Undisturbed plasma proteomics depth coverage analysis method

Abstract

The invention discloses an undisturbed plasma proteomics depth coverage analysis method. By introducing the parallel depth coverage reference spectrum library in the mixed plasma, the coverage depth of the undisturbed sample can be remarkably increased; and the method can also be used as an effective means for evaluating, monitoring and correcting the batch effect in the large-queue experiment process. Compared with the direct DIA technology, the cost is greatly reduced while the daily flux is doubled by multi-channel simultaneous detection based on the aminomethylation label technology; annotation analysis is carried out on low-abundance protein by adopting data-independent library searching software, false positive control is carried out at multiple levels of PSM, precursor ions and protein, and qualitative and quantitative accuracy is reliable; compared with other mass spectrometry methods, the analysis method has the advantages that the sample demand is greatly reduced, and the analysis method is suitable for detection of rare samples. Depth coverage of undisturbed plasma samples is realized, identification depth is remarkably increased, daily detection flux is doubled, and the method has great application potential for promoting clinical large-queue research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for deep coverage analysis of non-perturbation plasma proteomics. Background Art

[0002] Blood connects various tissues and organs and carries a large amount of biological information. It is considered to be the best sample for monitoring the nonlinear dynamic changes of systemic pathophysiology. Among the biological information carried by blood, proteins have become an important choice for mining blood biological information because they are closer to clinical phenotypes and exist in a more stable form. Many methods have been developed for plasma proteomics to achieve its deep coverage and precise quantification. Mainstream technologies include antibody affinity-based proteomics platforms (such as Olink), aptamer affinity-based proteomics platforms (such as SomaScan), and liquid chromatography–mass spectrometry (LC-MS)-based proteomics platforms. LC-MS-based blood proteomics plays an irreplaceable role in blood proteomics due to its low cost, high sensitivity, and unique post-translational modification information mining capabilities. Blood proteomics based on LC-MS can deplete the top 14 high-abundance proteins in the blood through antibody affinity columns or enrichment based on protein coronas to improve protein coverage depth to varying degrees, but the above methods all cause disturbances to the original samples, resulting in the random loss of low-abundance proteins and loss of biomarkers (such as immunoglobulins targeted by antibody affinity columns are also biomarkers in some cases). Despite the rapid development of mass spectrometry technology, both in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes, due to the serious masking effect of high-abundance proteins on low-abundance proteins, the depth of plasma protein coverage by DDA and DIA is less than 500, which seriously hinders its application in clinical research. Therefore, it is necessary to find some new analytical methods to achieve deep coverage of non-perturbation blood proteomics. Summary of the invention

[0003] To make up for the deficiencies of the prior art, the present invention provides a method for deep coverage analysis of non-perturbation plasma proteomics.

[0004] To achieve the above object, the present invention adopts the following technical solutions: A first aspect of the present invention provides a method for pre-processing samples for non-perturbation proteomics analysis, wherein the samples include parallel deep coverage reference spectrum library samples and samples to be tested, and the method comprises the following steps: The samples of the reference spectral library with deep coverage are co-incubated with the synergistic adsorption material in parallel, and the samples to be tested are digested by enzymes; The parallel deep coverage reference spectrum library samples and the samples to be tested are subjected to chemical labeling reaction.

[0005] Furthermore, the parallel deep coverage reference spectrum library sample is a mixed sample of all samples to be tested.

[0006] Furthermore, the sample includes serum and plasma.

[0007] Furthermore, the volume ratio of the parallel deep coverage reference spectrum library samples to the cooperative adsorption material is 1:(1-1.5).

[0008] Furthermore, the volume ratio of the parallel deep coverage reference spectrum library samples to the cooperative adsorption material is 1:1.

[0009] Furthermore, the synergistic adsorption material is prepared using wash buffer.

[0010] Furthermore, the wash buffer comprises KCl, CHAPS and tris-HCl.

[0011] Furthermore, the method also includes a step of incubating the samples of the parallel depth coverage reference spectrum library with the cooperative adsorption material and then cleaving the protein.

[0012] Furthermore, the components of the lysis solution used for lysing proteins include a buffer, a detergent, a reducing agent, and an alkylating agent.

[0013] Further, the buffer is selected from TEAB.

[0014] Furthermore, the detergent is selected from SDC.

[0015] Furthermore, the reducing agent includes TCEP and DTT.

[0016] Furthermore, the reducing agent is selected from TCEP.

[0017] Furthermore, the alkylating agent includes CAA and IAA.

[0018] Furthermore, the alkylating agent is selected from CAA.

[0019] Furthermore, the concentration of TEAB is 50 mM.

[0020] Furthermore, the concentration of SDC is 0.1%-0.5%.

[0021] Furthermore, the concentration of SDC is 0.5%.

[0022] Furthermore, the concentration of TCEP is 10 mM.

[0023] Furthermore, the concentration of the CAA is 40 mM.

[0024] Furthermore, the method also includes the step of cleaving the protein and then enzymatically cleaving it.

[0025] Further, trypsin was used for digestion.

[0026] Furthermore, the pancreatic enzyme also includes TEAB.

[0027] Furthermore, the method also includes a step of desalting after the enzyme cleavage.

[0028] Furthermore, the sample to be tested was digested using FASP.

[0029] Furthermore, the FASP enzyme digestion step includes reducing and alkylating the sample to be tested and then digesting it.

[0030] Furthermore, the reducing agent used in the reductive alkylation is selected from TCEP.

[0031] Furthermore, the alkylating agent used in the reductive alkylation is selected from CAA.

[0032] Further, trypsin was used for digestion.

[0033] Furthermore, the chemical labeling reaction is selected from an amine methylation labeling reaction.

[0034] Furthermore, the parallel deep coverage reference spectral library sample reacts with formaldehyde and a reducing agent.

[0035] Furthermore, the sample to be tested undergoes a labeling reaction with formaldehyde / formaldehyde derivatives and a reducing agent.

[0036] Furthermore, the formaldehyde derivative is selected from formaldehyde-12C, d2 or formaldehyde-13C, d2.

[0037] Furthermore, the reducing agent includes sodium (cyano-C) trihydroborate or its derivatives.

[0038] Furthermore, the derivative of sodium (cyano-C) trihydrogenborate is selected from sodium deuterated cyanoborohydride.

[0039] Furthermore, the concentration of formaldehyde / formaldehyde derivatives is 4%.

[0040] Furthermore, the concentration of the sodium (cyano-C) trihydrogenborate or its derivative is 37-39 μg / μL.

[0041] Furthermore, the concentration of sodium (cyano-C) trihydroborate was 37.7 μg / μL.

[0042] Furthermore, the concentration of deuterated sodium cyanoborohydride was 39.52 μg / μL.

[0043] Furthermore, the volume ratio of the formaldehyde / formaldehyde derivative to the reducing agent is 1:1.

[0044] The second aspect of the present invention provides a proteomic analysis sample prepared by the method described in the first aspect of the present invention.

[0045] The third aspect of the present invention provides the use of the proteomic analysis sample described in the second aspect of the present invention in detecting the protein expression profile in the sample.

[0046] The fourth aspect of the present invention provides a non-perturbation proteomics analysis pre-processing kit, the kit comprising the reagents used in the method described in the first aspect of the present invention.

[0047] Furthermore, the kit also includes a reaction container.

[0048] Further, the reaction container comprises a test tube.

[0049] Furthermore, the test tube includes an EP tube and a FASP tube.

[0050] Furthermore, the kit also includes instructions.

[0051] The fifth aspect of the present invention provides a method for deep coverage analysis of unperturbed plasma proteomics, the method comprising: mixing samples prepared by the method described in the first aspect of the present invention, and detecting the samples by mass spectrometry.

[0052] Furthermore, the method also includes the step of performing feature pairing on the parallel deep coverage reference spectrum library samples and the samples to be tested.

[0053] Furthermore, data-independent library search software was used to perform feature pairing between the parallel deep coverage reference spectral library samples and the samples to be tested.

[0054] Furthermore, the database used by the independent database search software is Uniprot Human.

[0055] A sixth aspect of the present invention provides a computer-readable storage medium, wherein the computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the method described in the first aspect of the present invention is implemented.

[0056] The seventh aspect of the present invention provides a system / device for pre-processing samples for non-perturbation proteomics analysis, wherein the system / device comprises a processor, and the processor is used to implement the steps of the method described in the first aspect of the present invention.

[0057] Furthermore, the system or device also includes a memory.

[0058] Furthermore, the memory stores a computer program.

[0059] Furthermore, the computer program is used to program the steps of the method described in the first aspect of the present invention.

[0060] Furthermore, the computer program is executed by a processor.

[0061] Advantages and beneficial effects of the present invention: The method of the present application can significantly increase the coverage depth of undisturbed samples by introducing a parallel deep coverage reference spectral library in mixed plasma; in addition, the parallel deep coverage reference spectral library can also be used as an effective means of batch effect evaluation, monitoring and correction in large cohort experiments. Compared with direct DIA technology, multi-channel simultaneous detection based on aminomethylation labeling technology doubles the daily throughput while significantly reducing costs; data-independent library search software is used to annotate and analyze low-abundance proteins, and false positives are controlled at multiple levels of PSM, precursor ions, and proteins, with reliable qualitative and quantitative accuracy; compared with other mass spectrometry methods, this analysis method greatly reduces the sample requirement and is suitable for the detection of rare samples. In short, the NEATSCOPE technology achieves deep coverage of undisturbed plasma samples, significantly increases the identification depth, and increases the daily detection throughput by 1 times, which has great application potential for promoting large clinical cohort studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the schematic diagram of the NEATSCOPE principle; Figure 2 NEATSCOPE, a comparison of protein coverage depth of Direct DIA and antibody affinity column high abundance removal strategies in unperturbed plasma samples; Figure 3 NEATSCOPE, a comparison of the spectral resolution of Direct DIA and antibody affinity column high abundance removal strategies for unperturbed plasma samples; Figure 4 NEATSCOPE, a qualitative comparison of the results of Direct DIA and antibody affinity column high abundance removal strategies for unperturbed plasma samples; Figure 5 It is a statistical graph of the marking reaction efficiency data; Figure 6 Quantitative accuracy and precision evaluation graph for NEATSCOPE. DETAILED DESCRIPTION

[0063] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] The present invention provides a method for pre-processing samples for non-disturbance proteomics analysis, wherein the samples include parallel deep coverage reference spectral library samples and samples to be tested, and the method includes the following steps: co-incubating the parallel deep coverage reference spectral library samples with a synergistic adsorption material, and enzymatically digesting the samples to be tested; and subjecting the parallel deep coverage reference spectral library samples and the samples to be tested to a chemical labeling reaction.

[0065] In some embodiments, the parallel deep coverage reference spectral library sample (also referred to as a mixed sample in this application) is an equal mixture of all samples to be tested, which may be plasma and / or serum, and the starting volume of the equal mixture of all samples to be tested is at least 50 μL.

[0066] The components of the lysis solution used for protein lysis include buffer, detergent, reducing agent, and alkylating agent.

[0067] In some embodiments, reducing agents include, but are not limited to, DTT, tris(2-carboxyethyl)phosphine (TCEP).

[0068] In a specific embodiment, the reducing agent is selected from tris(2-carboxyethyl)phosphine (TCEP).

[0069] In some embodiments, the alkylating agent includes but is not limited to iodoacetamide (IAA) and chloroacetamide (CAA).

[0070] In a specific embodiment, the alkylating agent is selected from chloroacetamide (CAA).

[0071] In some embodiments, the chemical labeling reaction includes an amine methylation tag reaction or a dimethyl labeling reaction.

[0072] In a specific embodiment, the chemical labeling reaction is selected from an amine methylation tag reaction.

[0073] The parallel deep coverage reference spectral library samples are reacted with formaldehyde and a reducing agent.

[0074] The sample to be tested undergoes a labeling reaction with formaldehyde / formaldehyde derivatives and a reducing agent.

[0075] In some embodiments, the formaldehyde derivatives include but are not limited to formaldehyde-12C, d2 and formaldehyde-13C, d2, wherein "d2" means that there are two deuterium (D) atoms replacing ordinary hydrogen (H) atoms in the molecule, and "12C" and "13C" represent carbon isotopes, respectively.

[0076] In some embodiments, the reducing agent comprises sodium (cyanoborohydride, NaCNBH 3 ) or its derivatives, borane-pyridine complex (BH 3 ·C 5 H 5 N) or its derivatives, lithium aluminum hydride (LiAlH 4 ) or its derivatives, triethylsilane (Et 3 SiH) or its derivatives.

[0077] In a specific embodiment, the reducing agent is selected from sodium (cyanoborohydride, NaCNBH 3 ) or its derivatives, sodium (cyanoborohydride, NaCNBH 3 ) is selected from deuterated sodium cyanoborohydride, which is a deuterated derivative of sodium cyanoborohydride.

[0078] The invention provides a non-disturbance proteomics analysis pre-processing kit, which comprises the reagents used in the above method.

[0079] In some embodiments, the components of the test kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the test kit usually include at least a vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will usually also include a second, third or other additional container, in which additional components are placed separately. However, the components of different combinations can be contained in a vial. The test kit of the present invention will also usually include a container for accommodating reactants, sealed for commercial sale. This container can include a plastic container of injection molding or blow molding, in which the required vial can be retained.

[0080] The solid support of the kit can be, for example, plastic, silicon, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate or slide.

[0081] In some embodiments, the kit further includes instructions, which record the usage and dosage of each reagent.

[0082] In some embodiments, the kit further comprises packaging material. Packaging material may refer to a physical structure that houses the components of the kit. The packaging material may maintain the sterility of the kit components and may be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, etc.). The kit may also contain a buffer, a preservative, or a protein / nucleic acid stabilizer.

[0083] The present invention provides a computer-readable storage medium, wherein the computer-readable storage medium stores a computer program, and the computer program implements the above method when executed by a processor.

[0084] In some embodiments, executable instructions may be in the form of a program, software, software module, script, or code, written in any form of programming language (including compiled or interpreted languages, or declarative or procedural languages), and may be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment.

[0085] As an example, executable instructions may, but need not, correspond to a file in a file system, may be stored as part of a file storing other programs or data, such as in one or more scripts in a HyperText Markup Language (HTML) document, in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files storing one or more modules, subroutines, or code portions).

[0086] By way of example, executable instructions may be deployed to be executed on one computing device, or on multiple computing devices located at one site, or on multiple computing devices distributed across multiple sites and interconnected by a communication network.

[0087] In some embodiments, the computer-readable storage medium includes, but is not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, device or device, or any combination thereof. Specifically, the computer-readable storage medium includes: an electrical connection with one or more wires, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination thereof. In the present invention, a computer-readable storage medium may be any tangible medium containing or storing a program that may be used by or in conjunction with an instruction execution system, device, or device.

[0088] The present invention provides a pre-processing system / device for non-disturbance proteomics analysis samples. The system / device comprises a processor, and the processor is used to implement the steps of the above method.

[0089] In some embodiments, the system and device include not only a structure connected by a communication medium such as a network (including a connection supporting one-to-one communication) of multiple computers, hardware or devices, but also a structure implemented by a single computer, hardware or device. In this application, device and system are used interchangeably.

[0090] When processing is performed by each module or whenever multiple processing is performed within a module, information to be processed is retrieved from the storage device, and the processing results are written back to the storage device after processing. Therefore, the description of retrieving from the storage device before processing and writing back to the storage device after processing may be shortened or omitted in some cases. It should be noted that the storage device here may include a hard disk, a random access memory (RAM), an auxiliary or external storage medium, a storage device accessed via a communication link, and a register in a central processing unit (CPU), etc.

[0091] In some embodiments, a processor is intended to include any integrated circuit or other electronic and mechanical device capable of operating at least one instruction language, such as executing instructions, codes, computer programs and scripts accessed from a storage medium. However, a processor should not be construed as limited to hardware capable of executing software, but rather refers to a processing device in a general manner.

[0092] The system or device also includes a memory.

[0093] In some embodiments, the memory includes any type of integrated circuit or other memory device suitable for storing digital data, including but not limited to random access memory (RAM), pseudo-static RAM (PSRAM), dynamic RAM (DRAM), synchronous dynamic RAM (SDRAM) including double data rate (DDR) class memory and graphics DDR (GDDR) and its variations, ferroelectric RAM (FeRAM), magnetic RAM (MRAM), resistive RAM (ReRAM), read-only memory (ROM), programmable ROM (PROM), electrically erasable PROM (EEPROM or E2PROM), DDR / 2SDR AM, EDO / FPMS, reduced latency DRAM (RLDRAM), static RAM (SRAM), flash memory (e.g., NAND / NOR), phase change memory (PCM), three-dimensional cross point memory (3DXpoint), stacked memory such as HBM / HBM2, and magnetoresistive RAM (MRAM), such as spin torque transfer RAM (STTRAM).

[0094] In some embodiments, the storage device includes, but is not limited to, a computer hard drive (e.g., a hard disk drive (HDD), a solid-state drive (SDD)), a flash drive, a DVR device, a memory, a RAID device or array, an optical media (e.g., a CD-ROM, laser disc, Blu-ray, etc.), or any other device or media capable of storing content or other information, including a semiconductor device capable of maintaining data in the absence of power.

[0095] In some embodiments, the system or device includes one or more processors, and a memory, wherein the memory is used to store a single or multiple computer programs, the computer programs are executed by the processors, the single computer program can be executed by a single or multiple processors, and the multiple computer programs can be executed by a single or multiple processors.

[0096] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0097] Example

[0098] Example 1. NEATSCOPE achieves deep coverage analysis of undisturbed plasma samples Step 1. Parallel deep coverage reference spectral library peptide preparation: Sample mixing: All samples to be tested were mixed in equal amounts. The samples to be tested were from the plasma of 10 healthy volunteers (collected at Beijing Protein Center on August 6, 2023).

[0099] Equal amount of mixed samples for low abundance enrichment: Take 100 μL of mixed plasma and add 100 μL of wash buffer (150 mM KCl, 0.05% CHAPS, 10 mM tris-HCl) prepared with laboratory-synergistic adsorption material (magnetic material synthesized in the laboratory, the magnetic material is the product of the following chemical modification of graphene structure in the laboratory: -NHCH 3 、-SO 3After the incubation, centrifuge at 15000×g for 10 minutes, discard the supernatant, add 200μL wash buffer, shake at 1500rpm, 37℃ for 5 minutes, centrifuge at 15000×g for 10 minutes, discard the supernatant, and repeat the washing three times; after the last washing and discarding the supernatant, add 20μL lysis buffer (50 mM TEAB, 0.5% SDC, 10 mMTCEP, and 40 mM CAA) to the synergistic adsorption material, sonicate for 10 minutes, centrifuge at 15000×g for 10 minutes, take the supernatant, repeat twice, combine the supernatants obtained twice, place in a metal bath at 95℃ for 10 minutes, cool to room temperature, add 10μL 50mM TEAB to the EP tube, add trypsin (20μg Trypsin / 40μL 20mM TEAB, 0.5 μg / μL) was incubated at 37°C for 12 hours; after the incubation, 2.63 μL of 20% TFA was added to the sample, and the sample was placed at -20°C for 10 minutes, and then centrifuged at 17000×g for 5 minutes, the supernatant was collected, and 50 μL of 1% TFA was added to the precipitate, sonicated for 10 minutes, and centrifuged at 17000 g for 5 minutes, the supernatant was collected, and the two supernatants were combined; Equal amount of mixed samples to enrich low abundance peptides and desalt: Prepare a 200μL Tip head and fill it with 3 layers of C18 Empore (3M) membrane. Before use, centrifuge with 100μL of pure acetonitrile at 300×g for 2 minutes, then centrifuge with 100μL of 50% acetonitrile (containing 0.1% TFA) at 300×g for 2 minutes, and finally centrifuge with 100μL of 0.1% TFA at 300×g for 2 minutes to complete the filler activation; add 100μL of the enriched sample supernatant obtained in the previous step to the Tip head, centrifuge at 300g for 2 minutes (mainly to avoid drying the column), repeat three times; then add 100μL of 0.1% TFA and centrifuge at 300×g for 2 minutes, repeat three times; finally, replace the EP tube, add 100μL of 50% acetonitrile (containing 0.1% TFA) to the Tip head and centrifuge at 300g for 2 minutes, repeat three times, collect the eluate, dry it in a vacuum centrifuge at 45℃, store it at -80℃ or 4℃ for use.

[0100] Step 2. Preparation of peptides from unperturbed plasma: The FASP sample preparation method assisted by ultrafiltration tubes with a cutoff value of 30 k Dalton was used for protease cleavage. 200 μL UA (8M Urea, 100 mM Tris / HCl, pH 8.0) was added to each tube to wash the FASP filter membrane, and the UA was removed by centrifugation at 14,000 × g for 15 minutes. 1 μL of plasma sample was added to 200 μL UA and mixed, then added to the FASP tube, and centrifuged at 14,000 × g for 15 minutes to remove UA. 10 mM TCEP and 40 Mm CAA were added to react at room temperature for 40 minutes. After the reaction was completed, the tube was centrifuged at 14,000 × g for 15 minutes, and then 200 μL UA was added, and the tube was centrifuged at 14,000 × g for 20 minutes to ensure that the residual reducing alkylating agent in the FASP tube was completely removed. Subsequently, 200 μL of 50 mM TEAB buffer was added, and the tube was centrifuged at 14,000 × g for 15 minutes to remove UA. This was repeated three times to ensure that the UA was completely replaced. A new cannula was replaced, and 100 mM TEAB dissolved with 2 μg of pancreatin was added. 200 μL, digested at 37℃ for 4 hours, and then 2 μg trypsin was added to continue the reaction for 8 hours; after the reaction, the filtrate was collected by centrifugation at 14000×g for 15 minutes, and then 200 μL H 2 O, centrifuge at 14000×g for 15 min, combine the two filtrates, dry them in a vacuum centrifuge at 45°C, and store them at -80°C or at 4°C for use.

[0101] Step 3. Preparation of peptides using the Top 14 antibody affinity column depletion high abundance strategy: Take 10 μL of a single plasma sample and add it to a Top14 antibody affinity column (Thermo Fisher), vortex mix at room temperature for 15 minutes, then centrifuge at 12,000 × g for 5 minutes, and take the filtrate for FASP digestion. The digestion operation is the same as in step 2 "Preparation of undisturbed plasma peptides".

[0102] Step 4. Aminomethylation labeling reaction of samples 20 μg of low-abundance peptides enriched in the mixed plasma sample obtained in step 1 were labeled with the mass_shift 1 channel, and 20 μg of non-perturbed peptides obtained in step 2 were labeled with mass shift 2 and mass shift 3; the labeling experiment was as follows: Dissolve the peptides obtained in step 1 and step 2 in 100 μL 100 mM TEAB, take 20 μg of each peptide, and make up to 100 μL.

[0103] Mass shift 1: Add 4 μL 4% formaldehyde and 4 μL 37.7 μg / μL (cyano-C) sodium trihydroborate to 20 μg of the enriched peptides obtained in step 1 (dissolved in 100 μL 100 mM TEAB).

[0104] Mass shift 2: 4 μL of 4% formaldehyde-12C, d2 solution and 4 μL of 37.7 μg / μL (cyano-C) sodium trihydroborate were added to 20 μg of the unperturbed peptide obtained in step 2 (dissolved in 100 μL of 100 mM TEAB).

[0105] Mass shift 3: Add 4 μL of 4% formaldehyde-13C, d2 solution and 4 μL of 39.52 μg / μL deuterated sodium cyanoborohydride to 20 μg of the unperturbed peptide obtained in step 2 (dissolved in 100 μL of 100 mM TEAB).

[0106] After adding the corresponding amine methylation labeling reagent, vortex mix, and place on a shaker at 22°C and 1000 rpm for 1 hour.

[0107] After the reaction was completed, 2 μL of 10% NH 3 .H 2 O, vortex to mix, and let stand for 5 minutes to terminate the reaction.

[0108] After the reaction was terminated, 15 μL of 10% TFA was added to each EP tube to acidify the sample.

[0109] The acidified samples were then desalted using a homemade Tip head combined with a C18 membrane, and the operation was the same as the "Equal amount of mixed samples to enrich low-abundance peptides and desalt" section in step 1.

[0110] The eluate obtained by desalting was dried in a vacuum centrifuge at 45°C, then reconstituted with 0.1% formic acid solution, and the concentration was determined by NanoDrop (Thermo Fisher). The corresponding channel labeled peptide was added according to the mass ratio of mass shift 1: mass shift 2: mass shift 3 = 2:1:1. The final concentration was prepared to be 0.1μg / μL and stored at -80°C or 4°C for use.

[0111] Step 5. Mass spectrometry detection and data analysis 200 ng of peptides obtained in steps 2, 3 and 4 were taken for chromatographic separation using the nanoliter liquid phase system EASY-nLC1200 and then detected by the Orbitrap Exploris 480 mass spectrometer. Mobile phase A and mobile phase B were 0.1% formic acid aqueous solution and 80% acetonitrile solution containing 0.1% formic acid, respectively.

[0112] The mass spectrometry data were searched using data-independent search software. The aminomethylation labeled samples obtained in step 4 were searched using the NEATSCOPE library search process. First, the Uniprot Human database (downloaded on 2024_02_17, containing 20398 reviewed sequences) was used to complete the mass spectrometry secondary spectrum library prediction assisted DIA spectrum analysis based on deep learning on the Python framework. The enzyme cleavage method was set to trypsin, the predicted library mass axis offset was 28.0313, the maximum missed cleavage site was 1, the fixed modification was 'Carbamidomethyl@C', and the variable modification was 'Oxidation@M'. Subsequently, the NEATSCOPE data analysis process was performed using data-independent search software. Import the above fasta file and its prediction library. The peptide length, precursor ion charge, mass range and fragment range are all default. The MBR function is turned on. Library Generation selects 'IDs, RT and IM Profiling'. The Speed ​​and RAM usage parameter selects 'Optimal results'. In addition, the command line needs to additionally indicate that the modification type is fixed modification, where mass shift 1 mass shift is +28.0313, mass shift 2 mass shift is +32.0564, and mass shift 3 mass shift is +36.0757. The peak translation function is turned on, and the command line indicates that the prediction library is a fixed modification prediction library.

[0113] The report.tsv file obtained by searching the library is then further filtered using an R-based script with the following filtering criteria: Lib.PG.Q.Value≤0.01 and Q.value≤0.01 and Channel.Q.Value≤0.15, the final result was obtained after filtering, at which time the FDR of protein and precursor ion levels were both less than 1%. The obtained results were further processed based on R language to obtain the final quantitative matrix.

[0114] The direct DIA test data of the unperturbed plasma sample obtained in step 2 and step 3 and the DIA test data of the plasma sample after depletion of the Top14 antibody affinity column were searched and processed according to the following standards: the Uniprot Human database (downloaded on 2024_02_17, containing 20398 reviewed sequences) was used as a fasta file, MBR was checked, the 'FASTA digest for library-free search' and 'Deep learning-basedspectra, RTs and IMs prediction' options were checked, and other options were left to default. No additional commands were added to the command line. The report.tsv file obtained by searching the library was then further filtered using an R-based script. The filtering criteria were as follows: Q.Value≤0.01, Lib.Q.Value≤0.01, Lib.PG.Q.Value≤0.01, PG.Q.Value≤0.01. After filtering, the final results were obtained. At this time, the FDR of the protein and precursor ion levels were both less than 1%. The results were further processed based on the R language to obtain the final quantitative matrix.

[0115] As a proof of concept, three samples were prepared to test the feasibility of NEATSCOPE to achieve deep coverage perturbation-free plasma proteomics. Figure 2 The results showed that the average number of precursor ions and proteins detected by direct DIA of traditional undisturbed plasma samples was 8104 and 459, while the depth after Top14 depletion increased to 12541 precursor ions and 907 proteins. After the detection and analysis of undisturbed samples based on the NEATSCOPE strategy, the number of precursor ions increased to about 19,000, and the protein identification depth reached 1800, which was 3.9 times higher than the traditional undisturbed plasma proteomics method. Even compared with the current commercially successful Top14 depletion strategy, the detection capability of NEATSCOPE technology for undisturbed plasma is twice that of Top14 perturbed plasma samples. Figure 3The results explain the principle of NEATSCOPE technology. The total number of spectra obtained from undisturbed plasma samples under the same DIA acquisition method is approximately 54,000, while the spectral resolution rates of the traditional undisturbed plasma proteomics analysis strategy and the high abundance removal strategy based on antibody affinity columns are only 13.78% and 20.01%, respectively. The introduction of the parallel deep coverage reference spectral library (mass shift 1) under the NEATSCOPE strategy effectively increases the spectral resolution rate of the undisturbed sample channels (here mass shift 2 and mass shift 3), and the spectral resolution rate reaches 45.11% under the same data acquisition method. Figure 4 The results showed that there was no significant difference in identification reproducibility between the NEATSCOPE technology and the traditional unperturbed plasma proteomics technology, proving that its identification results were repeatable.

[0116] Example 2. NEATSCOPE quantitative accuracy and precision evaluation Step 1. Preparation of peptides from unperturbed human plasma Take 1 μL of human plasma sample, use FASP enzymatic digestion method, and select pancreatic enzyme to prepare human plasma peptides. The detailed steps are the same as "Step 2. Preparation of undisturbed plasma peptides" in "Example 1. NEATSCOPE achieves deep coverage analysis of undisturbed plasma samples".

[0117] Step 2. Preparation of unperturbed bovine serum peptides Take 1 μL of bovine serum sample, use FASP enzymatic digestion method, and select pancreatic enzyme to prepare human plasma peptides. The detailed steps are the same as "Step 2. Preparation of undisturbed plasma peptides" in "Example 1. NEATSCOPE achieves deep coverage analysis of undisturbed plasma samples".

[0118] Step 3. Enrichment of human plasma pool samples 100 μL of human mixed plasma was taken for low-abundance protein capture. The detailed steps were the same as those in “Step 1. Parallel deep coverage spectrum library peptide preparation” in “Example 1. NEATSCOPE achieves deep coverage analysis of undisturbed plasma samples”.

[0119] Step 4. Sample amine methylation labeling reaction Two portions of 20 μg of the human plasma peptides and bovine serum peptides obtained in step 1 and step 2 were taken, and two channels (mass shift 2 and mass shift 3) were labeled respectively. The detailed steps were the same as those in "Step 4. Aminomethylation labeling reaction of samples" in "Example 1. NEATSCOPE achieves deep coverage analysis of non-perturbation plasma samples".

[0120] Step 5. Sample mixing on the machine Run 1: Take the plasma protein-enriched peptides labeled with mass shift 1 and the unperturbed human plasma peptides labeled with mass shift 3, mix them in the ratio of mass shift 1: mass shift 3 = 2:1, and dilute them to a final concentration of 100 ng / μL.

[0121] Run 2: Take the plasma protein enriched peptides labeled by mass shift 1, the unperturbed human plasma peptides labeled by mass shift 2 and the unperturbed bovine serum peptides labeled by mass shift 2, the unperturbed human plasma peptides labeled by mass shift 3 and the unperturbed bovine serum peptides labeled by mass shift 3, mix them into the loading solution according to the ratio of mass shift 1 (200 ng): mass shift 2 (40 ng unperturbed human plasma peptides + 60 ng unperturbed bovine serum peptides): mass shift 3 (20 ng unperturbed human plasma peptides + 80 ng unperturbed bovine serum peptides) and dilute them to a final concentration of 100 ng / μL.

[0122] Run 3: Take the plasma protein enriched peptides labeled by mass shift 1, the unperturbed human plasma peptides labeled by mass shift 2, the unperturbed bovine serum peptides labeled by mass shift 2, the unperturbed human plasma peptides labeled by mass shift 3, and the unperturbed bovine serum peptides labeled by mass shift 3, and mix them into the loading solution according to the ratio of mass shift 1 (200 ng): mass shift 2 (15 ng unperturbed human plasma peptides + 85 ng unperturbed bovine serum peptides): mass shift 3 (10 ng unperturbed human plasma peptides + 90 ng unperturbed bovine serum peptides) and dilute them to a final concentration of 100 ng / μL. The labeling reaction efficiency is as follows: Figure 5 shown.

[0123] Step 6. Mass spectrometry detection and data analysis Take 200 ng of each of Run 1, Run 2 and Run 3 in step 5., perform chromatographic separation using the nanoliter liquid phase system EASY-nLC1200, and then detect using the Orbitrap Exploris 480 mass spectrometer. Mobile phase A and mobile phase B are 0.1% formic acid aqueous solution and 80% acetonitrile solution containing 0.1% formic acid, respectively.

[0124] The mass spectrometry data was searched using data independent search software. The amine methylation reaction samples obtained in step 4 were searched using the NEATSCOPE library process. The detailed steps were the same as "Step 5. Mass spectrometry detection and data analysis" in "Example 1. NEATSCOPE achieves deep coverage analysis of non-perturbed plasma samples".

[0125] To further investigate the quantitative capability of the NEATSCOPE technique, bovine serum peptides were spiked in to simulate the high-abundance masking effect. The total ratio of mass shift 1:mass shift 2:mass shift 3=2:1:1 was kept constant, and the amount of undisturbed human plasma peptides loaded in each channel was changed to evaluate the quantitative capability of the data. Figure 6 The results on the left show that the quantitative CV of the NEATSCOPE technology is lower than 15% in the case of deep coverage, indicating high quantitative accuracy. Figure 6 The right result selected proteins that were identified in 10ng, 15ng, 20ng, 40ng and 100ng of unperturbed human plasma peptides, and calculated the multiple relationship of each protein with 100ng as the denominator, and finally drew a distribution diagram of the multiple relationship. The results showed that the multiple relationship based on the protein expression matrix in the NEATSCOPE technology was consistent with the incorporation ratio of the real unperturbed human plasma peptides, indicating that this technology is accurate in quantitative analysis. At the same time, different sample loads were selected to load different channels in different Runs, and their quantitative results still conformed to the real incorporation ratio, which also indicated that the quantitative performance of the NEATSCOP technology was not affected by the channel.

[0126] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for pre-treatment of samples for non-perturbation proteomics analysis, characterized in that: The samples include parallel deep coverage reference spectrum library samples and samples to be tested, and the method includes the following steps: The samples of the reference spectral library with deep coverage are co-incubated with the synergistic adsorption material in parallel, and the samples to be tested are digested by enzymes; Perform chemical labeling reaction on the parallel deep coverage reference spectrum library samples and the samples to be tested; Preferably, the parallel deep coverage reference spectrum library sample is a mixed sample of all samples to be tested; Preferably, the sample includes serum or plasma.

2. The method according to claim 1, characterized in that The volume ratio of the parallel deep coverage reference spectrum library sample to the cooperative adsorption material is 1:(1-1.5); Preferably, the cooperative adsorption material is prepared using wash buffer; Preferably, the wash buffer comprises KCl, CHAPS, and tris-HCl; Preferably, the method further comprises the step of cleaving the protein after incubating the parallel depth coverage reference spectrum library sample with the cooperative adsorption material; Preferably, the lysis solution used to lyse the protein includes a buffer, a detergent, a reducing agent, and an alkylating agent; Preferably, the buffer is selected from TEAB; Preferably, the detergent is selected from SDC; Preferably, the reducing agent is selected from TCEP; Preferably, the alkylating agent is selected from CAA; Preferably, the concentration of TEAB is 50 mM; Preferably, the concentration of SDC is 0.1%-0.5%; Preferably, the concentration of TCEP is 10 mM; Preferably, the concentration of CAA is 40 mM; Preferably, the method further comprises the step of cleaving the protein followed by enzymatic cleavage; Preferably, trypsin is used for enzymatic digestion; Preferably, the pancreatic enzyme further comprises TEAB; Preferably, the method further comprises a step of desalting after the enzyme cleavage.

3. The method according to claim 1, characterized in that Use FASP enzyme to digest the sample to be tested; Preferably, the FASP enzyme digestion step includes reductive alkylation of the sample to be tested followed by enzyme digestion; Preferably, the reducing agent used in the reductive alkylation is selected from TCEP; Preferably, the alkylating agent used in the reductive alkylation is selected from CAA; Preferably, trypsin is used for the digestion.

4. The method according to claim 1, characterized in that The chemical labeling reaction is selected from an amine methylation tag reaction; Preferably, the parallel deep coverage reference spectral library sample is reacted with formaldehyde and a reducing agent; Preferably, the sample to be tested undergoes a labeling reaction with formaldehyde / formaldehyde derivatives and a reducing agent; Preferably, the formaldehyde derivative is selected from formaldehyde-12C, d2 or formaldehyde-13C, d2; Preferably, the reducing agent comprises sodium (cyano-C) trihydroborate or a derivative thereof; Preferably, the derivative of sodium (cyano-C)trihydrogenborate is selected from sodium deuterated cyanoborohydride; Preferably, the concentration of formaldehyde / formaldehyde derivatives is 4%; Preferably, the concentration of the sodium (cyano-C) trihydroborate or its derivative is 37-39 μg / μL; Preferably, the volume ratio of the formaldehyde / formaldehyde derivative to the reducing agent is 1:

1.

5. A proteomic analysis sample prepared by the method according to any one of claims 1 to 4.

6. Use of the proteomic analysis sample according to claim 5 in detecting protein expression profiles in samples.

7. A non-perturbation proteomics analysis pre-treatment kit, characterized in that: The kit comprises the reagents used in the method according to any one of claims 1 to 4; Preferably, the kit further comprises a reaction container; Preferably, the reaction vessel comprises a test tube; Preferably, the test tube comprises an EP tube or a FASP tube; Preferably, the kit further comprises instructions.

8. A method for deep coverage analysis of unperturbed plasma proteomics, characterized in that: The method comprises: mixing samples prepared by the method according to any one of claims 1 to 4, and detecting the samples by mass spectrometry; Preferably, the method further comprises the step of performing feature pairing on the parallel deep coverage reference spectrum library samples and the samples to be tested; Preferably, data-independent library search software is used to perform feature pairing between the parallel deep coverage reference spectral library samples and the samples to be tested; Preferably, the database used by the independent database search software is Uniprot Human.

9. A computer-readable storage medium storing a computer program, wherein the computer program implements the method according to any one of claims 1 to 4 when executed by a processor.

10. A system / device for pre-processing samples for non-perturbation proteomics analysis, characterized in that: The system / device comprises a processor, wherein the processor is used to implement the steps of the method according to any one of claims 1 to 4; Preferably, the system or device further comprises a memory; Preferably, the memory stores a computer program; Preferably, the computer program is used to program the steps of the method according to any one of claims 1 to 4; Preferably, the computer program is operated by a processor.

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