A single-sample multi-omics component extraction method for multi-omics joint analysis

Abstract

This invention discloses a single-sample multi-omics component extraction method for multi-omics joint analysis. The method includes: adding nucleic acid extraction solution to the sample, transferring it to a DNA adsorption column, centrifuging, and collecting the filtrate; washing the DNA adsorption column; eluting DNA; adding ethanol to the filtrate, transferring it to an RNA adsorption column, centrifuging, and collecting the filtrate; washing the RNA adsorption column; eluting RNA; and extracting proteins. This method integrates centrifugation column-based DNA / RNA co-extraction technology, a zinc ion-mediated protein precipitation and capture strategy, and an efficient Fe-IMAC phosphorylated peptide enrichment process. It achieves efficient protein precipitation and in-situ washing, thoroughly removing chemical components that interfere with mass spectrometry detection. It simultaneously achieves high-quality nucleic acid extraction and deep proteomics and phosphorylated proteomics coverage in a single sample, effectively solving the bottlenecks of traditional methods such as limited sample size, complex procedures, and poor reproducibility.

Description

Technical Field

[0001] This invention belongs to the fields of proteomics and genomics, specifically relating to a method for extracting multi-omics components from a single sample for multi-omics joint analysis. Background Technology

[0002] The development of systems biology and precision medicine increasingly relies on multidimensional integrated analysis of the genome, transcriptome, proteome, and post-translational modifications (PTM) to achieve a systematic understanding of biological processes and disease mechanisms. Compared to separate assays from different samples, simultaneously acquiring multi-omics information from the same biological sample can significantly reduce systematic errors introduced by sample heterogeneity and improve the comparability and biological correlation between different omics levels, becoming an important development direction in current translational medicine and clinical research. However, in application scenarios such as clinical biopsy samples, laser microdissection (LCM) tissues, or rare cell populations, sample materials are usually extremely limited, severely restricting the routine application of multi-omics analysis methods.

[0003] Existing multi-component extraction methods for single samples mainly fall into two categories. One category is classic methods based on the phenol / chloroform system (such as TRIzol and RNA-Bee), which can achieve sequential separation of DNA, RNA, and proteins. However, these methods generally suffer from low protein recovery efficiency, demanding dissolution and enzymatic digestion conditions, and interference from phenol residues in mass spectrometry detection, making it difficult to meet the sample quality and reproducibility requirements of deep proteomics and PTM analysis. Although some studies have attempted to optimize these procedures and apply them to biopsy-grade samples, their core still relies on organic phase separation mechanisms, making the operation complex, reproducibility limited, and standardization and automation difficult to achieve.

[0004] Another type of method, based on phenol-free nucleic acid extraction kits, can efficiently obtain high-quality DNA and RNA, and is widely used in clinical and research applications. However, this type of method is designed to prioritize nucleic acid binding and elution, and the resulting effluent is usually rich in high concentrations of ionizing salts (such as guanidine isothiocyanate), strong denaturants, and surfactants. Because the aforementioned chemical environment severely inhibits protease activity, interferes with mass spectrometry ionization, and leads to instability in protein post-translational modifications, this effluent is generally considered "waste liquid" unsuitable for proteomics analysis and is discarded directly in current technologies.

[0005] However, the effluent after nucleic acid extraction actually retains the vast majority of the full-spectrum protein information in the sample. The key challenge is not insufficient protein content, but rather the lack of a protein capture and transformation method suitable for this high-salt, high-denaturation, and multi-interference system. Existing protein precipitation, magnetic bead capture, or sample cleaning techniques are mostly developed for relatively simple and controllable protein solution systems. When directly applied to the nucleic acid extraction residue system, they often lead to significant protein loss, PTM modification detachment, or excessive levels of residual interferences in the sample, thus making reliable proteomics analysis impossible under micro-sample conditions. Summary of the Invention

[0006] The purpose of this invention is to provide a reliable proteomics analysis method based on a nucleic acid extraction residue system to address the above-mentioned technical problems.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for extracting multi-omics components from a single sample for multi-omics joint analysis includes the following steps: S1, DNA extraction S11. Add nucleic acid extraction solution to the sample to be tested; S12. Transfer all the solutions in the S11 system to the DNA adsorption column CS23, centrifuge at 12000 rcf for 30-60 seconds, and collect the filtrate. S13. Wash the DNA adsorption column with buffer GD; S14. Wash the DNA adsorption column with PW rinse buffer and repeat once; S15. Place the DNA adsorption column at room temperature to allow the residual washing solution in the adsorption material to dry completely. S16. Elute the DNA-adsorbed column with elution buffer TB to obtain a DNA solution.

[0008] S2, RNA extraction S21. Add 1 volume of 70% v / v ethanol to the filtrate collected in S12, mix well, transfer to an RNA adsorption column, centrifuge at 12000 rcf for 30-60 seconds, and collect the filtrate. S22. Wash the RNA adsorption column with protein-removing buffer RW1; S23. Wash the RNA adsorption column with rinse buffer RW, and repeat once; S24. Place the RNA adsorption column at room temperature to allow the residual washing solution in the adsorption material to dry completely; S25. Elute the RNA adsorption column with RNase-Free dd H2O to obtain an RNA solution.

[0009] S3, Protein Extraction S31. Add ZASP precipitant to the filtrate collected in S21 to precipitate proteins; S32. Use detergent to clean protein deposits; S33. Resuspend the protein precipitate using 1% SDC lysis buffer and then sonicate it. S34. Heating the S33 system sample to reduce and alkylate the protein; S35. Add trypsin to the sample in system S34 to obtain the enzymatically digested sample; S36. Add the protease hydrolysis reaction terminator to the sample in system S35, then centrifuge and collect the supernatant.

[0010] Preferably, the method further includes the following steps: S4, peptide desalting S41. Activate and equilibrate the peptide desalting column using methanol, desalting buffer B, and desalting buffer A, respectively. S42. Add the supernatant collected in S36 to the peptide desalting column; S43. Wash the peptide desalting column with desalting buffer A, repeat once; S44. Elute the peptide desalting column with desalting buffer B to obtain a peptide solution; S45. A portion of the peptide solution from S44 was concentrated and evaporated to dryness under vacuum at 45 °C to obtain purified peptides.

[0011] Preferably, the method further includes the following steps: S5, phosphorylated peptide enrichment S51. Activate and equilibrate the phosphopeptide enrichment column using methanol and phosphopeptide enrichment buffer A; S52. Add a portion of the peptide solution from S44 to the phosphopeptide enrichment column; S53. Wash the phosphopeptide enrichment column with phosphopeptide enrichment buffer A; S54. Elute the phosphopeptide enrichment column with phosphopeptide enrichment buffer B to obtain a phosphopeptide solution; The phosphopeptide was concentrated and evaporated to dryness under vacuum at 55.45 °C to obtain purified phosphopeptide.

[0012] In the above method steps, both DNA and RNA extraction were performed using a DNA / RNA co-extraction kit, and the samples to be tested could be selected from cell samples or fresh frozen tissue samples.

[0013] In step S11 of the above method, if the sample to be tested is a cell sample, it is recommended to use 1×10⁻⁶ cells / mL. 5 Up to 1×10 7For each cell, add 100-600 μL of nucleic acid extraction solution; for tissue samples, it is recommended to use 10-20 mg of tissue sample and add 350-600 μL of nucleic acid extraction solution.

[0014] In step S11 of the above method, β-mercaptoethanol is added to the nucleic acid extraction solution to a final concentration of 1% before the operation, such as adding 10 μL of β-mercaptoethanol to 1 mL of nucleic acid extraction solution.

[0015] In step S13 of the above method, the components of buffer GD are: 4 M guanidine hydrochloride and 30% v / v ethanol solution.

[0016] In step S14 of the above method, the rinsing solution PW consists of 10 mM Tris-HCl, 10 mM NaCl, and 70% v / v ethanol solution.

[0017] In step S16 of the above method, the elution buffer TB consists of 10 mM Tris-HCl buffer.

[0018] In steps S12 and S21 of the above method, the centrifugation conditions are: 12000 rcf for 30-60 seconds.

[0019] In step S22 of the above method, the components of the protein removal solution RW1 are: 2 M guanidine isothiocyanate and 20% v / v ethanol solution.

[0020] In step S23 of the above method, the rinsing solution RW consists of 10 mM Tris-HCl and 80% v / v ethanol solution.

[0021] In step S31 of the above method, the ZASP precipitant is: 200 mM ZnCl2, 99.9% v / v methanol and 0.1% v / v formic acid.

[0022] In step S31 of the above method, the detergent consists of 95% ACN (v / v%), 0.1% FA (v / v%), and the remainder is water.

[0023] In step S33 of the above method, the 1% SDC lysis buffer is a mixed aqueous solution containing the following final concentrations of solute: 1% SDC (w / w), 100 mM Tris-HCl at pH 8.5, 40 mM TCEP-HCl, and 10 mM CAA. SDC is sodium deoxycholate; TCEP-HCl is tris(2-carboxyethyl)phosphonic acid hydrochloride; and CAA is 2-chloroacetamide.

[0024] In step S33 of the above method, preferably, the parameters of the non-contact ultrasonic crusher are set as follows: 1 min, 85% energy, 4℃, intermittent ultrasound (20 s ON / 20 s OFF).

[0025] In step S35 of the above method, the amount of trypsin is added according to a protein to trypsin mass ratio of 25:1.

[0026] In step S36 of the above method, the protease hydrolysis reaction terminator is pure formic acid; the amount of the protease hydrolysis reaction terminator is added according to a volume ratio of 100:1 between the enzymatic hydrolysate and the protease hydrolysis reaction terminator.

[0027] In step S36 of the above method, the centrifugation conditions are: 14000 rcf, centrifugation at 4℃ for 10 min.

[0028] In step S41 of the above method, the desalting buffer A can be a 0.1% (V / V%) aqueous solution of formic acid.

[0029] In step S41 of the above method, the desalting buffer B may be composed of the following substances in volume percentage: 80% ACN (acetonitrile), 0.1% TFA (trifluoroacetic acid), and purified water.

[0030] In step S51 of the above method, the phosphopeptide enrichment buffer A may be composed of the following substances in volume percentage: 80% ACN, 0.1% TFA and purified water.

[0031] In step S53 of the above method, the phosphopeptide enrichment buffer B may be composed of the following substances in volume percentage: 80% ACN, 3% ammonia water (concentration of 14.82 M) and purified water.

[0032] In the above method, the peptide desalting column is composed of several layers of C18 solid-phase extraction membrane filled in a 200 μL pipette tip.

[0033] In the above method, the phosphopeptide enrichment column consists of a 200 μL pipette tip filled with a single layer of C8 solid-phase extraction membrane and 10 μL of phosphopeptide enrichment material.

[0034] Preferably, the phosphopeptide enrichment material is PureCube Fe-NTA Agarose (CubeBiotech, catalog number 31403-Fe).

[0035] Preferably, the DNA, RNA, peptides, and phosphorylated peptides prepared by the method of the present invention can be directly applied to whole genome sequencing, transcriptome sequencing, proteomics, and phosphorylated proteomics analysis.

[0036] Preferably, the method for proteomics and phosphorylated proteomics analysis is LC-MS / MS.

[0037] To enable in-depth proteomics analysis from nucleic acid extraction residues, this invention proposes a single-sample multi-omics sample preparation method for nucleic acid extraction residues. This method uses the high-salt eluent after nucleic acid extraction via centrifugation column as the starting material and constructs an integrated multi-omics analysis workflow by introducing a zinc ion-mediated specific precipitation strategy and Fe-IMAC phosphorylation enrichment technology. This method abandons the traditional "divide and conquer" multi-omics strategy, significantly reducing sample requirements and shortening pretreatment time. This invention uses 1×10⁻⁶ samples. 4 Up to 5×10 6 The sensitivity of this method was tested using HEK 293T cells. This method, without the use of high-risk organic reagents such as phenol, chloroform, and acetone, achieves efficient protein capture, simultaneous removal of interfering components, and rapid conversion of proteins to a mass spectrometry-compatible state through systematic analysis of the complex chemical environment after nucleic acid extraction. This allows for the continuous acquisition of high-quality DNA, RNA, and proteomic data from the same sample source. The method can be performed at room temperature within a short time, and the depth of proteomic and phosphorylated proteomic identification obtained is comparable to that of in-solution enzymatic digestion methods.

[0038] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention innovatively utilizes zinc ion-mediated protein capture technology to successfully solve the problem of high salt content and interfering substances coexisting in nucleic acid extraction effluent, seamlessly integrating centrifugal column-based DNA / RNA extraction, effluent proteomics analysis, and Fe-IMAC phosphorylation enrichment. This process enables the simultaneous acquisition of genomic, transcriptomic, proteomic, and phosphorylated proteomic data in a single sample. 2. This invention achieves proteomic and phosphorylated proteomic identification depths comparable to traditional in-solution enzymatic digestion methods while ensuring efficient extraction of DNA and RNA, indicating that this technology successfully utilizes the residual nucleic acid extraction system without sacrificing data quality. 3. This invention can be applied to applications as low as 1×10⁻⁶. 5 Deep multi-omics joint analysis was achieved using HEK 293T cells, providing a feasible technical solution for systematic research on rare cell populations, clinical puncture biopsies, and other trace and precious samples; 4. This invention is applicable to a variety of different fresh frozen tissue samples, demonstrating good robustness and universality. Attached Figure Description

[0039] Figure 1 This is a flowchart illustrating the operational steps of the present invention.

[0040] Figure 2 A comparison diagram showing the identification of proteins, phosphopeptides, phosphopeptides, and phosphate sites after processing a starting amount of 200 μg of protein using the traditional in-solution enzymatic digestion process and the process of this invention.

[0041] Figure 3 Venn diagrams showing the proteins and phosphate sites identified by processing a starting amount of 200 μg of protein using both the traditional in-solution enzymatic digestion process and the process of this invention.

[0042] Figure 4 The method of this invention and Comparative Example 2 (TRIzol method) treated 3×10 6 A comparison of the number of proteins and peptides identified after processing HEK 293T cells.

[0043] Figure 5 The method of this invention and Comparative Example 2 (TRIzol method) were used to treat 3 × 10 6 Distribution of quantitative coefficient of variation (CV) of proteins identified in HEK 293T cells.

[0044] Figure 6 The process of this invention processes 1×10 4 Up to 5×10 6 DNA yield identified in HEK 293T cells.

[0045] Figure 7 The process of this invention processes 1×10 4 Up to 5×10 6 RNA production identified in HEK 293T cells.

[0046] Figure 8 The process of this invention processes 1×10 4 Up to 5×10 6 The number of proteins and peptides identified in each HEK 293T cell.

[0047] Figure 9 The process of this invention processes 1×10 4 Up to 5×10 6 The number of phosphoproteins, phosphopeptides, and phosphate sites identified in HEK 293T cells. Detailed Implementation

[0048] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0051] This invention proposes a proteomics analysis method based on a nucleic acid extraction residue system, the procedure steps of which are as follows: Figure 1 As shown. The overall sample preparation process is described as follows: (a) DNA / RNA extraction (1 h), (b) ZASP precipitation of proteins, washing and reductive alkylation (0.5 h), (c) protein digestion (16 h), (d) desalting (1 h), (e) Fe 3+ -NTA phosphopeptide enrichment (1 h), (f) drying (1 h). In this invention, each step was individually adjusted to simplify the operation as much as possible.

[0052] Original reagents for experiments 1. Sodium deoxycholate (SDC; Sigma-Aldrich, cat. no. D6750) 2. Tris(hydroxymethyl)aminomethane (Tris); Pierce, cat. no. 17926 3. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl; Pierce, cat. no. 20490) 4. 2-Chloroacetamide (CAA; Sigma-Aldrich, cat. no. C0267) 5. Trypsin (MS Grade; Pierce, cat. no. 90057) 6. β-Mercaptoethanol (BME; RHAWN, cat.no.R054186) 7. Anhydrous ethanol (Ethanol, EtOH; Fisher Scientific, cat.no.033361.M1) 8. Methanol (Metanol, MeOH; Fisher Scientific, cat.no. W6-4) 9. Zinc chloride (Chlorek cynku; RHAWN, cat.no R018471) 10. Formic acid (FA; Sigma-Aldrich, cat. no. 33015) 11. Trifluoroacetic acid (TFA; Merck, cat. no. 8082600100) 12. Acetonitrile (ACN; Fisher Scientific, cat. no. A955-4) 13. Ammonia solution (25% wt / Vol, NH4OH; Merck, cat. no. 5330030050) Experimental raw materials and consumables 1. Pipette tips (50–1250 μL; Eppendorf, cat. no. 0030073320) 2. C18 Solid Phase Extraction (SPE) membrane (C18 Solid Phase Extraction (SPE) disks; 3M Empore, CAT. No. 98060402173) 3. C8 Solid Phase Extraction (SPE) disks; 3M Empore, cat. no. 14386 Experimental instruments 1. Non-contact ultrasonic crusher (Qsonica, cat. no. Q800R3) 2. Vacuum centrifugal concentrator (SpeedVac Integrated Vacuum Concentrator; Fisher Scientific, cat. no. SPD1030A-230) Reagents used in the experiment 1. ZASP precipitant (200 mM ZnCl2, 99.9% v / v methanol and 0.1% v / v FA) 2. Detergent (95% ACN (v / v%), 0.1% FA (v / v%), and the remainder water) 3.1% SDC lysis buffer (1% SDC), 10 mM TCEP, 40 mM CAA, and the balance water) 4. Proteolytic reaction terminator (pure formic acid) 5. Desalting buffer A (0.1% FA aqueous solution, v / v%) 6. Desalination buffer B (80% ACN (v / v%), 0.1% FA (v / v%), and the balance water) 7. Phosphatopeptide enrichment material (PureCube Fe-NTA Agarose; Cube Biotech, cat. no. 31403-Fe) 8. Phosphatopeptide enrichment solution A (80% ACN (v / v%), 0.1% TFA (v / v%), and the balance water) 9. Phosphatopeptide enrichment solution B (60% ACN (v / v%), 3% ammonia (v / v%), and the balance water) 10. Buffer GD (4 M guanidine hydrochloride and 30% v / v ethanol solution) 11. The rinsing solution PW consists of: 10 mM Tris-HCl, 10 mM NaCl and 70% v / v ethanol solution.

[0053] 12. The elution buffer TB consists of 10 mM Tris-HCl buffer.

[0054] 13. The components of protein removal solution RW1 are: 2 M guanidine isothiocyanate and 20% v / v ethanol solution.

[0055] 14. The composition of rinsing solution RW is: 10 mM Tris-HCl and 80% v / v ethanol solution.

[0056] 15. DNA / RNA Co-extraction Kit: Tiangen Biotech DNA / RNA Co-extraction Kit (Catalog No. DP422) Consumables used in experiments 1. Peptide desalting column (composed of several layers of C18 solid-phase extraction membrane packed in a 200 μL pipette tip) 2. Phosphopeptide enrichment column (composed of a 200 μL pipette tip filled with a single layer of C8 solid-phase extraction membrane and 10 μL of phosphopeptide enrichment material) Example 1: A proteomics analysis method based on nucleic acid extraction residue system I. DNA Extraction 1. Add 350 μL of RLplus nucleic acid extraction buffer (1×10⁻⁶) to the sample to be tested. 5 Up to 5×10 6 Add 350 μL of each cell to 5 × 10⁶ cells. 6 Up to 1×10 7 Add 600 μL for 10 mg to 20 mg of tissue; add 350 μL for 10 mg to 20 mg of tissue; add 600 μL for more than 20 μg of tissue. Transfer all solutions to a DNA adsorption column CB3, centrifuge at 12000 rcf for 30-60 seconds, and collect the filtrate.

[0057] 2. Wash the DNA adsorption column with 500 μL of GD buffer, centrifuge at 12000 rcf for 30-60 seconds, and collect the filtrate.

[0058] 3. Wash the DNA adsorption column with 500 μL of PW wash buffer, centrifuge at 12000 rcf for 30-60 seconds, collect the filtrate, and repeat once.

[0059] 4. Allow the DNA adsorption column to stand at room temperature to allow the adsorption material to dry completely before adding it to the rinsing solution.

[0060] 5. Elute the DNA-adsorbed column with 100 μL of elution buffer TB, centrifuge at 12000 rcf for 2 min to obtain the DNA solution.

[0061] II. RNA Extraction 1. Add 1 volume of 70% ethanol to the filtrate after DNA extraction, mix well, transfer to an RNA adsorption column, centrifuge at 12000 rcf for 30-60 seconds, and collect the filtrate.

[0062] 2. Wash the RNA adsorption column with 700 μL of buffer RW1, centrifuge at 12000 rcf for 30-60 seconds, and collect the filtrate.

[0063] 3. Wash the RNA adsorption column with 500 μL of wash buffer (RW), centrifuge at 12000 rcf for 30-60 seconds, collect the filtrate, and repeat once.

[0064] 4. Allow the RNA adsorption column to stand at room temperature to allow the adsorption material to dry completely before adding it to the rinsing solution.

[0065] 5. Elute the DNA adsorption column with 100 μL RNase-Free dd H2O, centrifuge at 12000 rcf for 2 min to obtain RNA solution.

[0066] III. Protein Extraction 1. Add 1 volume of protein precipitant to the lysate after nucleic acid extraction to precipitate the protein. Incubate at room temperature for 10 min, then centrifuge at 19000 rcf for 10 min and remove the supernatant.

[0067] 2. After adding the same volume of detergent, place the sample in a non-contact sonicator for auxiliary washing (sonicator settings: 1 min, 85% energy, 4℃, and 20 s ON / 20 s OFF), centrifuge at 19000 rcf for 10 min, and remove the supernatant.

[0068] 3. Let stand at room temperature until the acetonitrile has completely evaporated.

[0069] 4. Add 100 μL of 1% SDC lysis buffer to the protein precipitate to resuspend the precipitate. Place the sample in a non-contact sonicator to assist in resuspending (sonicator settings: 1 min, 85% energy, 4℃ and 20 s ON / 20 s OFF), and incubate at 65°C for 10 min.

[0070] 5. After the sample has cooled to room temperature, add an appropriate amount of trypsin at a ratio of protein:trypsin = 25:1 (w:w) and incubate at 37°C for 16 hours.

[0071] 6. Add an appropriate amount of protease hydrolysis terminator according to the ratio of enzyme hydrolysate to protease hydrolysis terminator = 100:1 (v:v), centrifuge at 14000 rcf for 10 min at room temperature, and retain the supernatant.

[0072] Example 2 Based on Example 1 above, the following "peptide desalting" operation step is added: 1. The peptide desalting column was activated and equilibrated using methanol, desalting buffer B, and desalting buffer A in sequence.

[0073] 2. Add the supernatant to the peptide desalting column and repeat this operation once.

[0074] 3. Wash the peptide desalting column twice with 200 μL of desalting buffer A.

[0075] 4. Elute the peptide desalting column twice with 100 μL desalting buffer B.

[0076] 5. Concentrate a portion of the eluent under vacuum at 45°C until dry to obtain the purified peptide fragment of the sample to be tested, and store at -20°C.

[0077] Example 3 Based on Example 2 above, the following "phosphopeptide enrichment" operation steps are added: 1. Activate and equilibrate the phosphopeptide enrichment column using phosphopeptide enrichment solution A.

[0078] 2. Allow the remaining solution obtained from elution in step 4 of the peptide desalting operation in Example 2 to flow completely through the phosphopeptide enrichment column.

[0079] 3. Wash the phosphopeptide enrichment column 2-3 times with 200 μL of phosphopeptide enrichment solution A.

[0080] 4. Elute the phosphopeptide enrichment column twice with 50 μL of phosphopeptide enrichment solution B.

[0081] 5. Concentrate and evaporate to dryness under vacuum at 45 ℃ to obtain the enriched and purified phosphopeptides of the sample to be tested, and store at -20 ℃.

[0082] Comparative Example 1: Traditional in-solution methods for preparing proteomic and phosphorylated proteomic samples I. Protein Extraction 1. Add an appropriate amount of protein extraction solution to the sample to be tested and heat and incubate at 95°C for 5 minutes.

[0083] 2. After the sample has cooled to room temperature, place it in a non-contact sonicator (sonicator settings: 10 min, 85% energy, 4°C, and 3 s ON / 3 s OFF), centrifuge at 14000 rcf for 10 min, and retain the supernatant.

[0084] 3. Determination of protein concentration using the BCA method.

[0085] II. Protein Reduction Alkylation and Enzymatic Digestion 1. Determine the protein concentration according to the BCA method. Take 20 μg of protein and add 40 mM CAA and 10 mMTCEP to a final concentration. Make up to 100 μL and incubate at 65℃ and 1000 rcf for 10 min.

[0086] 2. After the sample has cooled to room temperature, add an appropriate amount of trypsin at a ratio of protein:trypsin = 25:1 (w:w) and incubate at 37°C for 16 hours.

[0087] 3. Add an appropriate amount of protease terminating agent according to the ratio of enzyme hydrolysate to protease terminating agent = 50:1 (v:v), centrifuge at 1,4000 rcf for 10 min at room temperature, and retain the supernatant.

[0088] III. Peptide Desalting 1. The peptide desalting column was activated and equilibrated using methanol, desalting buffer B, and desalting buffer A in sequence.

[0089] 2. Add the supernatant to the peptide desalting column and repeat this operation once.

[0090] 3. Wash the peptide desalting column twice with 200 μL of desalting buffer A.

[0091] 4. Elute the peptide desalting column twice with 100 μL desalting buffer B.

[0092] 5. Concentrate and evaporate a portion of the eluent under vacuum at 45 °C to obtain the purified peptide fragments of the sample to be tested, which can be stored at -20 °C.

[0093] IV. Phosphopeptide enrichment 1. Activate and equilibrate the phosphopeptide enrichment column using phosphopeptide enrichment solution A.

[0094] 2. Allow the remaining solution obtained from the peptide desalting step 4 to flow completely through the phosphopeptide enrichment column.

[0095] 3. Wash the phosphopeptide enrichment column 2-3 times with 200 μL of phosphopeptide enrichment solution A.

[0096] 4. Elute the phosphopeptide enrichment column twice with 50 μL of phosphopeptide enrichment solution B.

[0097] 5. Vacuum concentration and evaporation at 45 ℃ yields the enriched and purified phosphopeptides of the sample to be tested, which can be stored at -20 ℃.

[0098] Comparative Example 2: Proteomics Analysis Based on Phenol-Chloroform Method (TRIzol) I. RNA Extraction 1. Add 1 mL of TRIzol reagent to the sample to be tested, homogenize thoroughly and lyse, and incubate at room temperature for 5 min to allow the nucleoprotein complex to completely dissociate.

[0099] 2. Add 0.2 mL of chloroform to the lysis buffer, shake vigorously for 15 s, incubate at room temperature for 3 min, and then centrifuge at 4 ℃ and 12000 rcf for 15 min.

[0100] 3. Carefully aspirate the colorless aqueous phase from the top layer into a new centrifuge tube, add an equal volume of isopropanol to precipitate the RNA, wash with 75% v / v ethanol, and dry to obtain the RNA sample.

[0101] 4. Retain the middle and bottom red organic phases in the centrifuge tubes for subsequent DNA and protein extraction.

[0102] II. DNA Extraction 1. Add 0.3 mL of anhydrous ethanol to the intermediate and bottom organic phases retained in step one, mix by inversion, and incubate at room temperature for 3 min.

[0103] 2. Centrifuge at 4 ℃ and 2000 rcf for 5 min to precipitate DNA. Transfer the supernatant to a new centrifuge tube (for protein recovery). After washing and drying the precipitate, dissolve it to obtain the DNA sample.

[0104] 3. Protein extraction 1. Add 1 volume of protein precipitant to the supernatant organic solution to precipitate the protein, incubate at room temperature for 10 min, then centrifuge at 19000 rcf for 10 min and remove the supernatant.

[0105] 2. After adding the same volume of detergent, place the sample in a non-contact sonicator for auxiliary washing (sonicator settings: 1 min, 85% energy, 4℃, and 20 s ON / 20 s OFF), centrifuge at 19000 rcf for 10 min, and remove the supernatant.

[0106] 3. Let stand at room temperature until the acetonitrile has completely evaporated.

[0107] 4. Add 100 μL of 1% SDC lysis buffer to the protein precipitate to resuspend the precipitate. Place the sample in a non-contact sonicator to assist in resuspending (sonicator settings: 1 min, 85% energy, 4℃ and 20 s ON / 20 s OFF), and incubate at 65°C for 10 min.

[0108] 5. After the sample has cooled to room temperature, add an appropriate amount of trypsin at a ratio of protein:trypsin = 25:1 (w:w) and incubate at 37°C for 16 hours.

[0109] 6. Add an appropriate amount of protease hydrolysis terminator according to the ratio of enzyme hydrolysate to protease hydrolysis terminator = 100:1 (v:v), centrifuge at 14000 rcf for 10 min at room temperature, and retain the supernatant.

[0110] IV. Peptide Desalting 1. The peptide desalting column was activated and equilibrated using methanol, desalting buffer B, and desalting buffer A in sequence.

[0111] 2. Add the supernatant to the peptide desalting column and repeat this operation once.

[0112] 3. Wash the peptide desalting column twice with 200 μL of desalting buffer A.

[0113] 4. Elute the peptide desalting column twice with 100 μL desalting buffer B.

[0114] 5. A portion of the eluent was concentrated and evaporated to dryness under vacuum at 45°C to obtain the purified peptide fragment of the sample to be tested, which was then stored at -20°C.

[0115] Comparison of experimental results of the method in Example 3 with those of Comparative Examples 1 and 2 All whole proteomics results were obtained by analyzing a Tims TOF Pro mass spectrometer (Bruker) at a 60-min effective liquid chromatography gradient LC-MS / MS. The dia-PASEF files were retrieved using Spectronaut 18.2 for library search. All phosphorylated proteomics results were obtained by analyzing a Phosphorylated proteomics data by an Orbitrap Eclipse mass spectrometer (Fisher Scientific) at a 120-min effective liquid chromatography gradient LC-MS / MS (sensitivity testing at 60 min). The RAW files were retrieved using FragPipe 20.0 for library search.

[0116] Liquid chromatography conditions: (1) Mobile phase: Buffer A (0.1% FA, v / v%), Buffer B (80% ACN + 0.1% FA, v / v%).

[0117] (2) Tims TOF Pro 60 min effective gradient: Buffer B increases from 0% to 6% within 13 min, Buffer B increases from 6% to 30% within 45 min, Buffer B increases from 30% to 40% within 13 min, Buffer B increases from 40% to 99% within 1 second, and maintains 99% Buffer B for 3 min, Buffer B decreases from 99% to 1% within 1 second, and maintains 1% Buffer B for 2 min.

[0118] (3) Orbitrap Eclipse 60 min effective gradient: Buffer B increases from 3% to 6% within 3 min, Buffer B increases from 6% to 28% within 62 min, Buffer B increases from 28% to 40% within 5 min, Buffer B increases from 40% to 90% within 1 min, Buffer B remains at 90% for 5 min, and finally Buffer B decreases to 3% within the last 4 min.

[0119] (4) Orbitrap Eclipse 120 min effective gradient: Buffer B rises from 3% to 6% within 6 min, Buffer B rises from 6% to 28% within 104 min, Buffer B rises from 28% to 40% within 10 min, Buffer B rises from 40% to 90% within 2 min, Buffer B remains at 90% for 5 min, and Buffer B drops to 3% in the last 1 second.

[0120] Mass spectrometry conditions: (1) Tims TOF Pro: Full MS scan range: 300 ~ 1500 Da, resolution: , ion mobility range: 1 / K0 = 1.39 ~ 0.75 Vs / cm², accumulation time and ramp time are both set to 100 ms. dia-PASEF acquisition mode is used.

[0121] (2) Orbitrap Eclipse: Full MS scan range 350~1500 Da, resolution 60000; MS / MS isolation window set to 1.6 Da, resolution 30000, maximum injection time set to 45 ms. DDA acquisition mode used, cycle time set to 2 s. HCD fragmentation energy set to 30, dynamic exclusion time set to 15 s.

[0122] Experimental results are as follows Figures 2 to 9 As shown.

[0123] Figure 2 This is a comparison diagram showing the identification of proteins, phosphopeptides, phosphopeptides, and phosphate sites from a starting amount of 200 μg protein treated using the traditional in-solution enzymatic digestion procedure (Comparative Example 1) and the procedure of this invention. Figure 2 As can be seen, this invention can achieve an identification depth comparable to that of traditional in-solution enzymatic digestion processes while ensuring high-quality DNA / RNA extraction.

[0124] Figure 3 Venn diagrams showing the protein and phosphate sites identified using a 200 μg protein starting amount processed by the conventional procedure (Comparative Example 1) and the procedure of this invention. Figure 3 It can be seen that the process of this invention can cover more than 90% of the protein identification results and more than 70% of the phosphopeptide identification results of traditional in-solution enzymatic digestion processes. The higher the coverage, the more it proves that the results obtained by the two methods are similar and that the methods are highly substitutable.

[0125] Figure 4 The method of this invention and Comparative Example 2 (TRIzol method) treated 3×10 6 A comparison of the number of proteins and peptides identified after processing HEK 293T cells. Figure 4 As can be seen, the method of this invention identifies more proteins.

[0126] Figure 5 The method of this invention and Comparative Example 2 (TRIzol method) were used to treat 3 × 10 6 Distribution of coefficient of variation (CV) for protein quantification identified in HEK 293T cells. Figure 5As can be seen, the median CV of the protein identified by the process of this invention is only 6.34%, which is significantly lower than the 21.48% of Comparative Example 2 (TRIzol group). The high CV value indicates that organic solvents such as phenol in the TRIzol reagent are difficult to completely remove. The residual phenol will significantly interfere with the ionization efficiency in the mass spectrometry detection process, thereby introducing a large random error and resulting in poor reproducibility and precision of its quantification.

[0127] Figure 6 The process of this invention processes 1×10 5 Up to 5×10 6 DNA yield identified in HEK 293T cells.

[0128] Figure 7 The process of this invention processes 1×10 5 Up to 5×10 6 RNA production identified in HEK 293T cells.

[0129] Figure 8 The process of this invention processes 1×10 5 Up to 5×10 6 Proteins and peptides identified in HEK 293T cells.

[0130] Figure 9 The process of this invention processes 1×10 5 Up to 5×10 6 Phosphoproteins, phosphopeptides, and phosphate sites identified in HEK 293T cells.

[0131] Depend on Figures 6 to 9 As can be seen, the method of the present invention can not only achieve 1×10 5 Up to 5×10 6 High-quality DNA and RNA can be stably extracted from HEK 293T cells, and deep proteomics and phosphorylated proteomics analysis can be performed simultaneously.

[0132] This invention innovatively integrates column-based DNA / RNA co-extraction technology, a zinc ion-mediated protein precipitation and capture strategy, and a highly efficient Fe-IMAC phosphorylated peptide enrichment process. By introducing a zinc ion system into the high-salt / high-denaturation effluent after nucleic acid extraction, this method achieves efficient protein precipitation and in-situ washing, thoroughly removing chemical components that interfere with mass spectrometry detection. This allows for the simultaneous extraction of high-quality nucleic acids and deep proteomic and phosphorylated proteomic coverage in a single sample (especially micro-biopsy or rare cell samples), effectively overcoming the bottlenecks of traditional methods such as limited sample size, complex procedures, and poor reproducibility.

[0133] This method is a simple multi-omics consortium analysis approach that eliminates the need for toxic reagents such as phenol, chloroform, and acetone. It is easy to operate and highly suitable for clinical biopsy-grade samples. This workflow provides reliable and practical tool support for multi-omics integrated analysis in basic research and precision medicine. The method is simple, highly stable, and cost-effective, and is naturally compatible with automated operating platforms, having been successfully applied to 1×10⁻⁶ omics systems. 5 This technology enables multi-omics analysis of minute-scale cell and various mouse tissue samples. It provides a universal solution for achieving truly "single-sample, multi-omics, and scalable" analysis under minute sample conditions, and has broad prospects for scientific research and clinical applications.

[0134] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for extracting multi-omics components from a single sample for multi-omics joint analysis, characterized in that, Includes the following steps: S1, DNA extraction S11. Add nucleic acid extraction solution to the sample to be tested; S12. Transfer all solutions from the S11 system to a DNA adsorption column, centrifuge, and collect the filtrate. S13. Wash the DNA adsorption column with buffer GD; S14. Wash the DNA adsorption column with PW rinse buffer and repeat once; S15. Place the DNA adsorption column at room temperature to allow the residual washing solution in the adsorption material to dry completely. S16. Elute the DNA-adsorbed column with elution buffer TB to obtain a DNA solution; S2, RNA extraction S21. Add 1 volume of 70% v / v ethanol to the filtrate collected in S12, mix well, transfer to an RNA adsorption column, centrifuge, and collect the filtrate. S22. Wash the RNA adsorption column with protein-removing buffer RW1; S23. Wash the RNA adsorption column with rinse buffer RW, and repeat once; S24. Place the RNA adsorption column at room temperature to allow the residual washing solution in the adsorption material to dry completely; S25. Elute the RNA adsorption column with RNase-Free dd H2O to obtain an RNA solution; S3, Protein Extraction S31. Add ZASP precipitant to the filtrate collected in S21 to precipitate proteins; S32. Use detergent to clean protein deposits; S33. Resuspend the protein precipitate using 1% SDC lysis buffer and then sonicate it. S34. Heating the S33 system sample to reduce and alkylate the protein; S35. Add trypsin to the sample in system S34 to obtain the enzymatically digested sample; S36. Add the protease hydrolysis reaction terminator to the sample in system S35, then centrifuge and collect the supernatant.

2. The method according to claim 1, characterized in that, The method further includes the following steps: S4, peptide desalting: S41. Activate and equilibrate the peptide desalting column using methanol, desalting buffer B, and desalting buffer A, respectively. S42. Add the supernatant collected in S36 to the peptide desalting column; S43. Wash the peptide desalting column with desalting buffer A, repeat once; S44. Elute the peptide desalting column with desalting buffer B to obtain a peptide solution; S45. A portion of the peptide solution from S44 is concentrated and evaporated to dryness under vacuum at 45 °C to obtain purified peptides.

3. The method according to claim 2, characterized in that, The method further includes the following steps: S5, phosphorylated peptide enrichment S51. Activate and equilibrate the phosphopeptide enrichment column using methanol and phosphopeptide enrichment buffer A; S52. Add a portion of the peptide solution from S44 to the phosphopeptide enrichment column; S53. Wash the phosphopeptide enrichment column with phosphopeptide enrichment buffer A; S54. Elute the phosphopeptide enrichment column with phosphopeptide enrichment buffer B to obtain a phosphopeptide solution; The phosphopeptide was concentrated and evaporated to dryness under vacuum at 55.45 °C to obtain purified phosphopeptide.

4. The method according to any one of claims 1 to 3, characterized in that, In step S11, the sample to be tested is selected from any one of the following: cell sample, tissue sample; Preferably, the amount of the sample to be tested is 5~500 μg; Preferably, in step S11, DNA and RNA extraction are performed using a DNA / RNA co-extraction kit; Preferably, the amount of the cell sample used is 1×10⁻⁶. 5 Up to 1×10 7 For each cell, add 100-600 μL of nucleic acid extraction solution; or for tissue samples, add 350-600 μL of nucleic acid extraction solution to a volume of 10-20 mg.

5. The method according to any one of claims 1 to 3, characterized in that, In step S11, β-mercaptoethanol is added to the nucleic acid extraction solution before the operation to a final concentration of 1% v / v.

6. The method according to any one of claims 1 to 3, characterized in that, In step S31, the ZASP precipitant is a mixed solution containing the following final concentrations of solute: 200 mM ZnCl2, 99.9% v / v methanol and 0.1% v / v formic acid.

7. The method according to any one of claims 1 to 3, characterized in that, In step S33, the 1% SDC lysis buffer is a mixed aqueous solution containing the following final concentrations of solute: 1% w / w SDC, 100 mM Tris-HCl at pH 8.5, 40 mM MTCEP-HCl, and 10 mM CAA.

8. The method according to any one of claims 1 to 3, characterized in that, In step S35, the amount of trypsin is added according to a protein to trypsin mass ratio of 25:

1.

9. The method according to any one of claims 1 to 3, characterized in that, In step S36, the proteolytic reaction terminator is a 1% v / v aqueous formic acid solution; Alternatively, the amount of the protease hydrolysis reaction terminator is added according to a volume ratio of 100:1 between the hydrolysate and the protease hydrolysis reaction terminator. Alternatively, the centrifugation conditions are: 14000 rcf, 4℃ for 10 min.

10. The method according to claim 2 or 3, characterized in that, In step S41, the desalting buffer A is a 0.1% v / v formic acid aqueous solution; Alternatively, the desalting buffer B is composed of the following substances in volume percentages: 80% acetonitrile, 0.1% trifluoroacetic acid, and purified water; Preferably, in step S51, the phosphopeptide enrichment buffer A is composed of the following substances in volume percentage: 80% acetonitrile, 0.1% trifluoroacetic acid, and purified water; Alternatively, the phosphopeptide enrichment solution B may consist of the following substances in volume percentages: 80% ACN, 3% ammonia, and purified water.

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