with increased serum IGF-1 and IGF-2 levels.

[0187] Bias. In the IGF-2 assay split sample comparison, there is a 1-point SE (20.21) > TEa / 4 (20.03), but < TEa / 3 (26.70). For these comparisons, an SE less than TEa / 4 is preferred and an SE less than TEa / 3 is acceptable.

[0188] Conclusion. This validation study has been reviewed and the performance of this method is considered acceptable for patient testing.

Table 24

[0189] 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.

[0190] 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.

[0191] 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.

[0192] Other embodiments are within the scope of the following claims. Additionally, where features or aspects of the methods are described in terms of Markush groups, those of skill 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 an insulin-like growth factor-I (IGF-I) variant in a sample, comprising: a. ionizing IGF-I 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 ions having a mass to charge ratio selected from the group consisting of 1089.8±0.5, 1095±0.5, 1097.09±0.5, 1098.09±0.5; c) using the detected ions to determine the presence of an IGF1 variant in the sample.

2. Detection of m / z 1089.8±0.5 was P66A ４ , R55K ６ , or R36Q ６ The method of claim 1 , further comprising determining the presence of

3. Detection of m / z 1095.8±0.5 was A67S ４ Or T29I ８ The method of claim 1 , further comprising determining the presence of

4. Detection of m / z 1097.09±0.5 was S34N ２ , A70P ３ , A38V １ , M59R ４ , or A67S １３ The method of claim 1 , further comprising determining the presence of

5. Detection of m / z 1098.09±0.5 was S34N ９ , A70P １０ , A38V ８ , M59R １１ , V17 / 44M ４ , R50W ６ , T4M ６ , or A67 / 70T ６ The method of claim 1 , further comprising determining the presence of

6. The method of any one of claims 1 to 5, further comprising purifying the protein by high performance liquid chromatography (HPLC) prior to ionization.

7. The method of any one of claims 1-5, wherein the sample comprises plasma or serum.

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