Method for preparing solid phase extraction material and its application in detecting multi-components

Abstract

This invention discloses a method for preparing solid-phase extraction materials and their application in the detection of multiple components, including a method for preparing graphene oxide-iron tetroxide nanoparticle composite materials, and a method and kit for simultaneously detecting lipid and peptide components in a sample using the composite material.

Description

Technical Field

[0001] This disclosure relates to the field of bioassay. Specifically, this disclosure relates to a method for preparing a graphene oxide-iron oxide nanoparticle composite material, and a method, kit, and application of using the composite material to simultaneously detect peptide and lipid components in biological samples. Background Technology

[0002] Both peptides and lipids are important components and active substances in clinical samples.

[0003] The sources of polypeptide molecules in clinical samples, such as plasma and serum, can be broadly divided into two categories. One category consists of naturally occurring free polypeptides, which are mostly participants in biochemical reaction processes and perform certain biological functions, such as glutathione. The other category consists of polypeptides that are products of the degradation or formation of large molecular weight proteins in enzymatic reactions. Their levels often reflect changes in other specific biochemical indicators (such as enzymes) or physiological processes. Therefore, the changes and distribution of polypeptide components in clinical samples are an important part of the field of biochemical analysis. For clinical testing and diagnosis, many specific polypeptide molecules are important clinical indicators. For basic clinical research, the peptidomome itself is an important source of new biomarkers for diseases and physiological processes. In addition, the proteomics analysis techniques currently widely used in clinical practice are essentially peptidomics analyses of samples, and peptidomics analysis also plays an important role in the detection of proteins in clinical samples.

[0004] Lipid molecules in clinical samples are a class of small metabolic molecules, including different types of long-chain aliphatic molecules such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, triglycerides, cardiolipin, and sphingomyelin. These metabolic molecules participate in the structural building and energy storage of living organisms, and also participate in specific physiological processes through a series of biochemical reactions. Therefore, specific lipid molecules are important indicators of metabolism; for example, blood triglyceride levels represent the normality of the body's fat metabolism. Furthermore, different types of specific lipid molecules are also correlated with disease, nutrition, and physiological state, serving as important sources of physiological biomarkers. Thus, lipidomics analysis of clinical samples has become an important tool in clinical chemistry and physiological medicine research.

[0005] In recent years, with advancements in basic physiological and medical research and the development of systems biology research tools, the combined research and application of multiple omics in specific diseases has become increasingly important. For example, due to the significant correlation between peptides and lipids in physiological metabolic networks, peptidomics (or proteomics) and lipidomics are frequently used in combination for detection and application in different diseases. However, current traditional clinical proteomics and lipidomics analysis procedures in body fluids are two completely different technical processes, requiring different techniques for interferon separation, sample enrichment, sample purification, and sample detection. This increases the complexity of multi-omics joint research and raises research and detection costs. Furthermore, it significantly increases the waste of valuable clinical samples, as samples that have undergone one type of omics analysis are often no longer suitable for other types of omics analyses, thus multiplying the required sample volume for multi-omics analysis. These technical bottlenecks hinder the widespread application of multi-omics joint research and increase the economic and human costs of related clinical testing.

[0006] In the field of clinical mass spectrometry, matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) is a high-throughput, automated method with advantages such as high sensitivity, good stability, short experimental cycle, simple operation, and low sample and manpower consumption. It has significant potential value for the detection of clinical indicators. The applicable molecular weight range of MALDI-MS is well-suited for the simultaneous analysis of lipids and peptides, requiring only the selection of appropriate molecular weights and detection conditions during the instrument detection phase. However, clinical samples, such as plasma, are inherently complex, and the high abundance of large molecular weight proteins, salts, and other matrix components can severely interfere with the mass spectrometry detection process. Therefore, the core challenge in using MALDI-MS to detect lipidomics and peptidomics is to purify and enrich lipid and peptide components in the sample as much as possible during sample pretreatment, removing interference from other types of molecular matrices.

[0007] In summary, the development of combined lipidomics and peptidomics analysis protocols requires sample pretreatment methods that can simultaneously purify and enrich both lipidomics and peptidomics in a single sample, while ensuring that the analytical samples prepared by such methods are compatible with the MALDI MS instrument detection process. Summary of the Invention

[0008] To address one of the aforementioned technical problems in the prior art, this disclosure provides a method for preparing a solid-phase extraction material, and provides a novel analytical method for using this solid-phase extraction material to stepwise purify lipid and peptide components in liquid samples through solid-phase extraction, and for detection using MALDI MS. The method disclosed herein can be used to simultaneously determine lipid and peptide components in biological liquid samples (such as plasma, serum, urine, etc.).

[0009] According to one aspect of this disclosure, a method for preparing a graphene oxide-iron tetroxide nanoparticle composite material is provided, the method comprising: dispersing graphene oxide in water to obtain a first dispersion; dispersing iron tetroxide nanoparticles in water to obtain a second dispersion; mixing the first dispersion and the second dispersion, adding a dehydrating agent, and performing a dehydration condensation reaction to obtain the graphene oxide-iron tetroxide nanoparticle composite material, wherein the mass ratio of graphene oxide to iron tetroxide nanoparticles is 0.5:1 to 1:20.

[0010] When the mass ratio of graphene oxide to iron oxide nanoparticles is within the aforementioned range, it can form abundant micro / nano structures with nanopores and nanogrooves. Such structures exhibit good adsorption effects on peptide and lipid components. If the content of either graphene oxide or iron oxide is too low, it is impossible to form micro / nano structures with good adsorption properties. In some preferred embodiments of this disclosure, the mass ratio of graphene oxide to iron oxide nanoparticles is 1:1 to 1:20. In some preferred embodiments of this disclosure, the mass ratio of graphene oxide to iron oxide nanoparticles is 1:3.

[0011] According to some embodiments of this disclosure, the dehydrating agent may be N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide. The graphene oxide and the iron oxide nanoparticles undergo a dehydration condensation reaction with the assistance of the dehydrating agent.

[0012] According to some embodiments of this disclosure, the graphene oxide is in powder or sheet form. In some specific embodiments of this disclosure, the graphene oxide is a single-layer structure. According to some embodiments of this disclosure, the graphene oxide has functional groups such as hydroxyl and carboxyl groups on its surface. According to some embodiments of this disclosure, the size of the graphene oxide is 1–50 μm, preferably 5–10 μm. According to some embodiments of this disclosure, the size of the iron oxide nanoparticles is 20–200 nm. According to some embodiments of this disclosure, the surface of the iron oxide nanoparticles has carboxyl functional groups.

[0013] According to some embodiments of this disclosure, graphene oxide (GO) with functional groups such as hydroxyl and carboxyl groups on its surface undergoes a dehydration condensation reaction with magnetite nanoparticles (MNP) with carboxyl groups on their surface, forming a composite material through chemical bonding. The surface of the formed composite material GO-MNP exhibits numerous micro- and nanostructures, such as nanopores and nanogrooves, resulting from graphene sheet folding and covalent bonding with magnetic magnetite nanoparticles. These structures increase the surface area of ​​the composite material, and the nanopore walls, composed of hydrophobic reduced graphene oxide, effectively accommodate and adsorb small molecular weight targets with hydrophobic properties, such as peptides and lipids, through capillary action, making GO-MNP a good nano-adsorbent material. Furthermore, because GO-MNP contains magnetic magnetite, it can be separated by an external magnetic field. In some specific embodiments, the temperature of the dehydration condensation reaction can be 20°C to 40°C. In some specific embodiments, the reaction time of the dehydration condensation reaction is 2 to 20 hours.

[0014] On the other hand, this disclosure provides a method for simultaneously detecting lipid components and peptide components in a sample, the method comprising the following steps: adding a solid-phase extraction material to the sample to enrich the lipid components and peptide components in the sample; eluting the lipid components and peptide components from the solid-phase extraction material using an elution solvent; and detecting the eluted lipid components and peptide components using matrix-assisted laser desorption / ionization mass spectrometry, wherein the solid-phase extraction material is a graphene oxide-iron oxide nanoparticle (GO-MNP) composite material obtained by the above-described method for preparing graphene oxide-iron oxide nanoparticle composite materials.

[0015] According to some embodiments of this disclosure, the sample is a liquid sample. In some specific embodiments, the sample is a biological sample, such as blood, serum, plasma, saliva, urine, cerebrospinal fluid, or tissue cell extract. The method of this disclosure can use a single biological sample to simultaneously detect lipid and peptide components without the need to prepare separate samples and design separate analytical procedures for the two analytes.

[0016] According to some embodiments of this disclosure, the concentration of the solid-phase extraction material in the sample is 1.0–10.0 mg / mL. In some specific embodiments of this disclosure, the concentration of the solid-phase extraction material in the sample can be 2.5–7.5 mg / mL. For example, the concentration of the solid-phase extraction material in the sample can be 2.5 mg / mL.

[0017] According to some embodiments of this disclosure, after adding solid-phase extraction material to a sample, it can be incubated for 5 to 60 minutes to adsorb lipid and peptide components in the sample.

[0018] According to some embodiments of this disclosure, the step of eluting the lipid and peptide components from the solid-phase extraction material using an elution solvent includes: eluting the peptide component using a first elution solvent; and / or eluting the lipid component using a second elution solvent. The peptide component can be eluted first using the first elution solvent, and then the lipid component can be eluted using the second elution solvent. Alternatively, the lipid component can be eluted using the second elution solvent, and then the peptide component can be eluted using the first elution solvent. Alternatively, the solid-phase extraction material can be divided into two parts, and the first and second elution solvents can be used simultaneously to elute the peptide and lipid components.

[0019] According to some embodiments of this disclosure, the first eluent can be a highly polar mixed solution, such as one or more of water, acetonitrile, and trifluoroacetic acid. In some specific embodiments of this disclosure, the first eluent comprises water, acetonitrile, and trifluoroacetic acid. In some specific embodiments, the first eluent comprises 30-80 parts of acetonitrile, 0.05-0.5 parts of trifluoroacetic acid, and the balance being water. In some specific embodiments, the first eluent comprises acetonitrile, water, and trifluoroacetic acid in a volume ratio of 1:1:0.002.

[0020] According to some embodiments of this disclosure, the second eluent comprises one or both of methanol and dichloromethane. In some specific embodiments of this disclosure, the second eluent comprises methanol and dichloromethane. In some specific embodiments, the second eluent comprises methanol and dichloromethane in a volume ratio of 10:1 to 10:20. In some specific embodiments, the second eluent comprises methanol and dichloromethane in a volume ratio of 10:1 to 10:10, for example, a volume ratio of methanol and dichloromethane of 5:1.

[0021] According to some embodiments of this disclosure, the method further includes: performing ultrasonic treatment after adding the elution solvent. Ultrasonic treatment can facilitate the elution of lipid and peptide components from the solid-phase extraction material. In some specific embodiments, the ultrasonic treatment can last from 0.5 to 20 minutes, for example, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 minutes. In some specific embodiments of this disclosure, the ultrasonic treatment time after adding the first elution solvent can be the same as or different from the ultrasonic treatment time after adding the second elution solvent. The ultrasonic treatment can be performed at room temperature.

[0022] According to some embodiments of this disclosure, after elution, the solid-phase extraction material can be separated from the eluent by applying an external magnetic field or centrifugation. The resulting eluent can be directly used for subsequent detection without further separation and purification.

[0023] In another aspect, this disclosure provides a kit for simultaneously detecting lipid and peptide components in a sample, the kit comprising: a solid-phase extraction material, which is a graphene oxide-iron oxide nanoparticle (GO-MNP) composite material obtained according to the preparation method of this disclosure; and an elution solvent.

[0024] According to some embodiments of this disclosure, the elution solvent includes: a first eluent for eluting the polypeptide component; and a second eluent for eluting the lipid component. The first eluent and the second eluent are as described above.

[0025] According to some embodiments of this disclosure, the kit further includes standard samples for generating a standard curve. The standard samples may be standard peptides or standard lipid molecules. In some specific embodiments, a standard curve can be plotted based on the MS signal-to-noise ratio of the standard sample (e.g., a standard peptide or standard lipid molecule) to an internal standard, and the concentration of the added standard sample. Based on the plotted standard curve, the concentration of the analyzed peptide or lipid component in the sample can be calculated. In other words, the detection method and kit of this disclosure allow for the quantitative determination of lipid and peptide components in a sample.

[0026] In some specific embodiments, the method for simultaneously detecting lipid and peptide components in a sample according to this disclosure can perform qualitative detection of lipids and peptide components. In some specific embodiments, the method for simultaneously detecting lipid and peptide components in a sample according to this disclosure can perform quantitative detection of lipids and peptide components.

[0027] In some specific embodiments, the method of this disclosure can use standard samples to draw standard curves for quantitative detection.

[0028] In some specific embodiments, the method of this disclosure can employ an internal standard method for quantitative detection. The internal standard used can be another standard sample with a structure similar to the analyte or an isotopically labeled molecule of the same kind. In some specific embodiments, the internal standard is pre-added to the sample. In some specific embodiments, the internal standard is pre-added to the standard sample. In some embodiments, the internal standard is added to the biological sample or standard sample solution before sample processing. The internal standard, on the one hand, helps in the quantitative analysis of the target molecule in the sample, and on the other hand, can explain the loss of the target analyte during the experiment. Because interfering substances have a matrix effect, they may reduce the ionization efficiency of the target analyte in mass spectrometry.

[0029] In some specific implementations, a standard curve is plotted based on the MS signal-to-noise ratio of the standard sample and the internal standard, as well as the concentration of the added standard sample, to calculate the concentration of the polypeptide or lipid molecules to be analyzed in the biological sample.

[0030] According to some embodiments of this disclosure, the kit may further include reagents and consumables for mass spectrometry detection; and / or isotope-labeled internal standards or other internal standards for quantitative detection.

[0031] The method disclosed herein for the simultaneous detection of peptide and lipid components can be completed in a short time (less than 1 hour for plasma samples) and can simultaneously obtain peptidomics and lipidomics data using a very small sample size (no more than 40 μL for plasma samples). It is low-cost, fast, easy to operate, and offers good analytical sensitivity and quantification, making it suitable for widespread application in clinical laboratory testing and basic clinical research.

[0032] Current lipidomics and peptidomics analysis workflows use two completely different detection methods, making it impossible to efficiently acquire data from both omics sources simultaneously from a single sample. These analyses are costly, time-consuming, complex, and result in significant sample loss, hindering high-throughput analysis. Furthermore, existing methods such as chromatography-mass spectrometry (GC-MS) require complex method establishment, optimization, and validation processes, demanding strong chromatographic and mass spectrometry backgrounds and skilled operators—requirements many clinical institutions lack. The method disclosed herein can simultaneously analyze lipid and peptide components using a small biological sample within a short workflow. Moreover, this method boasts a high degree of integration; the prepared sample is directly applicable to MALDI MS analysis without additional chromatographic separation and purification. Additionally, this method offers low detection cost, short processing time, ease of operation, and small sample consumption, making it suitable for high-throughput micro-analysis of large-scale clinical samples. With appropriate equipment configuration, this method is also well-suited for automated applications.

[0033] definition

[0034] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0035] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0036] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0037] The numerical range used in this article is intended to include all values ​​within that range. For example, the numerical range 5 to 10 can be understood as including 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or other values ​​within that range.

[0038] The "internal standard" used in this paper can be selected from different molecules with similar structures and close molecular weights to the target molecules being analyzed; alternatively, it can be selected from those containing, for example, [missing information]. 15 N, Deuterium (D) 13 Identical molecules of C and other isotopes are used as internal isotope standards. These heavy isotope-labeled compounds are well known in the art and are available from multiple suppliers.

[0039] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention. Attached Figure Description

[0040] Figure 1 Infrared spectra of a graphene oxide-magnetic nanoparticle composite (GO-MNP) according to one embodiment of the present disclosure and infrared spectra of original graphene oxide (GO). (a) Infrared spectrum of original GO; (b) Infrared spectrum of GO-MNP.

[0041] Figure 2 Scanning electron microscope (SEM) images of a graphene oxide-magnetic nanoparticle composite (GO-MNP) obtained according to one embodiment of this disclosure. (a), (b), and (c) show GO-MNP surface images observed at different magnifications. Arrows indicate micro / nano structures, such as nanopores and nanogrooves, resulting from graphene sheet folding and covalent bonding with magnetic nanoparticles.

[0042] Figure 3Scanning electron microscope images of graphene oxide-magnetic nanoparticle composites (GO-MNP) obtained according to one embodiment of the present disclosure using different GO and MNP mass ratios. (a) shows a surface image of GO-MNP obtained with a GO and MNP mass ratio of 1:0.5; (b) shows a surface image of GO-MNP obtained with a GO and MNP mass ratio of 1:3; and (c) shows a surface image of GO-MNP obtained with a GO and MNP mass ratio of 1:20.

[0043] Figure 4 This is a bar chart showing the number of peptide signals collected under different processing conditions according to one embodiment of the present disclosure. Different GO / MNP mass ratios (a) and different GO-MNP extraction concentrations (C) are compared. HWGO (b) and (c) bar charts showing the number of peptide signals acquired at different ultrasound times (mg / mL).

[0044] Figure 5 This is a distribution map of low molecular weight polypeptide molecules in a human plasma sample collected according to one embodiment of the present disclosure. GPD: glyceraldehyde-3-phosphate dehydrogenase; His: histone, H2B type 1-K; APO: apolipoprotein (CI); CLU: aggregate protein; NHCP: non-histone chromosomal protein, HMG-17; IMUP: immortalized upregulated protein; ITIH: α-trypsin inhibitor heavy chain, H4; C4B: complement, C4-B; FGA: fibrinogen α chain; and TTR: thyroxine transporter.

[0045] Figure 6 This is a positive ion mass spectrometry spectrum of lipid molecules in a human plasma sample collected according to one embodiment of this disclosure. PC: phosphatidylcholine; TG: triglycerides.

[0046] Figure 7 This is a negative ion mass spectrometry spectrum of lipid molecules in a human plasma sample collected according to one embodiment of this disclosure. PE: phosphatidylethanolamine; PI: phosphatidylinositol.

[0047] Figure 8 This is a distribution map of low molecular weight polypeptide molecules in a human urine sample collected according to one embodiment of the present disclosure.

[0048] Figure 9 This is a quantitative curve of two standard peptides (A-II and ACTH-F) added to a plasma sample, collected according to one embodiment of this disclosure. The linearity r is analyzed. 2 =0.99, reproducibility CV < 20%.

[0049] Figure 10This is a quantitative curve for two lipid molecules, phosphatidylcholine (PC 32:0) and triglycerides (TAG 42:0), collected in a plasma sample according to one embodiment of this disclosure. The linearity of the analysis is shown in the graph. 2 >0.99, and reproducibility CV <20%. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0051] Example 1

[0052] The preparation process of solid-phase extraction materials and the characterization of their chemical and microstructures are described. The steps are as follows:

[0053] Graphene oxide (GO) with a scale of 5–10 μm was dispersed in deionized water to a final concentration of 5 mg / mL. Magnesium tetroxide (MNP) nanoparticles with carboxyl functional groups modified on their surface were also dispersed in deionized water to a final concentration of 15 mg / mL. 100 μL of each GO and MNP solution were mixed in a 1:1 ratio (GO to MNP mass ratio 1:3). Then, 5 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) was added, and the mixture was incubated at 25 °C with shaking for 4 hours. After the reaction was complete, the obtained solid particles were washed three times with deionized water under the assistance of an external magnetic field to obtain GO-MNP.

[0054] The original GO and the prepared GO-MNP were analyzed using infrared spectroscopy. The obtained infrared spectra are shown below. Figure 1 As shown. Figure 1 The original GO atlas shows 1730 and 1620 cm. -1 The spectral peaks shifted to 1739 and 1631 cm⁻¹ in GO-MNP. -1 This indicates that the original carboxyl group on GO underwent a chemical reaction and was consumed. Simultaneously, the hydroxyl peak of GO was at 3398 cm⁻¹. -1 A significant decrease was also observed in GO-MNP, indicating that some hydroxyl groups were consumed due to dehydration condensation. Furthermore, a Fe-O bond-related peak (575 cm⁻¹) was also observed in GO-MNP. -1 The above results demonstrate that under the preparation conditions of this embodiment, graphene oxide and surface carboxyl-modified magnetic nanoparticles can undergo a dehydration condensation reaction to form a large-volume composite material with covalent bonds.

[0055] In addition, the surface microstructure of GO-MNP was analyzed using scanning electron microscopy (SEM). The results are as follows: Figure 2 As shown in the image, SEM images reveal numerous micro- and nanostructures on the surface of GO-MNP, resulting from graphene sheet folding and covalent bonding with magnetic nanoparticles, such as nanopores and nanogrooves. These structures effectively increase the material's surface area. Furthermore, the nanopore walls, composed of hydrophobic reduced graphene oxide, can effectively accommodate and adsorb smaller, hydrophobic targets such as peptides and lipids through capillary action, making GO-MNP an excellent nano-adsorbent material.

[0056] Example 2

[0057] Following the procedure of Example 1, three types of GO-MNPs were prepared using different GO:MNP mass ratios of 1:0.5, 1:3, and 1:20. The surface microstructure of the three GO-MNPs was analyzed using scanning electron microscopy (SEM), and the results are shown below. Figure 3 middle. Figure 3 (a) shows GO-MNP prepared with a GO to MNP mass ratio of 1:0.5. Figure 3 (b) shows GO-MNP prepared with a GO to MNP mass ratio of 1:3. Figure 3 (c) shows GO-MNP prepared with a GO to MNP mass ratio of 1:20. The results indicate that only when the GO to MNP mass ratio is suitable (e.g., 1:3) can a wide range of micro / nanostructures such as nanopores and nanogrooves be formed. Insufficient MNP ( Figure 3 a) or too little GO ( Figure 3 c) None of them can form a microstructure with good adsorption performance.

[0058] Example 3

[0059] This embodiment compares the peptide-omics data obtained from materials prepared using different GO / MNP mass ratios. The steps are as follows:

[0060] 1) Following the procedure in Example 1, four types of GO-MNP were prepared with GO and MNP mass ratios of 1:0.5, 1:1, 1:3 and 1:20, respectively.

[0061] 2) Prepare four EP tubes and add 30 μL of the same plasma sample to each. Then add the four different GO-MNPs prepared in step 1) to each of the four tubes, with a final concentration of 2.5 mg / mL. Incubate at 25°C for 30 minutes with gentle shaking. Separate the GO-MNPs under an external magnetic field and discard the remaining plasma.

[0062] 3) Add 30 μL of deionized water to each of the four GO-MNPs, disperse the GO-MNPs with a pipette, separate the GO-MNPs under the action of an external magnetic field, and discard the remaining water. Repeat this process once.

[0063] 4) Add 10 μL of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution to each of the four GO-MNPs, disperse them, sonicate for 2 minutes, separate the GO-MNPs under the action of an external magnetic field, and transfer the acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution into four new EP tubes.

[0064] 5) Take 1.5 μL of the acetonitrile / water / trifluoroacetic acid eluent obtained in step 4), mix it with 1.5 μL of 2,5-dihydroxybenzoic acid [DHB, solvent is a mixed solution of acetonitrile / water / trifluoroacetic acid (volume ratio 1:1:0.002), DHB concentration 10 mg / mL], and spot 1.5 μL of the solution onto the surface of a stainless steel target plate. Peptide molecules in the m / z range of 850–5000 were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. The laser intensity for MALDI mass spectrometry was set to 100–120 using the instrument's built-in software, accumulated in cation mode, and averaged for 400 bombardments. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are as follows: Figure 4 As shown in (a).

[0065] The results showed that the highest number of highly sensitive peptide signals could be collected when the mass ratio of GO to MNP was 1:3. This indicates that the enrichment effect of the material is best when there is an abundance of micro- and nanostructures such as nanopores and nanogrooves; too little MNP or GO may affect the extraction effect.

[0066] Example 4

[0067] This embodiment compares the omics data obtained using different concentrations of solid-phase extraction materials, and the feasibility of the method disclosed herein for peptide omics analysis of plasma. The steps are as follows:

[0068] 1) Add 30 μL of the same plasma sample to each of six EP tubes, and then add 1.0, 2.5, 5.0, 7.5, and 10 mg / mL of GO-MNP to each of the six tubes respectively. Incubate at 25°C for 30 minutes with gentle shaking. Separate the GO-MNP under an external magnetic field and discard the remaining plasma.

[0069] 2) Add 30 μL of deionized water to each of the 6 tubes containing GO-MNP, disperse the GO-MNP with a pipette, separate the GO-MNP under the action of an external magnetic field, and discard the remaining water. Repeat this process once.

[0070] 3) Add 10 μL of a mixed solution of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) to each of the 6 tubes containing GO-MNP. After dispersion, sonicate for 2 minutes and separate the GO-MNP under the action of an external magnetic field. Transfer the mixed solution of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) into 6 new EP tubes.

[0071] 4) Take 1.5 μL of the acetonitrile / water / trifluoroacetic acid eluent obtained in step 3), mix it with 1.5 μL of 2,5-dihydroxybenzoic acid [DHB, solvent is a mixed solution of acetonitrile / water / trifluoroacetic acid (volume ratio 1:1:0.002), DHB concentration 10 mg / mL], and spot 1.5 μL of the solution onto the surface of a stainless steel target plate. Peptide molecules in the m / z range of 850–5000 were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI mass spectrometry was performed by accumulating and averaging 400 bombardments in cation mode with a laser intensity of 100–120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are shown below. Figure 4 As shown in (b).

[0072] The results showed that different concentrations of solid-phase extraction materials could all obtain good peptidomics results, and the number of peptide signals obtained was similar. The number of peptide signals obtained was relatively the highest when the GO-MNP concentration was 2.5 mg / mL.

[0073] Example 5

[0074] This embodiment compares the omics data obtained under different ultrasonic elution time conditions, and demonstrates the feasibility of the method disclosed herein for plasma peptidomics analysis. The steps are as follows:

[0075] 1) Add 30 μL of the same plasma sample to each of six EP tubes. Then add 2.5 mg / mL of GO-MNP to each of the six tubes and incubate at 25°C for 30 minutes with gentle shaking. Separate the GO-MNP under an external magnetic field and discard the remaining plasma.

[0076] 2) Add 30 μL of deionized water to each of the 6 tubes containing GO-MNP, disperse the GO-MNP with a pipette, separate the GO-MNP under the action of an external magnetic field, and discard the remaining water. Repeat this process once.

[0077] 3) Add 10 μL of a mixed solution of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) to each of the six EP tubes containing GO-MNP. After dispersion, sonicate the six EP tubes for 0.5, 2, 5, 10, and 20 minutes, respectively. Then, separate the GO-MNP under an external magnetic field and transfer the acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution into six new EP tubes.

[0078] 4) Take 1.5 μL of the acetonitrile / water / trifluoroacetic acid eluent obtained in step 3), mix it with 1.5 μL of 2,5-dihydroxybenzoic acid [DHB, solvent is a mixed solution of acetonitrile / water / trifluoroacetic acid (volume ratio 1:1:0.002), DHB concentration 10 mg / mL], and spot 1.5 μL of the solution onto the surface of a stainless steel target plate. Peptide molecules in the m / z range of 850–5000 were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI mass spectrometry was performed by accumulating and averaging 400 bombardments in cation mode with a laser intensity of 100–120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are shown below. Figure 4 As shown in (c).

[0079] The results showed that the method disclosed herein could acquire certain peptide omics signals under different ultrasound time conditions, with the highest number of peptide signals being acquired when the ultrasound time was 2 minutes.

[0080] Example 6

[0081] This embodiment verifies the feasibility of using the method of this disclosure to simultaneously analyze polypeptide and lipid components in human plasma samples. The steps are as follows:

[0082] 1) Take 30 μL of plasma sample, add 2.5 mg / mL of GO-MNP, and incubate at 25°C for 30 minutes with gentle shaking. Separate GO-MNP under an external magnetic field and discard the remaining plasma.

[0083] 2) Add 30 μL of deionized water to the GO-MNP, disperse the GO-MNP with a pipette, separate the GO-MNP under the action of an external magnetic field, and discard the remaining water. Repeat this process once.

[0084] 3) Add 10 μL of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution to GO-MNP, disperse and sonicate for 2 minutes. Separate GO-MNP under the action of an external magnetic field and transfer the acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution into a new EP tube.

[0085] 4) Add 10 μL of methanol / dichloromethane (5:1, v / v) mixed solution to the GO-MNP, disperse and sonicate for 8 minutes. Separate the GO-MNP under the action of an external magnetic field and transfer the methanol / dichloromethane (5:1, v / v) mixed solution into a new EP tube.

[0086] 5) Take 1.5 μL of the acetonitrile / water / trifluoroacetic acid eluent obtained in step 3), mix it with 1.5 μL of 2,5-dihydroxybenzoic acid [DHB, solvent is a mixed solution of acetonitrile / water / trifluoroacetic acid (volume ratio 1:1:0.002), DHB concentration 10 mg / mL], and spot 1.5 μL of the solution onto the surface of a stainless steel target plate. Peptide molecules in the m / z range of 850–5000 were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI mass spectrometry was performed by accumulating and averaging 400 bombardments in cation mode with a laser intensity of 100–120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are as follows: Figure 5 As shown. In Figure 5 The spectrum shown identifies some of the stronger peaks and their source proteins. This result demonstrates that the analytical method of this invention can effectively obtain peptidomics information from human plasma samples, and that the method can be used for peptidomics or proteomics analysis in complex samples.

[0087] 6) Spot 1.5 μL of the methanol / dichloromethane eluent obtained in step 4) onto the surface of a stainless steel target plate. After completely drying at room temperature, add 1.5 μL of 2,5-dihydroxybenzoic acid [DHB, solvent: acetonitrile / water / trifluoroacetic acid (volume ratio 1:1:0.002), DHB concentration 10 mg / mL] to the sample spot and mix. Analyze lipid molecules in the m / z range of 500-1000 using MALDI-MS. Identification was performed using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI MS mass spectra were acquired in cation mode, and spectral acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). Results are as follows: Figure 6 As shown. Figure 6 The results show that the method disclosed herein can obtain relatively complete information on lipid molecules and can analyze lipid molecules such as phosphatidylcholine (PC) and triglycerides (TG) in plasma samples with high sensitivity, thus proving that the method disclosed herein can be used for lipidomics analysis in complex samples.

[0088] 7) Spot 1.5 μL of the methanol / dichloromethane eluent obtained in step 4) onto the surface of a stainless steel target plate. After complete drying at room temperature, add 1.5 μL of 9-aminoacridine [9-AA, dissolved in isopropanol / acetonitrile (1:1, v / v), 10 mg / mL] to the sample and mix. Lipid molecules in the m / z range of 500-1000 were analyzed by MALDI-MS using a SHIMADZUAXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI MS mass spectra were acquired in anion mode, and the spectra were acquired and processed using Shimadzu Biotech Launchpad software (version 2.9). The results are as follows: Figure 7 As shown. Figure 7 The results show that the method disclosed herein can effectively obtain information on lipid molecules such as phosphatidylethanolamine (PE) and phosphatidylinositol (PI) in human plasma samples, and thus can be used for lipidomics analysis in complex samples.

[0089] Figure 6 and Figure 7 This demonstrates that the method disclosed herein can conveniently and stably acquire peptidomics and lipidomics data from a very small sample volume (e.g., 30 μL) in a very short time (total time < 1 hour), with high signal-to-noise ratio and good detection sensitivity. More than 150 peptide signals can be collected from this sample; simultaneously, 51 cationic lipid signals and 23 anionic lipid signals can be collected.

[0090] Example 7

[0091] This embodiment verifies the feasibility of using the method disclosed herein for peptidomics analysis of human urine samples. The steps are as follows:

[0092] 1) Take 50 μL of urine sample, add 6.0 mg / mL of GO-MNP, and incubate at 25°C for 30 minutes with gentle shaking. Separate the GO-MNP under an external magnetic field and discard the remaining sample.

[0093] 2) Add 50 μL of deionized water to the GO-MNP, disperse the GO-MNP with a pipette, separate the GO-MNP under the action of an external magnetic field, and discard the remaining water. Repeat this process once.

[0094] 3) Add 10 μL of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution to GO-MNP, disperse and sonicate for 2 minutes. Separate GO-MNP under the action of an external magnetic field and transfer the acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution into a new EP tube.

[0095] 4) Take 1.5 μL of the acetonitrile / water / trifluoroacetic acid eluent obtained in step 3), mix it with 1.5 μL of 2,5-dihydroxybenzoic acid [DHB, solvent is a mixed solution of acetonitrile / water / trifluoroacetic acid (volume ratio 1:1:0.002), DHB concentration 10 mg / mL], and spot 1.5 μL of the solution onto the surface of a stainless steel target plate. Peptide molecules in the m / z range of 850–5000 were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI mass spectrometry was performed by accumulating and averaging 400 bombardments in cation mode with a laser intensity of 100–120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). The results are shown below. Figure 8 As shown.

[0096] Figure 8 The results show that the method disclosed herein can conveniently and stably obtain urinary peptide data from a very small volume sample in a very short time (total time <1 hour) (signals of 57 peptides were detected in the range of m / z 1000 to 3000), and the signal-to-noise ratio is high and the detection sensitivity is good.

[0097] Example 8

[0098] This embodiment verifies the feasibility of the method disclosed herein for quantitative analysis of standard-added polypeptide molecules in human plasma samples. The steps are as follows:

[0099] 1) A synthetic standard peptide P14R (molecular weight 1532.9) was added as an internal standard to a mixed plasma matrix to a final concentration of 50 nM. Then, two standard peptides at different concentrations were added to the plasma matrix: angiotensin II (A-II, molecular weight 1045.5) and adrenocorticotropic hormone (ACTH) fragment 18-39 (ACTH-F, molecular weight 2464.2). The concentration range of both standard peptides was 1–100 nM. 30 μL of the plasma sample with the above standard added was taken, and 2.5 mg / mL of GO-MNP was added. The mixture was incubated at 25°C for 30 minutes with gentle shaking. GO-MNP was separated under an external magnetic field, and the remaining plasma was discarded.

[0100] 2) Add 30 μL of deionized water to the GO-MNP, disperse the GO-MNP with a pipette, separate the GO-MNP under the action of an external magnetic field, and discard the remaining water. Repeat this process once.

[0101] 3) Add 10 μL of acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution to GO-MNP, disperse and sonicate for 2 minutes. Separate GO-MNP under the action of an external magnetic field and transfer the acetonitrile / water / trifluoroacetic acid (1:1:0.002, v / v / v) mixed solution into a new EP tube.

[0102] 4) Take 1.5 μL of the acetonitrile / water / trifluoroacetic acid eluent obtained in step 3), mix it with 1.5 μL of 2,5-dihydroxybenzoic acid [DHB, solvent is a mixed solution of acetonitrile / water / trifluoroacetic acid (volume ratio 1:1:0.002), DHB concentration 10 mg / mL], and spot 1.5 μL of the solution onto the surface of a stainless steel target plate. Peptide molecules in the m / z range of 850–5000 were analyzed by MALDI-MS using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer in reflectance mode. MALDI mass spectrometry was performed by accumulating and averaging 400 bombardments in cation mode with a laser intensity of 100–120. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). A quantitative standard curve of the standard peptide was plotted with the ratio of the target peptide signal to the internal standard signal as the ordinate and the actual concentration of the target peptide as the abscissa. The results are shown below. Figure 9 As shown.

[0103] The results show that, using the method disclosed herein, ideal peptide standard curves can be obtained with the addition of an internal standard. The linearity r of all peptide standard curves is... 2All values ​​were around 0.99, and the response values ​​at each concentration showed good reproducibility, with CV% all below 25%, meeting the requirements for quantitative analysis. Based on the standard curves of the obtained standard peptides, the recoveries of standard peptide A-II at 50.3, 12.2, and 6.2 nM were 100.5%, 97.9%, and 100.0%, respectively, and the recoveries of standard peptide ACTH-F at 50.3, 12.2, and 6.2 nM were 111.1%, 90.3%, and 105.5%, respectively, meeting the analytical requirements.

[0104] Example 9

[0105] This embodiment verifies the feasibility of the method disclosed herein for quantitative analysis of standard-added lipid molecules in human plasma samples. The steps are as follows:

[0106] 1) Phosphatidylcholine 28:0 (PC 28:0, m / z 734.5) was added as an internal standard to a mixed plasma matrix, with a final concentration of 200 μg / mL. Then, two different concentrations of lipids were added to the plasma matrix as standards: phosphatidylcholine 32:0 (PC32:0, m / z 756.6) and triglycerides 42:0 (TAG 42:0, m / z 745.6). The concentration ranges of these two lipids were 15–500 μg / mL and 30–1000 μg / mL, respectively. 30 μL of the plasma sample containing the above standards was taken, and 2.5 mg / mL of GO-MNP was added. The mixture was incubated at 25°C for 30 minutes with gentle shaking. GO-MNP was separated under an external magnetic field, and the remaining plasma was discarded.

[0107] 2) Add 30 μL of deionized water to the GO-MNP, disperse the GO-MNP with a pipette, separate the GO-MNP under the action of an external magnetic field, and discard the remaining water. Repeat this process once.

[0108] 3) Add 10 μL of methanol / dichloromethane (5:1, v / v) mixed solution to the GO-MNP, disperse and sonicate for 8 minutes. Separate the GO-MNP under the action of an external magnetic field and transfer the methanol / dichloromethane (5:1, v / v) mixed solution into a new EP tube.

[0109] 4) Spot 1.5 μL of the methanol / dichloromethane eluent obtained in step 3) onto the surface of a stainless steel target plate. After completely drying at room temperature, add 1.5 μL of 2,5-dihydroxybenzoic acid [DHB, solvent: acetonitrile / water / trifluoroacetic acid (volume ratio 1:1:0.002), DHB concentration 10 mg / mL] to the sample spot and mix. Analyze lipid molecules in the m / z range of 500-1000 using MALDI-MS. A SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer was used, acquiring MALDI MS mass spectra in reflectance and cation modes. Spectrum acquisition and processing were performed using Shimadzu Biotech Launchpad software (version 2.9). A quantitative standard curve for lipid molecules was plotted with the ratio of lipid signal to internal standard signal as the ordinate and the actual concentration of added lipids as the abscissa. The results are shown below. Figure 10 As shown.

[0110] The results show that, using the method disclosed herein, ideal standard curves for specific lipids can be obtained with the addition of an internal standard. The r values ​​of all lipid standard curves are... 2 All values ​​were above 0.99, and the response values ​​at each concentration showed good reproducibility, with CV% all below 25%, meeting the requirements for quantitative analysis. Based on the standard curve, the recoveries of the two lipids at different concentrations ranged from 85% to 110%, satisfying the analytical requirements.

[0111] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for simultaneously detecting lipid and polypeptide components in a sample, characterized in that, The method includes the following steps: Solid-phase extraction materials are added to the sample to enrich the lipid and peptide components in the sample; The lipid and polypeptide components are eluted from the solid-phase extraction material using an elution solvent. The eluted lipid and peptide components were detected using matrix-assisted laser desorption / ionization mass spectrometry. The solid-phase extraction material is a graphene oxide-iron oxide nanoparticle composite material. The method for preparing the graphene oxide-iron tetroxide nanoparticle composite material includes: Graphene oxide was dispersed in water to obtain the first dispersion; Ferric oxide nanoparticles were dispersed in water to obtain a second dispersion; The first dispersion and the second dispersion were mixed, and a dehydrating agent was added to carry out a dehydration condensation reaction to obtain a graphene oxide-iron tetroxide nanoparticle composite material. The mass ratio of the graphene oxide to the iron oxide nanoparticles is 1:1 to 1:

20.

2. The method according to claim 1, characterized in that, The dehydrating agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; or The mass ratio of the graphene oxide to the iron oxide nanoparticles is 1:

3.

3. The method according to claim 1, characterized in that, The graphene oxide is in the form of a powder or sheet; and / or, the graphene oxide has hydroxyl and / or carboxyl functional groups on its surface; and / or, the surface of the iron oxide nanoparticles has carboxyl functional groups.

4. The method according to claim 3, characterized in that, The graphene oxide has a size of 1–50 μm; The size of the iron oxide nanoparticles is 20–200 nm.

5. The method according to claim 4, characterized in that, The graphene oxide has a size of 5–10 μm.

6. The method according to any one of claims 1 to 5, characterized in that, The sample is a liquid sample.

7. The method according to claim 6, characterized in that, The sample is a biological sample.

8. The method according to claim 7, characterized in that, The samples are blood, serum, plasma, urine, cerebrospinal fluid, or tissue cell extracts.

9. The method according to any one of claims 1 to 5, characterized in that, The concentration of the solid-phase extraction material in the sample is 1.0–10.0 mg / mL.

10. The method according to any one of claims 1 to 5, characterized in that, The step of eluting the lipid and polypeptide components from the solid-phase extraction material using an elution solvent includes: eluting the polypeptide components using a first elution solvent; and / or eluting the lipid components using a second elution solvent.

11. The method according to claim 10, characterized in that, The first elution solvent includes one or more of water, acetonitrile, and trifluoroacetic acid.

12. The method according to claim 11, characterized in that, The first elution solvent includes: 30-80 parts acetonitrile, 0.05-0.5 parts trifluoroacetic acid, and the balance water.

13. The method according to claim 10, characterized in that, The second elution solvent includes one or both of methanol and dichloromethane.

14. The method according to claim 13, characterized in that, The second elution solvent comprises methanol and dichloromethane in a volume ratio of 10:1 to 10:

20.

15. The method according to any one of claims 1 to 5, characterized in that, The method further includes: performing ultrasonic treatment after adding the elution solvent.

16. The method according to claim 15, characterized in that, The ultrasonic treatment lasts for 0.5 to 20 minutes.

17. The method according to claim 16, characterized in that, The ultrasonic treatment lasts for 0.5 to 5.0 minutes.

18. A kit for simultaneously detecting lipid and peptide components in a sample, characterized in that, The kit includes: Solid-phase extraction material, which is a composite material of graphene oxide and iron oxide nanoparticles; and Elution solvent; The method for preparing the graphene oxide-iron tetroxide nanoparticle composite material includes: Graphene oxide was dispersed in water to obtain the first dispersion; Ferric oxide nanoparticles were dispersed in water to obtain a second dispersion; The first dispersion and the second dispersion were mixed, and a dehydrating agent was added to carry out a dehydration condensation reaction to obtain a graphene oxide-iron tetroxide nanoparticle composite material. The mass ratio of the graphene oxide to the iron oxide nanoparticles is 1:1 to 1:

20.

19. The reagent kit according to claim 18, characterized in that, The dehydrating agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; or The mass ratio of the graphene oxide to the iron oxide nanoparticles is 1:

3.

20. The reagent kit according to claim 18, characterized in that, The graphene oxide is in the form of a powder or sheet; and / or, the graphene oxide has hydroxyl and / or carboxyl functional groups on its surface; and / or, the surface of the iron oxide nanoparticles has carboxyl functional groups.

21. The reagent kit according to claim 20, characterized in that, The graphene oxide has a size of 1–50 μm; The size of the iron oxide nanoparticles is 20–200 nm.

22. The kit according to claim 21, characterized in that, The graphene oxide has a size of 5–10 μm.

23. The kit according to any one of claims 18 to 22, characterized in that, The elution solvent includes: A first elution solvent used to elute the polypeptide components; and A second elution solvent is used to elute the lipid components.

24. The reagent kit according to claim 23, characterized in that, The first elution solvent includes one or more of water, acetonitrile, and trifluoroacetic acid.

25. The kit according to claim 24, characterized in that, The first elution solvent includes: 30-80 parts acetonitrile, 0.05-0.5 parts trifluoroacetic acid, and the balance water.

26. The reagent kit according to claim 23, characterized in that, The second elution solvent includes one or both of methanol and dichloromethane.

27. The reagent kit according to claim 26, characterized in that, The second elution solvent comprises methanol and dichloromethane in a volume ratio of 10:1 to 10:

20.

28. The kit according to any one of claims 18 to 22, characterized in that, The kit further includes standard samples for creating a standard curve.

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