Mass spectrometry system for IDH gene mutation marker detection and method for improving detection accuracy

Abstract

Disclosed are a mass spectrometry system for IDH gene mutation marker detection and a method for improving detection accuracy. The mass spectrometry system comprises a sampling kit and a small mass spectrometer; the sampling kit comprises a sampler and a testing cartridge, the sampler comprising a handle portion and a conductive sampling piece, the testing cartridge comprising a cartridge body and a capillary, and a sample recess being formed in the cartridge body; the sampler is detachably secured to the cartridge body in a snap-fit manner, and the conductive sampling piece extends into the sample recess; the small mass spectrometer comprises an ion source interface, an ion sample injection transmission system, and a vacuum system connected in sequence, wherein the sampling kit is prepared for insertion into the ion source interface of the small mass spectrometer, and high voltage electricity is applied to the conductive sampling piece, so as to enable an analytical solution to be sprayed out of the capillary to form a spray. In addition, the present invention also provides a method for improving the detection accuracy of the described mass spectrometry system. By setting processes such as sample injection volume adjustments, judging against a standard spectrum, thresholding and the like, the accuracy of analytical results can be effectively improved.

Description

A mass spectrometry system for detecting IDH gene mutation biomarkers and a method for improving detection accuracy. Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to a mass spectrometry system for detecting IDH gene mutation biomarkers and a method for improving detection accuracy. Background Technology

[0002] Genetic studies have shown that the mutation status of the IDH gene in gliomas is closely related to their occurrence, development, and prognosis, and plays an important role in the differential diagnosis, prognostic assessment, and treatment strategy selection of gliomas.

[0003] According to the latest World Health Organization (WHO) CNS5 guidelines for glioma classification, IDH mutation status is one of the important bases for glioma subtyping. Detecting the IDH gene mutation status helps doctors determine the type and characteristics of the tumor, which is particularly important in differential diagnosis. Furthermore, IDH mutation status may affect the choice of treatment strategy for gliomas. Some studies have shown that patients with IDH mutations are more sensitive to radiotherapy and chemotherapy drugs (such as fludarabine) compared to glioma patients without IDH mutations. Other related clinical studies have demonstrated that patients with IDH mutations benefit from maximum tumor resection and exhibit better prognostic outcomes. Therefore, detecting IDH mutation status can help doctors assess patient prognosis and provide a reference for treatment planning. Thus, IDH gene mutation can serve as an important molecular marker for gliomas, playing a significant role in diagnosis, treatment, and prognostic assessment.

[0004] However, current methods for detecting IDH gene mutations have some limitations and challenges. In current clinical practice, the most commonly used methods are immunohistochemical staining and PCR. Immunohistochemical staining uses IDH1 or IDH2-specific antibodies to stain tissue, thereby revealing cells that may have IDH mutations. However, because this test relies on visual inspection, the results are highly subjective, easily misinterpreted, or difficult to confirm, and have low sensitivity and specificity, thus it is not considered the gold standard. In contrast, PCR is the gold standard for IDH mutation detection. Typically, PCR takes at least several days to obtain results and requires trained professionals to operate the equipment. Furthermore, PCR has extremely high specificity for a single IDH mutation (1 or 2), and it is essential to simultaneously detect both major IDH mutation sites, which further prolongs the time to obtain results; therefore, this test is generally performed postoperatively. However, compared to intraoperative diagnosis, postoperative diagnosis of IDH mutations has some limitations and drawbacks, such as the inability to guide timely treatment decisions such as surgery or radiotherapy due to time delays, the risk of sample loss, and sample selection bias. Intraoperative diagnosis can provide real-time molecular information during surgery, helping to more accurately assess the biological characteristics of tumors and guide the selection of precision treatment. Therefore, there is a need to develop a rapid, accurate, and convenient method for intraoperative diagnosis of IDH mutations in gliomas.

[0005] Numerous studies have shown that 2-hydroxyglutaric acid (2-HG) accumulates in IDH-mutant glioma tissues, but is almost absent in normal brain tissue. Therefore, the detection of 2-HG can serve as an important and strongly correlated indicator for elucidating the IDH mutation status in gliomas. Among existing techniques for detecting 2-HG, mass spectrometry has advantages such as high sensitivity and specificity. However, traditional liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques are highly versatile but require complex and time-consuming sample pretreatment and have large instrument sizes, making them unsuitable for intraoperative pathological analysis. Harvard Medical School has previously used in-situ ionization techniques such as DESI to perform brain tumor subtyping, and researchers at the University of Texas have also developed in-situ ionization techniques such as the MassSpec Pen. However, these techniques are limited by the complexity of large-scale mass spectrometry systems and are difficult to use for intraoperative monitoring.

[0006] Summary of the Invention

[0007] The technical problem this invention aims to solve is to provide a mass spectrometry system for detecting IDH gene mutation biomarkers and a method for improving detection accuracy. This mass spectrometry system allows sampling, extraction, ionization, and analysis to be completed within minutes, and the accuracy of the analytical results can be effectively improved through process settings such as sample volume adjustment, target spectrum judgment, and threshold judgment. The mass spectrometry system and method described in this invention not only enable intraoperative diagnosis but also allow the detection of IDH gene mutation biomarkers based on this mass spectrometry system to be quickly and easily integrated into existing glioma surgical procedures.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] The first aspect of this invention provides a mass spectrometry system for detecting IDH gene mutation biomarkers, comprising:

[0010] A sampling kit includes a sampler and a detection box; the sampler includes a handle and a conductive sampling strip fixed to one end of the handle, the conductive sampling strip being used to collect a sample to be tested; the detection box includes a box body and a capillary tube, the box body having a sample slot; the sampler is detachably snapped onto the box body, and the conductive sampling strip extends into the sample slot; the sample slot is used to hold an elution solvent, the elution solvent being used to elute the sample to be tested collected on the conductive sampling strip to form an analytical solution; one end of the capillary tube extends into the sample slot, and the other end is located outside the sample slot; and

[0011] A small mass spectrometer includes an ion source interface, an ion sample delivery system, and a vacuum system connected in sequence; the vacuum system is equipped with a mass analyzer and an ion detector.

[0012] When the mass spectrometry system is in use, the sampling kit is configured to be plugged into the ion source interface of the miniature mass spectrometer, and a high voltage is applied to the conductive sampling plate so that the analytical solution in the sample cell is charged and enters the capillary, and is sprayed out from the other end of the capillary to form a spray; the spray enters the vacuum system via the ion sample introduction and transmission system.

[0013] Furthermore, the conductive sampling sheet includes a sampling part and a conductive part connected in sequence. The sampling part is used to collect brain tissue samples, and the conductive part is used to connect to high voltage. The box body is also provided with a handle mounting slot, and the handle part is detachably engaged and fixed in the handle mounting slot.

[0014] Furthermore, the sampling section is provided with a notch and a round hole, the notch and the round hole are in communication; the end of the capillary tube that extends into the sample groove is located in the notch.

[0015] Furthermore, the sampler also includes sampling fibers, which are detachably fixed to the sampling section.

[0016] Furthermore, the sampling fiber is a filter paper strip; the sampling part has a double-slit structure, and the round hole is located between the two slits of the double-slit structure; the filter paper strip passes through the double slits and is then tightened and fixed.

[0017] Furthermore, the handle is made of polypropylene material and has anti-slip texture; the conductive sampling sheet is made of metal sheet and is mounted on the handle via a bayonet.

[0018] Furthermore, the capillary is a quartz capillary with a polyimide coating on its surface.

[0019] Furthermore, the vacuum system includes a vacuum chamber, a molecular pump, and a vortex pump, with the mass analyzer and ion detector located within the vacuum chamber.

[0020] Furthermore, the ion sample delivery system includes a clamp valve, which is connected to the vacuum chamber.

[0021] Furthermore, when the mass spectrometry system is used to detect IDH gene mutation markers in brain tissue, the specific operation includes the following steps:

[0022] (1) Take a brain tissue sample and place it on a carrier. Wipe the brain tissue with the conductive sampling pad of the sampler and then fix it on the detection box. Add an elution solvent to the sample slot of the detection box to extract the analytes from the brain tissue sample to form an analytical solution. The elution solvent contains ethanol and water, and the volume percentage of ethanol in the elution solvent is 50%-90%. The analytes in the brain tissue sample include, but are not limited to, 2-hydroxyglutaric acid (2-HG) and glutamic acid (GLU).

[0023] (2) Insert the detection box into the ion source interface of the miniature mass spectrometer, set the test parameters of the miniature mass spectrometer, and apply voltage to the conductive sampling plate through the ion source. The conductive sampling plate transmits the voltage to the capillary tip through the analytical solution. The analytical solution forms a Taylor cone at the tip and generates an electrospray. The analyte is ionized by the electrospray to form ions, which enter the vacuum system through the ion sample introduction and transmission system of the miniature mass spectrometer for mass analysis and recording the signal intensity of each ion.

[0024] (3) After obtaining the mass spectrometry signal, the intensity ratio of the secondary fragment ion signals of the target fragment ions m / z 129 and m / z 128 is calculated based on the intensity signals of the target fragment ions. Then, by comparing with the embedded threshold, the IDH mutation state of the brain tissue sample is determined.

[0025] Furthermore, when the mass spectrometry system is used to detect IDH gene mutation biomarkers, the test parameters of the miniature mass spectrometer are as follows: the voltage applied to the conductive sampling plate is -4.5 kV, the target cascade precursor ion mass-to-nucleus ratio (m / z) is 146.5, the ion isolation range is 2 Da, the ion isolation energy is 0.5-2.5 V, the collision-induced dissociation energy is 1.1-4 V, and the mass spectrometry scan range is m / z 50-m / z 300.

[0026] Further, in step (3), the embedded threshold is obtained as follows: a clinical retrospective experimental study of a certain number of samples is compared with the gold standard Sanger sequencing to obtain the ratio of m / z 129 (2-HG) and m / z 128 (GLU) ion signal intensities at the maximum Yangden index, which is used as the embedded threshold. The embedded threshold is a certain range or a fixed value; the number of samples is preferably greater than 100.

[0027] A second aspect of the present invention provides a method for improving the detection accuracy of the mass spectrometry system described in the first aspect, comprising the following steps:

[0028] S1. Set the initial injection volume to Q1, execute the scan, and obtain the initial MS2 spectrum; determine whether the total number of target ions on the initial MS2 spectrum meets the set limit requirement; if it meets the set limit requirement, determine the optimal injection volume Q0 = Q1; if it does not meet the set limit requirement, execute the injection volume optimization and adjustment scan process until the total number of target ions on the scan spectrum meets the set limit requirement, which is the optimal injection volume Q0;

[0029] S2: Perform n scans with the optimal injection volume Q0 and collect MS2 spectra; during the collection of MS2 spectra, perform the following judgments:

[0030] First judgment: Determine whether the total number of target ions in the MS2 spectrum meets the set limit; if it meets the set limit, it is a compliant spectrum and proceeds to the second judgment process; if it does not meet the set limit, it is a non-compliant spectrum and proceeds to the third judgment process.

[0031] Second judgment: Determine whether the number of qualified spectra has reached the set upper limit N1; if the set upper limit N1 has been reached, the scan ends and the fourth judgment process begins; if the set upper limit N1 has not been reached, the third judgment process begins.

[0032] The third judgment: Check if the number of scans is greater than or equal to the set upper limit n1. If it is greater than or equal to the set upper limit n1, the scan ends and the process proceeds to the fourth judgment. If it is less than the set upper limit n1, the next scan is performed.

[0033] Fourth judgment: Determine whether the number of qualified spectra is greater than or equal to the set lower limit N2; if it is greater than or equal to the set lower limit N2, proceed to step S3; if it is less than the set lower limit N2, prompt for resampling.

[0034] S3: Combine the N qualified spectra obtained in S2 into one spectrum, determine the signal strength of the target object, calculate according to the preset calculation formula, compare the calculation result with the embedded threshold, and output the result;

[0035] Where N1≤n≤n1; N2≤N≤N1.

[0036] Furthermore, in S1, the specific steps of the sample volume optimization and adjustment scanning process are as follows:

[0037] Determine the relationship between the total number of target ions on the MS2 spectrum and the set limit; if it is less than the lower limit of the set limit, increase the injection volume based on the previous injection volume and then perform the scan; if it is greater than the upper limit of the set limit, decrease the injection volume based on the previous injection volume and then perform the scan.

[0038] Repeat the above operation until the total number of target ions on the scan spectrum meets the set limit requirement, which is the optimal injection amount Q0.

[0039] Furthermore, the injection volume optimization adjustment is performed within a set injection volume range; and / or, the initial injection volume Q1 is 45ms, and the set injection volume range is 20-60ms.

[0040] Furthermore, in S2, during the collection of MS2 spectra, a valid spectrum filtering operation is performed to determine whether the obtained MS2 spectra are valid spectra; if they are valid spectra, the first judgment process is initiated; if they are invalid spectra, they are discarded.

[0041] The effective spectrum filtering operation is specifically as follows: match the spectral peaks of the internal standard in the MS2 spectrum; if a match can be made, it is a valid spectrum; if a match cannot be made, it is an invalid spectrum.

[0042] Furthermore, in S3, the original data of the target analyte in the N compliant spectra are summed and the average value is calculated based on the number of compliant spectra to synthesize a single spectra; when the combined internal standard method is used for quantitative testing, the target analyte includes the analyte and the internal standard.

[0043] Furthermore, when the mass spectrometry system is used to detect the mutation status of the IDH gene in brain tissue, the target substances are 2-HG and glutamate, wherein:

[0044] The peak of the target analyte is located in the range of m / z 127.5-m / z 129.5;

[0045] The set limit value is preferably 100-5000;

[0046] The upper limit n1 is preferably 10;

[0047] The upper limit N1 is preferably 5, and the lower limit N2 is preferably 3.

[0048] Furthermore, when the target analyte is 2-HG and glutamic acid, the calculation formula is: M1 = (signal intensity of target ion m / z 129) / (0.94 × signal intensity of target ion m / z 128);

[0049] When the embedded threshold is set to a certain range, if M1 ≥ the upper limit of the embedded threshold, the output result is IDH mutation positive; if M1 < the lower limit of the embedded threshold, the output result is IDH mutation negative.

[0050] When the embedded threshold is set to a fixed value, if M1 ≥ the embedded threshold, the output result is IDH mutation positive; otherwise, the output result is IDH mutation negative.

[0051] Furthermore, the embedded threshold is determined by the following method:

[0052] Sampling and analysis were performed on IDH gene mutation biomarkers and non-IDH gene mutation biomarkers;

[0053] Establish a classification model;

[0054] By adjusting different thresholds to obtain ROC curves, the threshold corresponding to the largest area under the ROC curve is determined, which is the embedded threshold.

[0055] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0056] The mass spectrometry system provided by this invention integrates a direct sampling method and capillary electrospray technology in its sampling kit. With the assistance of a small portable mass spectrometer, sampling, extraction, ionization, and analysis can be completed in just minutes. Furthermore, this invention also provides a method to improve the detection accuracy of the aforementioned mass spectrometry system. Through process settings such as sample volume adjustment, target spectrum judgment, and threshold judgment, the unstable impact of rapid sampling and sample preparation using the kit on the analytical results is effectively reduced, thereby effectively improving the accuracy of the analytical results of this mass spectrometry system and enabling the rapid acquisition of highly accurate detection results.

[0057] The mass spectrometry system provided by this invention has high sensitivity and specificity. In conjunction with the above-mentioned methods, it can quickly obtain highly accurate detection results, making the system usable for intraoperative diagnosis and providing clinicians with reliable diagnostic results. For example, it can rapidly detect IDH gene mutation markers in existing glioma surgical procedures, thereby helping to guide individualized treatment of gliomas. Attached Figure Description

[0058] Figure 1 is a schematic diagram of the sampling kit in one embodiment of the present invention;

[0059] Figure 2 is a top view of the sampling kit in Figure 1;

[0060] Figure 3 is a schematic diagram of the mass spectrometry system in one embodiment of the present invention;

[0061] Figure 4 is a flowchart of the mass spectrometry system algorithm;

[0062] Figure 5 is a flowchart of a mass spectrometry system used to detect IDH gene mutation markers in brain tissue.

[0063] Figure 6 shows the detection results when the sample size of IDH mutant samples is too small;

[0064] Figure 7 shows the detection results when the sample size of non-mutated IDH samples is too small;

[0065] Figure 8 shows the detection results when the number of non-mutant IDH samples was too large;

[0066] Figure 9 shows the detection results when the sample size of IDH mutant samples is too large;

[0067] Figure 10 shows the detection results when the sample size of IDH mutant samples was too large;

[0068] Figure 11 shows the detection results obtained with and without performing the compliance spectrum judgment;

[0069] Among them: 100, sampler; 110, conductive sampling sheet; 111, sampling part; 1111, round hole; 1112, notch; 1113, double slit structure; 112, conductive part; 114, sampling fiber; 120, handle part; 121, anti-slip texture;

[0070] 210. Box body; 211. Sample groove; 212. Tentacle; 220. Capillary tube;

[0071] 300. Miniature mass spectrometer; 310. Ion source interface. Detailed Implementation

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0073] This invention provides a mass spectrometry system for detecting IDH gene mutation biomarkers, including a sampling kit and a miniature mass spectrometer.

[0074] Please refer to Figures 1-2. The sampling kit includes a sampler and a detection box. The sampler includes a handle and a conductive sampling pad fixed to one end of the handle. This conductive sampling pad is used to collect the sample to be tested. The detection box includes a body and a capillary tube. The body is detachably engaged with the handle of the sampler. The body has a sample slot, and when the sampler is engaged with the body, the conductive sampling pad extends into the sample slot. The sample slot holds the elution solvent, which is used to elute the sample collected on the conductive sampling pad to form an analytical solution. One end of the capillary tube extends into the sample slot, and the other end is outside the sample slot.

[0075] In the mass spectrometry system of this invention, since the sampler is detachably snapped onto the detection box, the sampler can be removed from the detection box for sampling when collecting the sample to be tested. After sampling, the sampler is snapped back onto the detection box and then assembled onto the miniature mass spectrometer for detection.

[0076] The sampling kit of the present invention integrates sampling, extraction, and electrospray ionization processes, effectively reducing the complex sample transfer steps in the mass spectrometry pretreatment process and significantly improving the analysis efficiency of trace samples.

[0077] In some embodiments of the present invention, the handle is made of an insulating material, and the conductive sampling piece includes a sampling part and a conductive part connected in sequence. The sampling part is used to collect brain tissue samples, and the conductive part is used to connect to a high-voltage circuit. In some embodiments, the handle may be made of polypropylene material by 3D printing, and its surface may be provided with anti-slip textures for easy handheld operation. The conductive sampling piece may be made of conductive materials such as metal sheets to achieve conductivity; for example, it may be integrally stamped from a thin stainless steel sheet. The conductive sampling piece can be mounted on the handle via a bayonet.

[0078] In some embodiments of the present invention, the conductive portion of the conductive sampling sheet protrudes upward, forming a stepped shape, which facilitates placing the sampling portion into the bottom of the sample slot of the reagent kit after sampling, while ensuring that the conductive portion is in full contact with the external high voltage, thus ensuring the stability of the electrical connection.

[0079] In some embodiments of the present invention, the sampling section is provided with a notch and a circular hole, the notch communicating with the circular hole; and the end of the capillary extending into the sample cell is located in the notch. The purpose is twofold: firstly, to avoid interference between the capillary and the sampling section during installation; and secondly, the capillary action generated by the notch and the circular hole can converge the analytical solution, thereby promoting the entry of the analytical solution into the capillary.

[0080] In this invention, sampling fibers are detachably fixed to the sampling section. The sampling fibers facilitate sample collection, such as brain tissue, and effectively adsorb whole blood from the tissue, preventing hemoglobin in the whole blood from denaturing and clogging the spray capillary in an organic solvent environment. In some embodiments of this invention, the sampling section has a double-slit structure, with a circular hole located between the two slits of the double-slit structure. The sampling fibers are filter paper strips that cross-pass through the double slits and are then tightened and fixed. Preferably, the sampling fibers are cellulose chromatography paper (Waterman Grade 1) with a thickness of 0.18 mm and length and width of 7 mm and 3 mm, respectively.

[0081] In some embodiments of the present invention, a handle mounting slot is provided on the box body, and the handle of the sampler is detachably engaged and fixed in the handle mounting slot. Furthermore, buckles are provided on both sides of the handle mounting slot, through which the handle can be engaged in the handle mounting slot, facilitating the assembly and disassembly of the sampler. The box body can be made of plastic; for example, in some embodiments, the box body is prepared from polypropylene material by 3D printing.

[0082] In some embodiments of the present invention, the capillary is a quartz capillary, preferably with a polyimide coating on its surface. Preferably, the outer diameter of the capillary is 150 μm and the inner diameter is 75 μm. One end of the capillary passes through the mounting hole into the housing and extends into the sample cell, preferably inserted against the bottom of the sample cell. This ensures that the end of the capillary can fully contact the analytical solution in the sample cell, allowing the analytical solution to enter the capillary through capillary action.

[0083] In some embodiments of the present invention, after one end of the capillary is inserted into the sample slot, the capillary is fixed to the box body with adhesive to prevent the capillary from moving. The adhesive is preferably an epoxy resin adhesive.

[0084] In some embodiments of the present invention, the front end of the box is provided with multiple tentacles, which are distributed around the capillary tube at intervals. Their function is to protect the capillary tube and prevent it from breaking during operation.

[0085] In this invention, the miniature mass spectrometer is characterized by its small size and portability, enabling rapid on-site detection. Specifically, the miniature mass spectrometer includes an ion source interface, an ion sample delivery system, and a vacuum system connected in sequence, with a mass analyzer and an ion detector installed in the vacuum system.

[0086] Please refer to Figure 3. In this invention, the ion source interface and the sampling kit are fully compatible in mechanical structure. Therefore, the sampling kit can be inserted into the ion source interface. At the same time, the ion source interface can provide a stable and reliable high-voltage electrical connection for the conductive sampling sheet of the sampling kit.

[0087] In some embodiments of the present invention, the vacuum system includes a vacuum chamber, a molecular pump, and a vortex pump, wherein the molecular pump and the vortex pump can provide 10 Ω·cm for the mass spectrometry system. -5 Torr operates in a vacuum environment, with the mass analyzer and ion detector housed within the vacuum chamber.

[0088] In some embodiments of the present invention, the ion transport system employs a discontinuous atmospheric pressure interface technology, which includes a clamp valve connecting atmospheric pressure and a vacuum chamber. The clamp valve remains normally closed during non-sample injection. When ion injection occurs, the clamp valve opens for 10–20 ms, thereby introducing electrospray ions generated by the capillary of the sampling kit into the vacuum chamber.

[0089] In this invention, the miniature mass spectrometer uses a linear ion trap as a mass analyzer. When performing mass analysis, the linear ion trap selectively captures specific precursor ions and further dissociates them into secondary fragment ions by applying a certain electric field and excitation energy. These fragment ions are then introduced into the ion detector to record and calculate the intensity signal of each ion.

[0090] In a preferred embodiment, the miniature mass spectrometer, including the vacuum system and battery, weighs a total of 8.5 kg and has external dimensions of 33 cm × 23 cm × 15 cm, allowing for easy on-site transport. The instrument's peak power consumption is less than 85 W, and the built-in battery supports continuous operation for over 3 hours. The ion source, sample inlet, and sample inlet tubing are held in place by 3D-printed fixtures. The vacuum chamber has internal dimensions of 11.3 cm × 9.3 cm × 10.1 cm and houses a linear ion trap and an electron multiplier tube detector. The vacuum pump system consists of a turbomolecular pump and a vortex pump. When DAPI is off, the pressure can be below 1 × 10⁻⁶. -5The instrument operates using a radio frequency (RF) voltage with a peak amplitude greater than 5 kVp-p and a frequency of 955 kHz to drive the linear ion trap. Furthermore, the main RF signal for the ion trap is generated using inductive-capacitive resonant amplification. The instrument also includes a control system comprising a host ARM processor and a slave field-programmable gate array (FPGA). The ARM processor runs a Linux operating system, driving a 7-inch capacitive touchscreen, and features a user-friendly interface for mass spectrometry analysis and data processing. Additionally, the instrument is equipped with a 4G module and a WiFi module for network connectivity and data transmission.

[0091] Using the mass spectrometry system of this invention, 2-HG and another endogenous metabolite, glutamate, can be used as markers to directly analyze and determine the IDH mutation status of brain tissue samples. Specifically, in the detection of IDH mutations in gliomas, the preferred mass spectrometry parameters are: electrospray voltage -4.5 kV, target cascade precursor ion mass-to-nucleus ratio (m / z) of 146.5, ion isolation range of 2 Da, ion isolation energy of 0.5-2.5 V, collision-induced dissociation energy of 1.1-4 V, and mass spectrometry scan range of m / z 50-m / z 300.

[0092] Because the analysis targets two metabolites, glutamate and 2-hydroxyglutarate, a negative ion mode was used for the spray voltage selection, which showed better response compared to the positive mode. Through spray voltage optimization experiments, 4.5 kV was selected as the optimal voltage. At lower spray voltages, point spraying was not possible, while at higher voltages, tip discharge would affect the analysis. In negative mode, both glutamate and 2-hydroxyglutarate exhibit a hydrogen loss ion peak [MH]-, with mass-to-nucleus ratios (M / N ratios) of 146 and 147, respectively. The designed tandem mass spectrometry parameters targeting the parent ion M / N ratio (m / z) were 146.5, the ion isolation range was 2 Da, the ion isolation energy was 1.8 V, and the collision-induced dissociation energy was 1.2 V, thus yielding tandem mass spectrometry results. The fragments of glutamate and 2-hydroxyglutarate are m / z 128 and m / z 129, respectively; therefore, a mass spectrometry scan range of m / z 50–m / z 300 can obtain complete mass spectrum data.

[0093] The steps for detecting IDH gene mutation markers in glioma tissue using the mass spectrometry system of this invention are as follows:

[0094] 1. Take a human brain tissue sample, wipe the brain tissue with a sampler, and then fix it on the test box; then add 50 μL of special reagent (90% chromatographic grade ethanol and 10% chromatographic grade pure water) to the sample slot of the test box.

[0095] 2. Insert the detection box directly into the ion source interface of the miniature mass spectrometer. Set the mass spectrometer to negative voltage mode and apply a 4.5kV spray voltage to the conductive sampling plate. The metal plate transmits the voltage to the capillary tip through the analytical solvent. The solution forms a Taylor cone at the tip and generates an electrospray. The analyte is ionized by the electrospray to form ions. The injection of sample ions is controlled by the discontinuous atmospheric pressure interface of the miniature mass spectrometer.

[0096] 3. After obtaining the mass spectrometry signal, the intensity ratio of the secondary fragment ions of the target ions at m / z 129 and m / z 128 can be calculated based on the intensity signals of the target fragment ions. Then, by comparing with the embedded threshold, the IDH mutation state of the sample can be determined.

[0097] Each step of the above procedure is very easy to perform in the operating room. The entire workflow takes less than 2 minutes and can be easily completed by the surgeon in the operating room, with an immediate report on the IDH mutation status.

[0098] In this invention, the 2-HG / GLU ratio detection employs a relative value quantitative method, primarily involving a single spectral scan and two data matching steps (one scan simultaneously obtains MS2 spectra containing fragments of the target analyte 2-HG and the internal standard GLU; data matching is then performed on the fragment lists of both substances to determine the peak intensities of the target analyte and the internal standard). When analyzing samples with complex matrices, compared to MS1, tandem mass spectrometry (MS / MS) not only enables qualitative identification of molecules through fragmentation spectra but also effectively improves the signal-to-noise ratio for accurate quantitative detection. Specifically, in this invention, tandem mass spectrometry (MS / MS) is implemented by applying an alternating current (AC) excitation signal corresponding to the resonance frequency of the target ion in the ion trap to enhance the kinetic energy of the target ion; and by introducing a small amount of neutral gas via DAPI to collide with the ions, thus breaking the chemical bonds within the ions. This invention selects GLU, whose mass-to-charge ratio is close to that of 2-HG, as an internal standard. Centering on the average mass-to-charge ratio of 2-HG and GLU negative mode at m / z 146.5, segmented ion isolation and scanning are achieved in ionized hydrazine. Then, collision-induced dissociation technology is used to realize MS / MS analysis, which can achieve simultaneous fragmentation of two substances in one MS2 spectrum, resulting in a more stable spectral signal.

[0099] In this invention, the above-mentioned embedded threshold is obtained as follows: a clinical retrospective experimental study of a certain number of samples is compared with the gold standard Sanger sequencing to obtain the ratio of m / z 129 (2-HG) and m / z 128 (GLU) ion signal intensities at the maximum Yangden index, which is used as the embedded threshold. The embedded threshold can be a certain range or a fixed value; in some preferred embodiments, the number of samples is 114.

[0100] Because the mass spectrometry system provided by this invention can quickly sample and prepare samples, it cannot guarantee that an appropriate amount of sample can be collected each time. Therefore, it cannot automatically adjust the injection volume to match the mass spectrometry detection concentration range, and cannot guarantee the quality of the spectrum, resulting in low accuracy of the results. In addition, due to the inherent defects of the reagent kit, the spray stability cannot guarantee that the MS2 spectrum will meet the standard 100%.

[0101] To address the problems existing in the aforementioned mass spectrometry system, this invention also provides a method for improving the detection accuracy of the aforementioned mass spectrometry system, as shown in Figure 4, comprising the following steps:

[0102] S1: Set the initial injection volume to Q1, execute the scan, and obtain the initial MS2 spectrum; determine whether the total number of target ions on the initial MS2 spectrum meets the set limit requirement; if it meets the set limit requirement, determine the optimal injection volume Q0 = Q1; if it does not meet the set limit requirement, execute the injection volume optimization and adjustment scan process until the total number of target ions on the scan spectrum meets the set limit requirement, which is the optimal injection volume Q0;

[0103] S2: Perform n scans with the optimal injection volume Q0 and collect MS2 spectra; during the collection of MS2 spectra, perform the following judgments:

[0104] First judgment: Determine whether the total number of target ions in the MS2 spectrum meets the set limit; if it meets the set limit, it is a compliant spectrum and proceeds to the second judgment process; if it does not meet the set limit, it is a non-compliant spectrum and proceeds to the third judgment process.

[0105] Second judgment: Determine whether the number of qualified spectra has reached the set upper limit N1; if the set upper limit N1 has been reached, the scan ends and the fourth judgment process begins; if the set upper limit N1 has not been reached, the third judgment process begins.

[0106] The third judgment: Check if the number of scans is greater than or equal to the set upper limit n1. If it is greater than or equal to the set upper limit n1, the scan ends and the process proceeds to the fourth judgment. If it is less than the set upper limit n1, the next scan is performed.

[0107] Fourth judgment: Determine whether the number of qualified spectra is greater than or equal to the set lower limit N2; if it is greater than or equal to the set lower limit N2, proceed to step S3; if it is less than the set lower limit N2, prompt for resampling.

[0108] S3: Combine the N qualified spectra obtained in S2 into one spectra, determine the signal strength of the target object, calculate according to the preset calculation formula, compare the calculation result with the embedded threshold, and output the result;

[0109] Where N1≤n≤n1; N2≤N≤N1.

[0110] In this invention, the sample volume optimization and adjustment scanning process specifically includes:

[0111] Determine the relationship between the total number of target ions on the MS2 spectrum and the set limit; if it is less than the lower limit of the set limit, increase the injection volume based on the previous injection volume and within the set injection volume range, and then perform the scan; if it is greater than the upper limit of the set limit, decrease the injection volume based on the previous injection volume and within the set injection volume range, and then perform the scan.

[0112] Repeat the above operation until the total number of target ions on the scan spectrum meets the set limit; this injection volume is the optimal injection volume Q. o .

[0113] In this invention, the injection volume optimization adjustment is performed within a set injection volume range, including adjusting by increasing or decreasing the injection volume by the same amount based on the previous step; or, adjusting by re-dividing the original injection volume range into new injection volume ranges, where one end of the new injection volume range is the value of the previous step injection volume, and the middle value of the new injection volume range is the new injection volume. In some preferred embodiments, the initial injection volume Q1 is 45ms, and the set injection volume range is 20-60ms.

[0114] In this invention, during the collection of MS2 spectra, a valid spectrum filtering operation is performed to determine whether the obtained MS2 spectra are valid spectra. Specifically, the spectral peaks of the internal standard are matched in the MS2 spectra. If a match can be made, the spectrum is valid; if a match cannot be made, the spectrum is invalid. If the spectrum is valid, the first judgment process is initiated; if the spectrum is invalid, it is discarded.

[0115] In some preferred embodiments of the present invention, the original data of the analyte and the standard in N qualified spectra are summed and the average value is calculated based on the number of qualified spectra to synthesize a single spectra.

[0116] In some embodiments of the present invention, a mass spectrometry system is used to detect IDH gene mutation markers, using 2-HG and glutamate as markers to analyze the IDH mutation status of samples, wherein: the peak of the target ion is located in the range of m / z 127.5-m / z 129.5; the set limit is preferably 100-5000; the set upper limit n1 is preferably 10; the set upper limit N1 is preferably 5; the set lower limit N2 is preferably 3; the preset calculation formula is: M1 = (signal intensity of target ion m / z 129) / (0.94 × signal intensity of target ion m / z 128), where (0.94 × signal intensity of target ion m / z 128) is used to detect IDH gene mutation markers, using 2-HG and glutamate as markers to analyze the IDH mutation status of samples, wherein: the peak of the target ion is located in the range of m / z 127.5-m / z 129.5; the set limit is preferably 100-5000; the set upper limit n1 is preferably 10; the set upper limit N2 is preferably 5; the set lower limit N2 is preferably 3; the preset calculation formula is: M1 = (signal intensity of target ion m / z 129) / (0.94 × signal intensity of target ion m / z 128), where (0.94 × signal intensity of target ion m / z 129) is used to detect IDH gene mutation markers, using 2-HG and glutamate as markers to analyze the IDH mutation status of samples, wherein: the peak of the target ion is located in the range of m / z 127.5-m / z 129.5; the set lower limit N2 is preferably 3; the set lower limit N2 is preferably 3; the set lower limit N2 is preferably 3; the set lower limit N2 is preferably 3; the set lower limit N2 is preferably 3; The signal intensity of 128 is calculated as follows: (Signal intensity of internal standard glutamate) = (Signal intensity of glutamate isotope) - (Signal intensity of glutamate isotope). The theoretical ratio of the signal intensity of glutamate isotope to that of internal standard glutamate is 0.06. When the embedded threshold is set to a certain range, if M1 ≥ the upper limit of the embedded threshold, the output result is IDH mutation positive; if M1 < the lower limit of the embedded threshold, the output result is IDH mutation negative. When the embedded threshold is set to a fixed value, if M1 ≥ the embedded threshold, the output result is IDH mutation positive; otherwise, the output result is IDH mutation negative.

[0117] In some embodiments of the present invention, for example, a mass spectrometry system is used to detect IDH gene mutation biomarkers, and the aforementioned embedded threshold is determined by the following method:

[0118] Sampling and analysis were performed on IDH gene mutation biomarkers and non-IDH gene mutation biomarkers;

[0119] Establish a classification model;

[0120] By adjusting different thresholds to obtain ROC curves, the threshold corresponding to the largest area under the ROC curve is determined, which is the embedded threshold.

[0121] Example 1

[0122] This embodiment provides a method for detecting IDH gene mutation markers in glioma tissue. The detection process is shown in Figure 5, and the specific steps are as follows:

[0123] (1) Prepare glioma tissue samples. This experiment collected 228 brain tissue samples from different patients.

[0124] (2) Remove the sampler from the test box and press the paper fiber part of the sampler onto the extracted brain tissue to ensure that only flesh-colored, uniform tissue with a thickness of no more than 0.1 mm is visible on the paper fiber after sampling, with no uneven brain tissue sample residue. Next, attach the sampler to the test box and add 5 drops of the built-in special solvent into the sample slot of the test box (soaking the paper fiber part).

[0125] (3) After waiting for 10 seconds, insert the detection box into the ion source interface of the mass spectrometer until it can no longer be inserted. When the words "Start Detection" appear on the display screen of the mass spectrometer, click "Start Detection".

[0126] (4) After obtaining an MS2 spectrum by scanning with an initial injection volume of 45ms, the instrument calculates the total number of ions in the target peaks (glutamic acid m / z 128 and 2-HG m / z 129) to determine if it meets the limit (100-5000). If the total number of ions is less than 100 (low response signal intensity, prone to false negatives), the instrument automatically increases the injection volume and scans again; if the total number of ions is greater than 5000 (high response signal intensity, risk of oversaturation and false positives), the instrument automatically decreases the injection volume and scans again. After obtaining the optimal injection volume (meeting the total ion number limit), the instrument will use this injection volume for subsequent scanning analysis. After the second stage MS2 scan of the analyte begins, it is similarly determined whether the total number of ions in the obtained MS2 spectrum of the target peaks meets the limit requirements. Once the number of compliant spectra (total ion count meets the limit of 100-5000) reaches the upper limit (e.g., 5 spectra), spectrum acquisition stops for subsequent analysis. If the number of compliant spectra is 4, scanning continues until the upper limit of spectra or the upper limit of scanning times (e.g., 10 times) is reached, then spectrum acquisition stops. The instrument checks if the number of compliant spectra has reached the lower limit (e.g., 3 spectra). If it has, the instrument can proceed to the next stage; if not, the instrument prompts "Please resample" (this algorithm is a protective mechanism to reduce false negatives and false positives caused by poor spectrum quality due to reagent kit spray instability). After obtaining a sufficient number of compliant spectra, the instrument obtains an MS2 spectrum through overlay, calculates the signal intensity ratio of the target peak, and compares the ratio result with a set threshold. If the ratio is greater than or equal to the threshold or the upper limit of the threshold, it is judged as IDH mutation positive, and the final test report will appear on the display screen.

[0127] The threshold was determined by sampling and analyzing 114 clinically diagnosed IDH-mutant and non-IDH-mutant brain tissue samples, establishing a classification model, plotting ROC curves, and determining the embedded threshold to be 0.13–1.21. The determined threshold was then used for clinical diagnosis of IDH gene mutation in brain tissue.

[0128] The clinical diagnostic results of the above-mentioned test samples are shown in Table 1. Of the 228 brain tissue samples, 93 were IDH-mutant brain tissue samples, accounting for 40.79%, and 135 were non-IDH-mutant brain tissue samples (including wild-type brain tumor tissue and adjacent normal tissue), accounting for 59.21%.

[0129] A comparison was made between the IDH gene mutation detection mass spectrometry system of this invention and the clinical diagnostic results of Sanger sequencing. The results showed that the sensitivity / positive concordance rate was 96.77%, the specificity / negative concordance rate was 98.52%, the accuracy / total concordance rate was 97.81%, and the Kappa was 0.9546. It is worth noting that clinical diagnostic results using Sanger sequencing typically take more than 2 hours to complete and can only be obtained in a laboratory, with a report turnaround time usually exceeding 3 working days. However, the method of this invention can be performed at the bedside, with an analysis time of only 2 minutes.

[0130] Table 1 Clinical diagnostic results of the test samples

[0131] Comparative Example 1

[0132] The mass spectrometry system in Example 1 was used to detect IDH mutant and non-mutant samples. The difference from Example 1 is that when the sample volume is too small or too large, a fixed injection volume of 45ms is used for injection without adjusting the injection volume, and the MS2 spectrum is collected directly.

[0133] When the sampling volume is too small, the detection results of the IDH mutant sample are shown in Figure 6. When the sampling volume of the IDH mutant sample (positive) is too small, the total number of target ions (2-HG and GLU) obtained by the fixed injection volume cannot meet the statistical requirements of mass spectrometry detection (the lower limit of the target ion count is set to 100). The spectrum will show random distribution characteristics, and it is easy to see non-normal distribution of GLU peak shape and no signal of 2-HG MS2 spectrum. After calculating such spectrum, the ratio of 2-HG / GLU is 0, which is less than the lower limit of the embedded threshold. The system judges it as a negative result, which is contrary to the positive sample.

[0134] When the sampling volume is too small, the detection results for IDH non-mutant samples are shown in Figure 7. When the sampling volume of IDH non-mutant samples (negative) is too small, the total number of target ions obtained by the fixed injection volume does not meet the statistical requirements of mass spectrometry detection. It is easy to have an MS2 spectrum with the signal intensity of GLU isotope peak (the same as the mass-to-charge ratio m / z 129 of 2-HG) being greater than the theoretical value (GLU isotope peak signal intensity = GLU signal intensity × 0.06). It cannot be completely subtracted by the established algorithm formula (intensity ratio = target peak intensity / (internal standard peak intensity - isotope peak intensity)). When calculating the final ratio, the GLU isotope peak that was not subtracted is identified as 2-HG. The calculated result is 1.78, which is greater than the upper limit of the embedded threshold. The system judges it as a positive result, which is contrary to the negative sample.

[0135] When the sampling volume is too large, the detection results of IDH non-mutant samples are shown in Figure 8. When the sampling volume of IDH non-mutant samples (negative) is too large, the total number of target ions obtained by the fixed injection volume exceeds the statistical requirements of mass spectrometry detection (the upper limit of the target total ion number is set at 5000), which easily leads to GLU signal saturation. The GLU peak intensity in the spectrum does not truly reflect the GLU content (which is actually larger), but the GLU isotope peaks are not saturated and can normally reflect the true intensity. The algorithm calculates by multiplying the GLU peak intensity in the spectrum by 0.06, which cannot completely subtract the actual isotope peak intensity (actual GLU signal intensity × 0.06). The unsubtracted GLU isotope peaks are identified as 2-HG and included in the ratio calculation. The calculation result is 1.82, which is greater than the upper limit of the embedded threshold. The system judges it as a positive result, which is contrary to the negative sample.

[0136] When the sampling volume is too large, the detection results of IDH mutant samples are shown in Figures 9 and 10. When the sampling volume of IDH mutant samples (positive) is too large, the total number of target ions obtained by the fixed injection volume exceeds the statistical requirements of mass spectrometry detection, which can easily cause the GLU and 2-HG peaks in the spectrum to overlap. The algorithm cannot identify GLU (as shown in Figure 9) or 2-HG (as shown in Figure 10). During the calculation, the signal intensity of GLU or 2-HG is set to 0, thus indicating "no internal standard (GLU) detected" or because the 2-HG / GLU ratio is 0, which is less than the lower limit of the embedded threshold, the system judges it as a negative result, which is contrary to the positive sample.

[0137] Comparative Example 2

[0138] The same IDH mutation samples were detected using the mass spectrometry system described in Example 1, with and without standard spectrum determination.

[0139] The test results are shown in Figure 11. When testing the IDH mutant sample (positive), due to the limited stability of the reagent kit, three out of the first five MS2 spectra obtained from the scan (MS2 spectra 1, 2, 3, 4, and 5) showed no signal, and one spectrum showed a low 2-HG signal intensity. As a result, the 2-HG signal intensity was low in the spectrum synthesized from the original data of these five spectra, and the 2-HG / GLU ratio was 0.12, which was less than the lower limit of the embedded threshold. The system judged it as a negative result, which contradicted the positive sample.

[0140] After judging whether the MS2 spectrum meets the standard using the method described in Example 1, the spectrum synthesized from MS2 spectra 1, 2, 4, 6 and 7, which meet the requirements for each spectrum, has a 2-HG / GLU ratio greater than the upper limit of the embedded threshold. The system judges it as a positive result, which is consistent with the positive sample.

[0141] In summary, because the mass spectrometry system provided by this invention can rapidly sample and prepare samples, it cannot guarantee that an appropriate amount of sample can be collected each time. Therefore, it cannot automatically adjust the injection volume to match the mass spectrometry detection concentration range, and cannot guarantee the quality of the spectrum, resulting in low accuracy of the results. Furthermore, due to the inherent defects in the reagent kit, the spray stability cannot guarantee 100% compliance of the MS2 spectrum. Based on the above problems, the method provided by this invention can automatically adjust the injection volume to obtain a qualified spectrum, improve the dynamic concentration range of mass spectrometry detection, have a wider sampling range, higher fault tolerance, and higher accuracy. Moreover, it can control the quality of each MS2 spectrum, only merging qualified spectra for calculation after obtaining a sufficient number of qualified spectra, improving the stability of the detection and effectively ensuring the accuracy of the detection results of this mass spectrometry system.

[0142] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A mass spectrometry system for detecting IDH gene mutation biomarkers, characterized in that, include: A sampling kit includes a sampler and a detection box. The sampler includes a handle and a conductive sampling pad fixed to one end of the handle, the conductive sampling pad being used to collect a sample to be tested. The detection box includes a box body and a capillary tube, the box body having a sample slot. The sampler is detachably snapped onto the box body, and the conductive sampling pad extends into the sample slot. The sample slot is used to hold an elution solvent, the elution solvent being used to elute the sample to be tested collected on the conductive sampling pad to form an analytical solution. One end of the capillary tube extends into the sample slot, and the other end is located outside the sample slot. as well as A small mass spectrometer includes an ion source interface, an ion sample delivery system, and a vacuum system connected in sequence; the vacuum system is equipped with a mass analyzer and an ion detector. When the mass spectrometry system is in use, the sampling kit is configured to be plugged into the ion source interface of the miniature mass spectrometer, and a high voltage is applied to the conductive sampling plate so that the analytical solution in the sample cell is charged and enters the capillary, and is sprayed out from the other end of the capillary to form a spray; the spray enters the vacuum system via the ion sample introduction and transmission system.

2. The mass spectrometry system for detecting IDH gene mutation biomarkers according to claim 1, characterized in that, The conductive sampling sheet includes a sampling part and a conductive part connected in sequence. The sampling part is used to collect the sample to be tested, and the conductive part is used to connect to a high voltage. The sampling section is provided with a notch and a round hole, the notch and the round hole are in communication; the end of the capillary tube that extends into the sample groove is located in the notch; The sampler also includes sampling fibers, which are detachably fixed to the sampling section.

3. The mass spectrometry system for detecting IDH gene mutation markers according to claim 1, characterized in that, The vacuum system includes a vacuum chamber, a molecular pump, and a vortex pump, with the mass analyzer and ion detector located in the vacuum chamber. The ion sample delivery system includes a clamp valve, which is connected to the vacuum chamber.

4. The mass spectrometry system for detecting IDH gene mutation markers according to claim 1, characterized in that, When the mass spectrometry system is used to detect IDH gene mutation markers in brain tissue, the specific operation includes the following steps: (1) Take a brain tissue sample and place it on the carrier. Wipe the brain tissue with the conductive sampling pad of the sampler and then fix it on the detection box. Add elution solvent to the sample slot of the detection box to extract the analyte from the brain tissue sample to form an analytical solution. The elution solvent comprises ethanol and water, with ethanol accounting for 50%-90% of the volume in the elution solvent; the analytes in the brain tissue sample include, but are not limited to, 2-HG and GLU. (2) Insert the detection box into the ion source interface of the miniature mass spectrometer, set the test parameters of the miniature mass spectrometer, and apply voltage to the conductive sampling plate through the ion source. The conductive sampling plate transmits the voltage to the capillary tip through the analytical solution. The analytical solution forms a Taylor cone at the tip and generates an electrospray. The analyte is ionized by the electrospray to form ions, which enter the vacuum system through the ion sample introduction and transmission system of the miniature mass spectrometer for mass analysis and recording the signal intensity of each ion. (3) After obtaining the mass spectrometry signal, the intensity ratio of the secondary fragment ion signals of the target fragment ions m / z 129 and m / z 128 is calculated based on the intensity signals of the target fragment ions. Then, by comparing with the embedded threshold, the IDH mutation state of the brain tissue sample is determined.

5. A method for improving the detection accuracy of the mass spectrometry system according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1: Set the initial injection volume to Q1, perform the scan, and obtain the initial MS2 spectrum; Determine whether the total number of target ions on the initial MS2 spectrum meets the set limit requirement; if it meets the set limit requirement, determine the optimal injection volume Q0 = Q1; if it does not meet the set limit requirement, execute the injection volume optimization and adjustment scanning process until the injection volume that meets the set limit requirement on the scan spectrum is obtained, which is the optimal injection volume Q0. S2: Perform n scans with the optimal injection volume Q0 and collect MS2 spectra; during the collection of MS2 spectra, perform the following judgments: First judgment: Determine whether the total number of target ions in the MS2 spectrum meets the set limit; if it meets the set limit, it is a compliant spectrum and proceeds to the second judgment process; if it does not meet the set limit, it is a non-compliant spectrum and proceeds to the third judgment process. Second judgment: Determine whether the number of qualified spectra has reached the set upper limit N1; If the set upper limit N1 has been reached, the scan ends and the fourth judgment process begins or step S3 is entered. If the set upper limit N1 is not reached, proceed to the third judgment process; The third judgment: Check whether the number of scans is greater than or equal to the set upper limit n1. If it is greater than or equal to the set upper limit n1, the scan ends and the process proceeds to the fourth judgment. If the value is less than the set upper limit n1, then proceed to the next scan; Fourth judgment: Determine whether the number of qualified spectra is greater than or equal to the set lower limit N2; if it is greater than or equal to the set lower limit N2, proceed to step S3; if it is less than the set lower limit N2, prompt for resampling. S3: Combine the N qualified spectra obtained in S2 into one spectrum, determine the signal strength of the target object, and calculate according to the preset parameters. The formula is used to perform calculations, the results are compared with the embedded threshold, and the results are output. Where N1≤n≤n1; N2≤N≤N1.

6. The method according to claim 5, characterized in that, In S1, the specific steps of the injection volume optimization and adjustment scanning process are as follows: Determine the relationship between the total number of target ions on the MS2 spectrum and the set limit; if it is less than the lower limit of the set limit, increase the injection volume based on the previous injection volume and then perform the scan; if it is greater than the upper limit of the set limit, decrease the injection volume based on the previous injection volume and then perform the scan. Repeat the above operation until the total number of target ions on the scan spectrum meets the set limit requirement, which is the optimal injection amount Q0.

7. The method according to claim 6, characterized in that, The injection volume optimization adjustment is performed within the set injection volume range; And / or, the initial injection volume Q1 is 45ms, and the set injection volume range is 20-60ms.

8. The method according to claim 5, characterized in that, In S2, when using the combined internal standard method for quantitative testing, a valid spectrum filtering operation is performed during the collection of MS2 spectra to determine whether the obtained MS2 spectra are valid. If they are valid, the first judgment process is initiated; if they are invalid, they are discarded. The effective spectrum filtering operation is specifically as follows: match the spectral peaks of the internal standard in the MS2 spectrum; if a match can be made, it is a valid spectrum; if a match cannot be made, it is an invalid spectrum.

9. The method according to claim 5, characterized in that, In S3, the original data of the target object in N qualified spectra are summed and the average value is calculated based on the number of qualified spectra to synthesize a single spectra. When using the combined internal standard method for quantitative testing, the target analyte includes the analyte and the internal standard.

10. The method according to claim 5, characterized in that, When the mass spectrometry system is used to detect the mutation status of the IDH gene in brain tissue, the target substances are 2-HG and glutamate, wherein: The peak of the target analyte is located in the range of m / z 127.5-m / z 129.5; The set limit value is 100-5000; The upper limit n1 is set to 10; The upper limit N1 is set to 5, and the lower limit N2 is set to 3.

11. The method according to claim 10, characterized in that, The calculation formula is: M1 = (signal intensity of target ion m / z 129) / (0.94 × signal intensity of target ion m / z 128); When the embedded threshold is within a certain range, if M1 ≥ the upper limit of the embedded threshold, the output result is IDH mutation positive; if M1 < the lower limit of the embedded threshold, the output result is IDH mutation negative. When the embedded threshold is a fixed value, if M1 ≥ the embedded threshold, the output result is IDH mutation positive; otherwise, the output result is IDH mutation negative.

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