Three-dimensional nuclear magnetic resonance method and device for deeply annotating biological extract

Abstract

The invention discloses a three-dimensional nuclear magnetic resonance method and device for deeply annotating a biological extract. The method comprises the following steps: acquiring a 3D TOCSY J-reduced atlas of a target metabolite from a nuclear magnetic resonance spectrometer by setting a pulse sequence; the 3D TOCSY J-reduced atlas is processed through NMRPipe software, and processed three-dimensional nuclear magnetic data are obtained; analyzing and calculating the processed three-dimensional nuclear magnetic data through an R program to obtain a J coupling constant and chemical shift; carrying out characterization through a J coupling constant, chemical shift and peak multiplicity to obtain a spin system of the target metabolite; searching and matching the information of the spinning system in a nuclear magnetic database, identifying a target metabolite, and determining the attribution of the spinning system; and mapping a single peak of a high-resolution direct dimension of the belonging 3D TOCSY J-reduced atlas to a single peak in a high-resolution 1D < 1 > H NMR, and completing annotation of isolated and staggered multiple proton peaks of the 1D < 1 > H NMR. According to the method, the identification efficiency and accuracy of the metabolites in the complex biological sample are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of metabolomics, and in particular to a three-dimensional nuclear magnetic resonance method and device for deeply annotating biological extracts. Background Art

[0002] Metabolomics aims to identify and quantify metabolites in complex mixtures at high throughput, gain a deeper understanding of metabolic pathways, and explore biomarkers associated with specific phenotypes. Nuclear magnetic resonance (NMR) is a powerful tool in metabolomics, with advantages such as accurate quantification, good reproducibility, and the ability to detect a variety of chemicals without bias. At the same time, NMR can provide rich structural information. 1 H NMR is more widely used in metabolomics research due to its high sensitivity and short acquisition time, and it can accurately attribute 1D 1 Signal peaks in H NMR metabolic profiles are crucial for the correct identification of biomarkers. For complex metabolomics samples, due to similar chemical shifts and complex homonuclear J coupling, 1D 1 There is serious signal overlap in the H NMR spectrum, and multiple signals are detected in the high-resolution 1D 1 The H NMR spectrum shows staggered or closely spaced multiple peaks. At the same time, the chemical shift is extremely sensitive to changes in sample conditions (pH value, salt concentration, temperature, etc.), which may cause the observed chemical shift to deviate from the standard value in the database. At the same time, the multiple peaks with strong coupling effect peaks vary on spectrometers with different magnetic field strengths. These factors all affect the 1D 1 Accurate attribution and annotation of H NMR metabolic profiles poses challenges.

[0003] In recent years, a variety of NMR techniques have been introduced into metabolomics research, including 2D 1 H- 13 C HSQC spectra and pure chemical shift experiments (Zangger and Sterk (ZS), bilinear rotation decoupling (BIRD), chirpexcitation (PSYCHE)). Although these techniques have alleviated the signal overlap problem to a certain extent, they generally have the disadvantages of low sensitivity and long data acquisition time, and pure chemical shift spectra may also introduce additional strong coupling artifacts. In addition, the current methods used for 1D 1 Two-dimensional nuclear magnetic techniques for H NMR spectral assignment, such as 2D J-resolved, 2D 1 H- 13 C HSQC, 2D 1 H- 1 H TOCSY and 2D 1 H- 13C HSQC-TOCSY, due to insufficient resolution or self-overlapping problems, is still difficult to 1D 1 H NMR spectra were deeply annotated.

[0004] Therefore, a method to assist 1D 1 The nuclear magnetic resonance method of H NMR spectral attribution has important practical significance for metabolomics research and the discovery of clinical disease biomarkers. Summary of the Invention

[0005] To this end, the present invention provides a three-dimensional nuclear magnetic resonance method and device for deeply annotating biological extracts. By using the J-resolved plane analysis peak multiple modes, combined with the TOCSY dimensional correlated spin system, and using the R program to automatically calculate the J coupling constant, the problems of insufficient resolution, phase distortion and low sensitivity of traditional two-dimensional technology are solved. Through the dual verification of the BMRB and HMDB databases, accurate annotation of the staggered multiple peaks is achieved, and high-resolution 1D 1 The in-depth analysis of H NMR spectra significantly improves the efficiency and accuracy of metabolite identification in complex biological samples.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts, comprising:

[0007] Obtain 3D TOCSY J-resolved spectra of target metabolites from the NMR spectrometer by setting the pulse sequence;

[0008] The 3D TOCSYJ-resolved spectrum was processed using NMRPipe software to obtain processed three-dimensional nuclear magnetic resonance data; the processed three-dimensional nuclear magnetic resonance data were analyzed and calculated using R program to obtain J coupling constants and chemical shift information; the spin system of the target metabolite was obtained by characterizing the J coupling constants, the chemical shifts, and the peak multiplicity;

[0009] Searching and matching the information of the spin system in the nuclear magnetic resonance database to identify the target metabolite and determine the ownership of the spin system;

[0010] The assigned individual peaks of the high-resolution direct dimension of the 3D TOCSYJ-resolved spectrum were mapped onto the high-resolution 1D 1 A single peak in H NMR completes the 1D 1 Annotation of isolated and staggered multiple proton peaks in H NMR.

[0011] As a preferred embodiment of a three-dimensional nuclear magnetic resonance method for deeply annotating biological extracts, in the process of obtaining the 3D TOCSYJ-resolved spectrum of the target metabolite, the parameters of the pulse sequence are set as follows:

[0012] Spectral width: J-resolved plane 12ppm, TOCSY dimension 40Hz, direct dimension 11.706ppm;

[0013] Number of sampling points: Sampling points: t1 = 64 × 2, t2 = 64 × 1, t3 = 4096 × 2;

[0014] Relaxation time: 1 second, number of scans: 4 times.

[0015] As a preferred solution for a three-dimensional nuclear magnetic resonance method for deeply annotating biological extracts, in the process of processing the 3D TOCSYJ-resolved spectrum by the NMRPipe software, the J dimension and the direct dimension are processed by the Lorentz-Gaussian transform window function to eliminate phase distortion interference.

[0016] As a preferred solution for a three-dimensional nuclear magnetic resonance method for deeply annotating biological extracts, in the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the nuclear magnetic resonance database includes: the biological magnetic resonance database BMRB and the human metabolome database HMDB.

[0017] As a preferred solution for a three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts, in the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the information of the spin system is matched with the standard parameters of the metabolites in the nuclear magnetic resonance database, and screened by the chemical shift error threshold to obtain a preliminary candidate metabolite list; based on the preliminary candidate metabolite list, the consistency of the spin system is verified by TOCSY dimension signal correlation; if the J-resolved peaks of the same metabolite show the same signal bar in the TOCSY indirect dimension, the attribution of the spin system of the target metabolite is confirmed, and the interference of pseudo-peaks is eliminated.

[0018] The present invention also provides a three-dimensional nuclear magnetic resonance apparatus for deeply annotating biological extracts, based on the above three-dimensional nuclear magnetic resonance method for deeply annotating biological extracts, comprising:

[0019] 3D TOCSYJ-resolved spectrum acquisition module, used to obtain 3D TOCSYJ-resolved spectra of target metabolites from a nuclear magnetic resonance spectrometer by setting a pulse sequence;

[0020] A spin system characterization module is used to process the 3D TOCSYJ-resolved spectrum using NMRPipe software to obtain processed three-dimensional nuclear magnetic resonance data; analyze and calculate the processed three-dimensional nuclear magnetic resonance data using R program to obtain J coupling constants and chemical shift information; and characterize the spin system of the target metabolite using the J coupling constants, chemical shifts, and peak multiplicity;

[0021] A target metabolite matching and identification module is used to search and match the information of the spin system in the nuclear magnetic resonance database, identify the target metabolite, and determine the ownership of the spin system;

[0022] Deep Annotation Module for mapping individual peaks in the high-resolution direct dimension of the assigned 3D TOCSYJ-resolved atlas into high-resolution 1D 1 A single peak in H NMR completes the 1D 1 Annotation of isolated and staggered multiple proton peaks in H NMR.

[0023] As a preferred embodiment of a three-dimensional nuclear magnetic resonance apparatus for deeply annotating biological extracts, in the 3D TOCSY J-resolved spectrum acquisition module, in the process of acquiring the 3D TOCSY J-resolved spectrum of the target metabolite, the parameters of the set pulse sequence are:

[0024] Spectral width: J-resolved plane 12ppm, TOCSY dimension 40Hz, direct dimension 11.706ppm;

[0025] Number of sampling points: t1 = 64 × 2, t2 = 64 × 1, t3 = 4096 × 2;

[0026] Relaxation time: 1 second, number of scans: 4 times.

[0027] As a preferred solution for a three-dimensional nuclear magnetic resonance device for deep annotation of biological extracts, in the spin system characterization module, during the process of processing the 3D TOCSYJ-resolved spectrum by the NMRPipe software, the J dimension and the direct dimension are processed by the Lorentz-Gaussian transform window function to eliminate phase distortion interference.

[0028] As a preferred solution for a three-dimensional nuclear magnetic resonance device for deeply annotating biological extracts, in the target metabolite matching and identification module, in the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the nuclear magnetic resonance database includes: the biological magnetic resonance database BMRB and the human metabolome database HMDB.

[0029] As a preferred solution for a three-dimensional nuclear magnetic resonance device for deep annotation of biological extracts, in the target metabolite matching and identification module, during the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the information of the spin system is matched with the metabolite standard parameters in the nuclear magnetic resonance database, and screened by a chemical shift error threshold to obtain a preliminary candidate metabolite list; based on the preliminary candidate metabolite list, the consistency of the spin system is verified by TOCSY dimension signal correlation; if the J-resolved peaks of the same metabolite show the same signal bar in the TOCSY indirect dimension, the attribution of the spin system of the target metabolite is confirmed, and the interference of pseudo-peaks is eliminated.

[0030] The present invention has the following advantages: the present invention obtains a 3D TOCSYJ-resolved spectrum of a target metabolite from a nuclear magnetic resonance spectrometer by setting a pulse sequence; the 3D TOCSYJ-resolved spectrum is processed by NMRPipe software to obtain processed three-dimensional nuclear magnetic resonance data; the processed three-dimensional nuclear magnetic resonance data is analyzed and calculated by R program to obtain J coupling constants and chemical shift information; the spin system of the target metabolite is obtained by characterizing the J coupling constants, the chemical shift parameters and the peak multiplicity; the information of the spin system is searched and matched in a nuclear magnetic resonance database to identify the target metabolite and determine the attribution of the spin system; the high-resolution direct dimension single peak of the assigned 3D TOCSYJ-resolved spectrum is mapped on the high-resolution 1D 1 A single peak in H NMR completes the 1D 1 The present invention significantly improves the interpretation of isolated and interleaved multiple proton peaks in H NMR. 1 Analytical efficiency and accuracy of H NMR spectra. Compared with traditional two-dimensional nuclear magnetic techniques (such as HSQC, TOCSY), the present invention solves the signal ambiguity problems caused by multiple peak overlap, chemical shift deviation and J coupling effect by associating three-dimensional nuclear magnetic data collection with multidimensional parameters. The R program is innovatively introduced to automatically calculate the J coupling constant, and the Lorentz-Gaussian transform window function is combined to optimize the phase distortion, thereby achieving accurate separation and attribution of staggered peaks. The present invention not only ensures the quantitative accuracy of metabolite identification (chemical shift error ≤ 0.02ppm) through the dual verification mechanism of BMRB and HMDB databases, but also strengthens biological rationality (such as excluding non-physiological interference peaks). In addition, the present invention does not need to rely on standard product spectra, which greatly reduces experimental cost and time cost, and provides efficient and reliable analysis tools for metabolomics research and clinical biomarker discovery, with significant scientific research and industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can, without inventive effort, derive other implementation drawings based on the provided drawings.

[0032] The structures, proportions, sizes, etc. illustrated in this specification are intended solely to complement the contents disclosed herein and to facilitate understanding and reading by persons skilled in the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall remain within the scope of the technical contents disclosed herein.

[0033] Figure 1 Schematic diagram of a process for a three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts provided in Example 1 of the present invention;

[0034] Figure 2 Schematic diagram of the pulse sequence of 3D TOCSY J-resolved in a three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts provided in Example 1 of the present invention;

[0035] Figure 3 Schematic diagram of 3D TOCSY J-resolved spectrum analysis in a possible embodiment provided in Example 1 of the present invention; wherein (A) at high resolution 1D 1 In the H NMR spectrum, there is ambiguity in the assignment of two closely spaced proton triplets at 3.246 ppm and 3.213 ppm (their J coupling constants are nearly identical, approximately 6.9 Hz); (B) 2D J-resolved spectrum of the model mixture; (C) 3D TOCSY J-resolved spectrum helps characterize the spin systems of the two triplets at 3.246 ppm and 3.213 ppm, which show TOCSY correlations with four and five chemical shifts, respectively.

[0036] Figure 4 Schematic diagram of 3D TOCSY J-resolved in a three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts provided in Example 1 of the present invention; wherein (A) is a high-resolution 1D 1Complex multiple staggered proton multiplets in H NMR; (B) 2D J-resolved spectrum of multiple staggered proton multiplets, where the peaks marked in blue and black have strongly coupled artifacts; (C) 3D TOCSY J-resolved spectrum of multiple staggered proton multiplets.

[0037] Figure 5 This is a schematic diagram of the architecture of a three-dimensional nuclear magnetic resonance device for deep annotation of biological extracts provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0039] Example 1

[0040] See also Figure 1 Embodiment 1 of the present invention provides a three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts, comprising the following steps:

[0041] S1. Obtain the 3D TOCSY J-resolved spectrum of the target metabolite from the NMR spectrometer by setting the pulse sequence;

[0042] S2. Process the 3D TOCSYJ-resolved spectrum using NMRPipe software to obtain processed three-dimensional nuclear magnetic resonance data; analyze and calculate the processed three-dimensional nuclear magnetic resonance data using R program to obtain J coupling constants and chemical shift information; characterize the spin system of the target metabolite using the J coupling constants, chemical shifts, and peak multiplicity;

[0043] S3. Searching and matching the information of the spin system in the nuclear magnetic resonance database to identify the target metabolite and determine the ownership of the spin system;

[0044] S4. Mapping the assigned individual peaks of the high-resolution direct dimension of the 3D TOCSYJ-resolved spectrum onto the high-resolution 1D 1 A single peak in H NMR completes the 1D 1 Annotation of isolated and staggered multiple proton peaks in H NMR.

[0045] In this embodiment, in step S1, a 3D TOCSYJ-resolved spectrum of the target metabolite is obtained from a nuclear magnetic resonance spectrometer by setting a pulse sequence;

[0046] Specifically, the pulse sequence is set as follows: Figure 2 As shown, the acquisition parameters are:

[0047] The NMR spectrometer was a Bruker Avance III 700M NMR spectrometer equipped with a QCI-F cryogenic probe, controlled by TopSpin software. Data were acquired at 298 K using a sampling frequency of 64 × 2 (t1), 64 × 1 (t2), and 4096 × 2 (t3). The spectral widths in the t1, t2, and t3 dimensions were set to 12 ppm, 40 Hz, and 11.706 ppm, respectively. The relaxation time was set to 1 s to reduce sampling time, and the number of scans was four.

[0048] In this embodiment, the collection parameters can be appropriately adjusted according to the sample concentration and metabolite types.

[0049] In this embodiment, in step S2, the 3D TOCSY J-resolved spectrum is processed using NMRPipe software to obtain processed three-dimensional nuclear magnetic resonance data; the processed three-dimensional nuclear magnetic resonance data is analyzed and calculated using R program to obtain J coupling constants and chemical shifts; the spin system of the target metabolite is obtained by characterizing the J coupling constants, the chemical shifts, and the peak multiplicity;

[0050] Specifically, the 3D TOCSY J-resolved spectrum was processed using NMRPipe software to obtain processed 3D NMR data. The J and direct dimensions were processed using a Lorentz-Gaussian transform window function to eliminate phase distortion. The processed 3D NMR data were analyzed and calculated using the R program to obtain J coupling constants and chemical shifts. The spin system of the target metabolite was characterized using the J coupling constants, chemical shifts, and peak multiplicity.

[0051] In this embodiment, in step S3, the information of the spin system is searched and matched in the nuclear magnetic resonance database to identify the target metabolite and determine the ownership of the spin system;

[0052] The nuclear magnetic resonance database includes the biological magnetic resonance database BMRB and the human metabolome database HMDB.

[0053] Specifically, in the process of searching and matching the information of the spin system in the NMR database, the information of the spin system is matched with the standard parameters of the metabolites in the NMR database, and screened by the chemical shift error threshold to obtain a preliminary candidate metabolite list; based on the preliminary candidate metabolite list, the consistency of the spin system is verified by TOCSY dimension signal correlation; if the J-resolved peaks of the same metabolite show the same signal bar in the TOCSY indirect dimension, the attribution of the spin system of the target metabolite is confirmed, and the interference of pseudo-peaks is eliminated.

[0054] In this embodiment, in step S4, the single peak of the high-resolution direct dimension of the assigned 3D TOCSYJ-resolved spectrum is mapped on the high-resolution 1D 1 A single peak in H NMR completes the 1D 1 Annotation of isolated and staggered multiple proton peaks in H NMR.

[0055] Specifically, the individual peaks of the assigned 3D TOCSYJ-resolved spectrum in the high-resolution direct dimension are precisely mapped onto the high-resolution 1D 1 Single peak in H NMR, annotated 1D 1 Isolated and interlaced multiple proton peaks in H NMR.

[0056] In a possible embodiment, a specific verification example is provided as follows:

[0057] In this example, a model mixture containing five human metabolite standards was first prepared:

[0058] Stock solutions of five metabolite standards (leucine, isoleucine, lysine, homoarginine, and arginine) were prepared with concentrations of 53.14 mM, 41.19 mM, 30.46 mM, 26.32 mM, and 42.08 mM, respectively, in NMR sample buffer.

[0059] 100 μL of each metabolite stock solution was taken, vortexed and mixed, and then the mixture was transferred to a 5 mm diameter NMR tube for measurement.

[0060] The collected 3D TOCSYJ-resolved spectra were processed using NMRpipe. The processed spectra were analyzed, such as Figure 3As shown in the figure, the chemical shifts of the two metabolites of arginine-5CH2 and homoarginine-6CH2 in the model mixture are very close, 3.246ppm and 3.213ppm respectively, both of which are triplets, and the coupling constant J is almost the same, about 6.9Hz. If we only rely on the displacement information in the NMR database (3.234ppm and 3.189ppm), it is difficult to determine the identity of these two triplets with 100% certainty. Figure 3 (C) The 3D TOCSYJ-resolved spectrum shows TOCSY-related triplet peaks of four and five chemical shifts, corresponding to the structural formulas of arginine and homoarginine, and the identities of the two triplet peaks can be accurately identified. It can be seen that the 3DTOCSYJ-resolved spectrum can greatly help distinguish and attribute metabolites with similar structures, helping to achieve 1D 1 In-depth annotation of complex adjacent similar peaks in H NMR facilitates subsequent statistical analysis and biological interpretation.

[0061] This example also provides 1D 1 Examples of metabolite identification and peak annotation by HNMR are as follows:

[0062] Sample preparation: Human lung adenocarcinoma cell line PC9 was cultured in complete medium (RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin). Cells were cultured at 37°C, 95% humidity, and 5% CO2. Cells were plated at 5×10 6 Cells were seeded at an initial density of 100 cells / dish in a 15 cm diameter culture dish and cultured to a cell density of approximately 90%. The culture medium was removed and the cells were quickly rinsed with 10.0 mL of 37°C PBS and then immediately frozen in liquid nitrogen to terminate cell activity. The frozen cells were scraped twice with a rubber-tipped cell scraper in a -20°C pre-cooled acetonitrile / water (V / V = 1:1) extraction solvent, 2 mL each time. The cell suspensions were combined and mixed (approximately 4 mL) and then divided into two Eppendorf tubes for metabolite extraction. The cell suspension samples were ground using a tissue grinder and centrifuged at 4°C and 15,000 rpm for 10 minutes. The supernatant was transferred to a 15 mL centrifuge tube. The precipitate of each sample was extracted twice with 1.6 mL of extraction solvent, and the extraction process was the same each time. All supernatants were combined and mixed (a total of approximately 10.4 mL). Take 9000 μL of the supernatant, dry it with nitrogen, resuspend it in 530 μL of NMR sample buffer, vortex for 60 seconds, sonicate for 10 minutes, and finally centrifuge at 15,000 rpm for 10 minutes at 4°C. Transfer 500 μL of the supernatant to a 5 mm diameter NMR tube for NMR spectrum acquisition.

[0063] The collected 3D TOCSYJ-resolved spectra were processed using NMRpipe. Figure 4 As shown in the figure, 3DTOCSYJ-resolved has significant advantages in annotating adjacent staggered peaks in 1D 1HNMR of complex real cell extracts. According to the metabolite identification and annotation process based on 3D TOCSYJ-resolved of the present invention, a total of 51 metabolites were identified in the cell extracts, and 242 peaks were clearly annotated, including different multiple peaks that were staggered or closely spaced.

[0064] In summary, the present invention obtains a 3D TOCSYJ-resolved spectrum of a target metabolite from a nuclear magnetic resonance spectrometer by setting a pulse sequence; processes the 3D TOCSYJ-resolved spectrum by using NMRPipe software to obtain processed three-dimensional nuclear magnetic data; analyzes and calculates the processed three-dimensional nuclear magnetic data by using R program to obtain J coupling constant and chemical shift information; characterizes the spin system of the target metabolite by using the J coupling constant, the chemical shift and the peak multiplicity; searches and matches the information of the spin system in the nuclear magnetic database to identify the target metabolite and determine the attribution of the spin system; maps the high-resolution direct dimension single peak of the assigned 3D TOCSYJ-resolved spectrum to the high-resolution 1D 1 A single peak in H NMR completes the 1D 1 The present invention significantly improves the interpretation of isolated and interleaved multiple proton peaks in H NMR. 1 Analytical efficiency and accuracy of H NMR spectra. Compared with traditional two-dimensional nuclear magnetic techniques (such as HSQC, TOCSY), the present invention solves the problem of signal fuzzy attribution caused by multiple peak overlap, chemical shift deviation and J coupling effect through three-dimensional nuclear magnetic data acquisition and multi-dimensional parameter association. Innovatively introduce the R program to automatically calculate the J coupling constant, and combine the Lorentz-Gaussian transform window function to optimize phase distortion, thereby achieving accurate separation and attribution of staggered peaks. The present invention not only ensures the quantitative accuracy of metabolite identification (chemical shift error ≤ 0.02ppm) through the dual verification mechanism of BMRB and HMDB databases, but also strengthens biological rationality (such as excluding non-physiological interference peaks). In addition, the present invention does not need to rely on standard product spectra, which greatly reduces experimental cost and time cost, and provides efficient and reliable analysis tools for metabolomics research and clinical biomarker discovery, with significant scientific research and industrial application potential.

[0065] It should be noted that the method of the embodiments of the present disclosure can be performed by a single device, such as a computer or server. The method of the embodiments of the present disclosure can also be applied in a distributed scenario, where multiple devices cooperate to perform the method. In such a distributed scenario, one of the multiple devices may only perform one or more steps of the method of the embodiments of the present disclosure, and the multiple devices will interact with each other to complete the method.

[0066] It should be noted that the above description is limited to some embodiments of the present disclosure. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps recited in the claims may be performed in an order different from that described in the above embodiments and still achieve the desired results. Furthermore, the processes depicted in the accompanying drawings do not necessarily require the specific order or sequential order shown to achieve the desired results. In certain embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0067] Example 2

[0068] See also Figure 5 Embodiment 2 of the present invention further provides a three-dimensional nuclear magnetic resonance apparatus for deeply annotating biological extracts, comprising:

[0069] 3D TOCSYJ-resolved spectrum acquisition module 001, used to acquire the 3D TOCSYJ-resolved spectrum of the target metabolite from the nuclear magnetic resonance spectrometer by setting a pulse sequence;

[0070] Spin system characterization module 002 is used to process the 3D TOCSY J-resolved spectrum using NMRPipe software to obtain processed three-dimensional nuclear magnetic resonance data; analyze and calculate the processed three-dimensional nuclear magnetic resonance data using R program to obtain J coupling constant and chemical shift information; characterize the spin system of the target metabolite by the J coupling constant, the chemical shift and the peak multiplicity;

[0071] Target metabolite matching and identification module 003, used to search and match the information of the spin system in the nuclear magnetic resonance database, identify the target metabolite, and determine the ownership of the spin system;

[0072] The deep annotation module 004 is used to map the individual peaks of the high-resolution direct dimension of the assigned 3D TOCSYJ-resolved atlas to the high-resolution 1D 1 A single peak in H NMR completes the 1D 1 Annotation of isolated and staggered multiple proton peaks in H NMR.

[0073] In this embodiment, in the 3D TOCSYJ-resolved spectrum acquisition module 001, in the process of acquiring the 3D TOCSYJ-resolved spectrum of the target metabolite, the parameters of the set pulse sequence are:

[0074] Spectral width: J-resolved plane 12ppm, TOCSY dimension 40Hz, direct dimension 11.706ppm;

[0075] Number of sampling points: Sampling points: t1 = 64 × 2, t2 = 64 × 1, t3 = 4096 × 2;

[0076] Relaxation time: 1 second, number of scans: 4 times.

[0077] In this embodiment, in the spin system characterization module 002, when the 3DTOCSYJ-resolved spectrum is processed by the NMRPipe software, the J dimension and the direct dimension are processed by the Lorentz-Gaussian transform window function to eliminate phase distortion interference.

[0078] In this embodiment, in the target metabolite matching and identification module 003, in the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the nuclear magnetic resonance database includes: the biological magnetic resonance database BMRB and the human metabolome database HMDB.

[0079] In this embodiment, in the target metabolite matching and identification module 003, in the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the information of the spin system is matched with the metabolite standard parameters in the nuclear magnetic resonance database, and screened by the chemical shift error threshold to obtain a preliminary candidate metabolite list; based on the preliminary candidate metabolite list, the consistency of the spin system is verified by the TOCSY dimension signal correlation; if the J-resolved peaks of the same metabolite show the same signal bar in the TOCSY indirect dimension, the attribution of the spin system of the target metabolite is confirmed, and the interference of pseudo peaks is eliminated.

[0080] It should be noted that the information interaction, execution process, etc. between the modules of the above-mentioned system are based on the same concept as the method embodiment in Example 1 of the present application, and the technical effects they bring are the same as those of the method embodiment of the present application. For specific contents, please refer to the description in the method embodiment shown above in the present application, and no further details will be given here.

[0081] Example 3

[0082] Embodiment 3 of the present invention provides a non-transitory computer-readable storage medium, in which a program code for a three-dimensional nuclear magnetic resonance method for deeply annotating biological extracts is stored. The program code includes instructions for executing a three-dimensional nuclear magnetic resonance method for deeply annotating biological extracts of embodiment 1 or any possible implementation thereof.

[0083] Computer-readable storage media can be any available medium that can be accessed by a computer or a data storage device such as a server or data center that includes one or more available media. The available media can be magnetic media (e.g., floppy disks, hard disks, magnetic tapes), optical media (e.g., DVDs), or semiconductor media (e.g., solid-state drives (SSDs)).

[0084] Example 4

[0085] Embodiment 4 of the present invention provides an electronic device, including: a memory and a processor;

[0086] The processor and the memory communicate with each other via a bus; the memory stores program instructions that can be executed by the processor, and the processor calls the program instructions to execute a three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts according to Example 1 or any possible implementation thereof.

[0087] Specifically, the processor can be implemented by hardware or by software. When implemented by hardware, the processor can be a logic circuit, an integrated circuit, etc.; when implemented by software, the processor can be a general-purpose processor, which is implemented by reading software code stored in a memory. The memory can be integrated into the processor or located outside the processor and exist independently.

[0088] In the above embodiments, it can be implemented in whole or in part by software, hardware, firmware or any combination thereof. When implemented using software, it can be implemented in whole or in part in the form of a computer program product. The computer program product includes one or more computer instructions. When the computer program instructions are loaded and executed on a computer, the process or function described in the embodiment of the present invention is generated in whole or in part. The computer can be a general-purpose computer, a special-purpose computer, a computer network, or other programmable systems. The computer instructions can be stored in a computer-readable storage medium, or transmitted from one computer-readable storage medium to another computer-readable storage medium. For example, the computer instructions can be transmitted from a website, computer, server or data center to another website, computer, server or data center via a wired (e.g., coaxial cable, optical fiber, digital subscriber line (DSL)) or wireless (e.g., infrared, wireless, microwave, etc.) mode.

[0089] Obviously, those skilled in the art will appreciate that the various modules or steps of the present invention described above can be implemented using a general-purpose computing system. They can be centralized on a single computing system or distributed across a network of multiple computing systems. Alternatively, they can be implemented using program code executable by a computing system, and thus, they can be stored in a storage system and executed by the computing system. In some cases, the steps shown or described herein can be performed in a different order than that shown, or they can be fabricated into separate integrated circuit modules, or multiple modules or steps can be fabricated into a single integrated circuit module. Thus, the present invention is not limited to any particular combination of hardware and software.

[0090] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts, characterized in that include: Obtain 3D TOCSY J-resolved spectra of target metabolites from the NMR spectrometer by setting the pulse sequence; The 3D TOCSYJ-resolved spectrum was processed using NMRPipe software to obtain processed three-dimensional nuclear magnetic resonance data; the processed three-dimensional nuclear magnetic resonance data were analyzed and calculated using R program to obtain J coupling constants and chemical shift information; the spin system of the target metabolite was obtained by characterizing the J coupling constants, the chemical shifts, and the peak multiplicity; Searching and matching the information of the spin system in the nuclear magnetic resonance database to identify the target metabolite and determine the ownership of the spin system; The assigned individual peaks of the high-resolution direct dimension of the 3D TOCSYJ-resolved map were mapped onto the high-resolution 1D 1 A single peak in H NMR completes the 1D 1 Annotation of isolated and staggered multiple proton peaks in H NMR.

2. A three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts according to claim 1, characterized in that In the process of obtaining the 3D TOCSYJ-resolved spectrum of the target metabolite, the parameters of the pulse sequence are set as follows: Spectral width: J-resolved plane 12ppm, TOCSY dimension 40Hz, direct dimension 11.706ppm; Number of sampling points: t1 = 64 × 2, t2 = 64 × 1, t3 = 4096 × 2; Relaxation time: 1 second, number of scans: 4 times.

3. A three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts according to claim 2, characterized in that: In the process of processing the 3D TOCSYJ-resolved atlas by the NMRPipe software, the J dimension and the direct dimension are processed by the Lorentz-Gaussian transform window function to eliminate phase distortion interference.

4. A three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts according to claim 3, characterized in that In the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the nuclear magnetic resonance database includes: a biological magnetic resonance database BMRB and a human metabolome database HMDB.

5. A three-dimensional nuclear magnetic resonance method for deep annotation of biological extracts according to claim 4, characterized in that: In the process of searching and matching the information of the spin system in the NMR database, the information of the spin system is matched with the standard parameters of the metabolites in the NMR database, and screened by the chemical shift error threshold to obtain a preliminary list of candidate metabolites; based on the preliminary list of candidate metabolites, the consistency of the spin system is verified by TOCSY dimension signal correlation; if the J-resolved peaks of the same metabolite show the same signal bar in the TOCSY indirect dimension, the attribution of the spin system of the target metabolite is confirmed, and the interference of pseudo-peaks is eliminated.

6. A three-dimensional nuclear magnetic resonance device for deep annotation of biological extracts, characterized in that include: 3D TOCSYJ-resolved spectrum acquisition module, used to obtain 3D TOCSYJ-resolved spectra of target metabolites from a nuclear magnetic resonance spectrometer by setting a pulse sequence; A spin system characterization module is used to process the 3D TOCSYJ-resolved spectrum using NMRPipe software to obtain processed three-dimensional nuclear magnetic resonance data; analyze and calculate the processed three-dimensional nuclear magnetic resonance data using R program to obtain J coupling constants and chemical shift information; and characterize the spin system of the target metabolite using the J coupling constants, chemical shifts, and peak multiplicity; A target metabolite matching and identification module is used to search and match the information of the spin system in the nuclear magnetic resonance database, identify the target metabolite, and determine the ownership of the spin system; Deep Annotation Module for mapping individual peaks in the high-resolution direct dimension of the assigned 3D TOCSYJ-resolved atlas into high-resolution 1D 1 A single peak in H NMR completes the 1D 1 Annotation of isolated and staggered multiple proton peaks in HNMR.

7. A three-dimensional nuclear magnetic resonance apparatus for deep annotation of biological extracts according to claim 6, characterized in that: In the 3D TOCSYJ-resolved spectrum acquisition module, in the process of acquiring the 3D TOCSYJ-resolved spectrum of the target metabolite, the parameters of the set pulse sequence are: Spectral width: J-resolved plane 12ppm, TOCSY dimension 40Hz, direct dimension 11.706ppm; Number of sampling points: t1 = 64 × 2, t2 = 64 × 1, t3 = 4096 × 2; Relaxation time: 1 second, number of scans: 4 times.

8. The three-dimensional nuclear magnetic resonance apparatus for deep annotation of biological extracts according to claim 7, characterized in that: In the spin system characterization module, when the 3DTOCSY J-resolved spectrum is processed by the NMRPipe software, the J dimension and the direct dimension are processed by the Lorentz-Gaussian transform window function to eliminate phase distortion interference.

9. The three-dimensional nuclear magnetic resonance apparatus for deep annotation of biological extracts according to claim 8, characterized in that: In the target metabolite matching and identification module, in the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the nuclear magnetic resonance database includes: the biological magnetic resonance database BMRB and the human metabolome database HMDB.

10. The three-dimensional nuclear magnetic resonance apparatus for deep annotation of biological extracts according to claim 9, characterized in that: In the target metabolite matching and identification module, during the process of searching and matching the information of the spin system in the nuclear magnetic resonance database, the information of the spin system is matched with the metabolite standard parameters in the nuclear magnetic resonance database, and screened by the chemical shift error threshold to obtain a preliminary candidate metabolite list; based on the preliminary candidate metabolite list, the consistency of the spin system is verified by the TOCSY dimension signal correlation; if the J-resolved peaks of the same metabolite show the same signal bar in the TOCSY indirect dimension, the attribution of the spin system of the target metabolite is confirmed, and the interference of pseudo-peaks is eliminated.

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