Weak acid buffer and its use in immuno-peptide component separation reagents

Abstract

The present application relates to the biological field, and particularly to a weak acid buffer and its use in an immunopeptidome separation reagent, the weak acid buffer comprising amino acids and / or polypeptides, the mass-to-charge ratio of the amino acids and / or polypeptides being theoretically predictable and experimentally reproducible, or the mass-to-charge ratio being beyond the preset acquisition range of a mass spectrometer; the weak acid buffer further comprising a volatile acid, the volume concentration of the volatile acid being 0.01% to 10%; and the pH of the weak acid buffer being 3.0 to 3.5. The weak acid buffer of the present application has high compatibility, can maximally avoid pollution and signal interference to an analysis system, has a simple experimental procedure, and the overall operation can be completed within tens of minutes, thus having low technical threshold and high acceptability; can improve the number of immunopeptide groups identified, thus improving the efficiency of the experiment; and the reagent is cheap and easy to obtain, thus the method has low cost, is suitable for large-scale or even industrial use, has high universality, and the like.

Description

Technical Field

[0001] This invention relates to the field of biology, and in particular to a weak acid buffer and its use in an immunopeptide separation reagent. Background Technology

[0002] 1. Definition and application of immune peptides

[0003] The immunopeptidome refers to the collection of peptides presented by the major histocompatibility complex (MHC) molecules on the cell surface. These peptides originate from the degradation products of endogenous proteins and are one of the core mechanisms by which the immune system distinguishes between "self" and "non-self." In virology, immunopeptidome results provide important information on virus-specific antigenic epitopes, thus helping to elucidate host-virus interaction mechanisms and providing key targets for vaccine design. Furthermore, immunopeptidome has elucidated viral escape mechanisms, such as influenza virus and hepatitis C virus evading immune recognition by mutating MHC binding sites, providing theoretical support for the development of broad-spectrum vaccines. In tumor immunotherapy, immunopeptidome helps identify tumor-specific neoantigens for the development of personalized vaccines and T-cell therapies, demonstrating significant efficacy in diseases such as melanoma and lung cancer. In addition, immunopeptidome also has important applications in the diagnosis of infectious diseases and research on autoimmune diseases, such as early diagnosis by detecting virus-specific MHC peptides or revealing disease mechanisms by analyzing abnormally presented self-antigen peptides.

[0004] 2. Existing immunopeptide assay methods and their limitations

[0005] Currently, sample collection for immunopeptidomics mainly relies on immunoaffinity assays: enriching peptides with MHC-specific antibodies. However, this method is costly, complex, and prone to non-specific binding due to antibody cross-reactivity. As an alternative, Mild Acid Elution (MAE) dissociates the MHC-peptide complex using a weakly acidic buffer, directly releasing the peptides. This method is simple to operate, low-cost, and economical, making it suitable for large-scale experiments, and has been used in high-quality papers in recent years.

[0006] Current MAE methods generally use a citrate-Na2HPO4 buffer system as the weak acid. While citrate is a commonly used buffer in biochemical experiments, its use in immunopeptidomics experiments has several unavoidable drawbacks, as follows:

[0007] 1) Difficulty in purification: Citric acid is a non-volatile acid and cannot be removed by means of rotary evaporation; the C18 desalting column commonly used in omics experiments also needs to be gradually diluted through multiple rounds of washing, and the residual citric acid will still interfere with subsequent experiments.

[0008] 2) Poor compatibility of analytical instruments:

[0009] 2.1) In immunopeptidome experiments, it is often necessary to perform high-performance liquid chromatography (HPLC) fractionation and NanoDrop instrument to determine the peptide concentration of the obtained peptide samples: Citric acid and its complexes with peptides and other salts in the sample have strong ultraviolet absorption at 214 nm and 280 nm, which will mask the signal of the target peptide, resulting in baseline drift and abnormal peak shape.

[0010] 2.2) In mass spectrometry analysis, the components of residual buffer solution and the complexes formed can inhibit the ionization of peptides, generating unpredictable mass spectrometry interference signals, which reduces the quality of the acquired mass spectrometry data. After multiple consecutive analyses, the unionizable complexes that accumulate at the nozzle may even damage the ion source.

[0011] Therefore, current MAE experiments require multiple washes in the cleaning step to reduce background interference. However, multiple washes may also lead to the loss of low-abundance peptides, resulting in a decrease in the number of peptides identified and a reduction in experimental efficiency.

[0012] Some studies have attempted to add other components such as EDTA to the buffer to enhance the capture of immune peptides, but none of them have fundamentally solved the problem of interference from buffer components to downstream analysis.

[0013] In summary, traditional immunopeptidomics techniques, such as weak acid elution based on citrate buffer, suffer from bottlenecks such as loss of low-abundance peptides, mass spectrometry signal interference, and low experimental efficiency, which limit their translational and clinical applications. Summary of the Invention

[0014] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a weak acid buffer and its use in an immunopeptide separation reagent to solve the problems in the prior art.

[0015] The core innovation of this invention lies in proposing a novel weak acid buffer system based on amino acids or short peptides, which can improve the overall experimental efficiency of immunopeptides.

[0016] To achieve the above and other related objectives, the present invention first provides a weak acid buffer, wherein the weak acid buffer comprises amino acids and / or polypeptides, wherein the mass-to-charge ratio of the amino acids and / or polypeptides is theoretically predictable and experimentally reproducible, or the mass-to-charge ratio exceeds the preset acquisition range of the mass spectrometer; the weak acid buffer further comprises a volatile acid, wherein the volume concentration of the volatile acid is 0.01%-10%; and the pH of the weak acid buffer is 3.0 to 3.5.

[0017] In some embodiments of the present invention, the amino acid or polypeptide has no ultraviolet absorption at 280 nm.

[0018] In some embodiments of the present invention, the amino acids are selected from histidine, arginine, alanine, and other amino acids that are hydrophilic and hydrophobic, have a defined molecular weight, and generally do not complex with salts such as metal ions. The polypeptide contains three or fewer amino acids, for example, three amino acids (i.e., a tripeptide) or two amino acids (i.e., a dipeptide).

[0019] The volatile acid is selected from formic acid, acetic acid, hydrochloric acid, etc.

[0020] The weak acid buffer also includes one or more of the following: protease inhibitors, chloride ions, sodium ions, magnesium ions, potassium ions, etc. Based on the total volume of the weak acid buffer, the final concentration of chloride or sodium ions is, for example, 100–300 mM.

[0021] The present invention also provides the use of the weak acid buffer in the preparation of protein separation reagents.

[0022] The protein is selected from the immune peptide group, antigen, or antibody.

[0023] This invention also provides a method for isolating immune peptide samples, the method comprising the following experimental steps:

[0024] 1) Mix the weak acid buffer solution with the sample and pipette to elute the crude immunopeptide product from the sample;

[0025] 2) Remove amino acids and / or peptides from the weak acid buffer solution in the product obtained in step 1);

[0026] 3) Concentrate or enrich the product obtained in step 2) to obtain the immune peptide group sample.

[0027] In some embodiments of the present invention, in step 1), the weak acid buffer is a freshly prepared weak acid buffer; and / or, the sample is selected from tissues or cells.

[0028] In some embodiments of the present invention, after step 1), centrifugation can be performed to remove cell or tissue precipitates and collect the supernatant. Preferably, the supernatant can also be passed through a molecular rejection column to remove impurities such as protein fragments.

[0029] As described above, the weak acid buffer solution of the present invention and its use in immunopeptide separation reagents have the following beneficial effects:

[0030] 1. High compatibility. The buffer solution primarily consists of amino acids or short peptides, which, when combined with volatile acids, form a weakly acidic buffer system. The types of amino acids and short peptides can be flexibly configured according to experimental requirements such as buffering capacity. Amino acids, such as histidine, exhibit no UV absorption at 280 nm, ensuring the accuracy of HPLC fractionation and concentration detection of immunopeptide samples. Furthermore, these amino acids and short peptides are either outside the mass spectrometry acquisition range or have a stable and predictable mass-to-charge ratio (m / z), minimizing contamination and signal interference in the analytical system. Volatile acids such as formic acid or acetic acid are common liquid chromatography and mass spectrometry buffers and do not cause additional interference to the analytical system signal.

[0031] 2. The experimental procedure is simple. Formic acid or acetic acid are volatile acids and can be easily removed by rotary evaporation or similar operations. Amino acids and short peptides can be rapidly removed using a C18 desalting column, reducing the number of washes and simplifying the process. Even if small amounts remain, as mentioned above, they will not affect the analytical system. The entire operation can be completed within tens of minutes, thus having a low technical threshold and high acceptability.

[0032] 3. Increased number of identified peptides. Fewer washing cycles reduce the risk of sample loss, ensuring that low-abundance peptides are retained and included in the analysis system. Additionally, amino acids and peptides can act as carrier proteins, stabilizing hydrophobic MHC peptides and improving detection sensitivity. Overall, this leads to an increase in the number of peptides identified in the results, thereby improving experimental efficiency.

[0033] 4. High economic efficiency. Amino acids, short peptides, formic acid, acetic acid, etc., are all commonly used industrial products, inexpensive and readily available. Therefore, this method has low cost and is suitable for large-scale or even industrial-grade use. In addition, these components have almost no chemical damage to the analytical system, will not cause corrosion or clogging, thus extending the service life of the analytical system.

[0034] 5. Applicable to the elution of products in other immunoassays. The basic principle of the immunopeptide elution method is to disrupt the non-covalent interactions between peptides and MHC molecules. Therefore, this method is also applicable and has the potential to be applied to the collection of other antigen products, achieving efficient elution of antigens by disrupting hydrogen bonds and other interactions between complexes; especially for target products such as IgA that are difficult to obtain through immunoaffinity precipitation. Therefore, this method also has high versatility. Attached Figure Description

[0035] Figure 1 The diagram shown is an assessment chart of ultraviolet absorption pollution in Embodiment 1 of the present invention.

[0036] Figure 2 The diagram shown is a mass spectrometry signal interference evaluation diagram of Embodiment 1 of the present invention.

[0037] Figure 3 The image shown is a graph illustrating the elution evaluation results of a single immune peptide in Example 2 of this invention.

[0038] Figure 4 The figure shown is a graph of the peptide identification quantity evaluation results of Example 3 of the present invention.

[0039] Figure 5 The image shown is an evaluation chart of the elution effect of immune peptides in Example 3 of the present invention. Detailed Implementation

[0040] This invention aims to improve the efficiency and reliability of immunopeptide assays by developing an innovative weak acid buffer system, thereby accelerating its translational application in virology, vaccine development, and precision medicine.

[0041] The present invention first provides a weak acid buffer, wherein the weak acid buffer comprises amino acids and / or peptides, wherein the mass-to-charge ratio (m / z) of the amino acids and / or peptides is theoretically predictable and experimentally reproducible, or the mass-to-charge ratio exceeds the preset acquisition range of the mass spectrometer; the weak acid buffer further comprises a volatile acid, wherein the volume concentration of the volatile acid is 0.01%-10%; and the pH of the weak acid buffer is 3.0 to 3.5.

[0042] The amino acid has a buffering effect, used to adjust the pH of the weak acid buffer solution. The concentration of the amino acid is not specifically limited, but the goal is to achieve a pH of 3.0 to 3.5 after mixing the amino acid with the volatile acid. For example, under typical experimental requirements, 5 mM of neutral amino acid dissolved in 0.1% volatile acid can achieve the target pH range. In some cases, a buffer solution with stronger pH stability may be needed, requiring an increase in the concentration of the amino acid or acid. In other cases, a buffer system with stronger "carrier protein" capabilities may also be required, necessitating an increase in the concentration of hydrophobic amino acids in the buffer system.

[0043] The mass-to-charge ratio (m / z) of the amino acid or polypeptide is theoretically predictable and experimentally reproducible, minimizing contamination and signal interference to the analytical system.

[0044] In some embodiments of the present invention, the amino acids or peptides exhibit no UV absorption at 280 nm. This ensures the accuracy of HPLC fractionation and concentration detection of the immunopeptide samples.

[0045] The types of amino acids and peptides can be flexibly configured according to the experimental requirements such as buffering capacity. For example, any amino acid or its derivatives that are compatible with the experimental needs can be selected, including but not limited to its isomers and isomers of its derivatives. Similarly, any dipeptide or tripeptide that is compatible with the experimental needs can be selected, including but not limited to their isomers. Amino acids and short peptides can also be used in combination.

[0046] In some embodiments of the present invention, the amino acid is selected from histidine, arginine, alanine, and other amino acids that are hydrophilic and hydrophobic, have a definite molecular weight, and do not complex with metal ions or other salts. The polypeptide has three or fewer amino acids, for example, three amino acids (i.e., a tripeptide) or two amino acids (i.e., a dipeptide).

[0047] The volatile acid is selected from formic acid, acetic acid, hydrochloric acid, etc.

[0048] In some experiments, a buffer system may be required to maintain cell viability for a longer period. In such cases, the weak acid buffer may include additional components. For example, in some embodiments of the invention, the weak acid buffer may further include one or more of the following: protease inhibitors, chloride ions, sodium ions, magnesium ions, potassium ions, etc. The final concentration of chloride or sodium ions, based on the total volume of the weak acid buffer, is, for example, 100–300 mM.

[0049] Unless otherwise specified, all reagents used in this invention are of analytical grade or higher purity.

[0050] The present invention also provides the use of the weak acid buffer in the preparation of protein separation reagents.

[0051] The protein separation reagent may also be called a protein enrichment reagent or a protein collection reagent.

[0052] The protein is selected from the immune peptide group, antigen, or antibody.

[0053] The immunopeptide separation reagent refers to a reagent used to separate MHC-peptide complexes from tissues or cell lines.

[0054] This invention also provides a method for isolating immune peptide samples, the method comprising the following experimental steps:

[0055] 1) Mix the weak acid buffer solution with the sample and pipette to elute the crude immunopeptide product from the sample;

[0056] 2) Remove amino acids and / or peptides from the weak acid buffer solution in the product obtained in step 1);

[0057] 3) Concentrate or enrich the product obtained in step 2) to obtain the immune peptide group.

[0058] In some embodiments of the present invention, in step 1), the weak acid buffer is a freshly prepared weak acid buffer; and / or, the sample is selected from isolated tissues or cells.

[0059] In some embodiments of the present invention, in step 1), the freshly prepared weak acid buffer solution is mixed with the sample, and the immunopeptides are eluted by slow pipetting. This can also be achieved through other methods such as microfluidic chips or robotic arm operating systems, which are also within the scope of this patent.

[0060] Under normal experimental conditions, elution of the immunopeptide group can be completed by blowing the device 20 times at 4°C for less than 1 minute. In some cases, extending or reducing the number of blowing cycles and the duration may meet additional experimental needs. Researchers can choose appropriate processing methods according to experimental requirements, all of which are within the scope of this patent.

[0061] In some embodiments of the present invention, after step 1), centrifugation can be performed to remove cell or tissue precipitates and the supernatant can be collected. The supernatant is then passed through a molecular rejection column to remove impurities such as protein fragments.

[0062] In step 2), amino acids and / or peptides in the weak acid buffer solution can be removed by means of C18 column desalting, online desalting or low molecular weight molecular cutoff column, and non-peptide portions in the solution can also be removed.

[0063] The desalting procedure for the C18 column can be performed according to the manufacturer's instructions, but only 1-2 washes are usually sufficient to effectively remove buffer components. In some cases, more cell debris may be generated during the experiment, or a purer peptide product may be required. In such cases, it may be necessary to increase or extend the centrifugation or molecular trapping column purification, or increase the number of C18 column washes. Researchers can choose the appropriate treatment method according to the experimental requirements, and all of these are within the scope of this patent.

[0064] In step 3), concentration can be achieved using a rotary evaporator or other methods such as pre-column enrichment.

[0065] For the immunopeptide group obtained in step 3), subsequent experiments can be performed according to the specific experimental objectives. For example, after fractionation by HPLC, the absorbance at 280 nm can be measured to estimate the concentration. After gradient separation and elution of the mixed peptides by liquid chromatography, signal data can be acquired by mass spectrometry. Liquid chromatography can use a pre-column-analytical column tandem mode or analytical column mode, and can use any gradient, mobile phase, flow rate, and packing material required for the experiment. The charged ions of the peptides that have been eluted and enter the mass spectrometer through the ion source are usually acquired by tandem mass spectrometry, and can use any acquisition mode and resolution required for the experiment.

[0066] The above steps use immune peptides as an example, but this method can also be applied to the collection of other antigen products such as immunoglobulins, which are also within the scope of protection of this patent.

[0067] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0068] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0069] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of the present invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of the present invention may be used to implement the present invention.

[0070] Example 1

[0071] Experimental objective: To investigate the effects of several novel buffer systems on interference in the analytical system and to compare them with the control group (the existing immunopeptide assay protocol).

[0072] Experimental steps:

[0073] 1) Preparation of eluent.

[0074] For the formic acid-histidine buffer group, a 0.1% formic acid solution was prepared using HPLC-grade water, and a protease inhibitor (Pierce, catalog number A32955) was added to a final concentration of 1, followed by NaCl to a final concentration of 150 mM. Histidine powder was then added to the solution in small, repeated additions, dissolved and mixed thoroughly. The pH of the solution was measured using a pH meter until it reached a concentration between 3.30 and 3.35 at 23°C. The prepared eluent was filtered through a 0.22 μm filter and pre-cooled on ice.

[0075] For the acetate-arginine buffer group, a 1.5% acetic acid solution was prepared using HPLC-grade water, and the same protease inhibitor as in the formate-histidine buffer group was added to a final concentration of 1, and NaCl was added to a final concentration of 150 mM. Then, arginine powder was added to the solution in small, repeated additions, dissolved and mixed thoroughly. The pH of the solution was measured using a pH meter until it reached a pH between 3.30 and 3.35 at 23°C. After filtering the prepared eluent through a 0.22 μm filter membrane, the solution was pre-cooled on ice.

[0076] For the control group, citric acid powder and disodium hydrogen phosphate (Na₂HPO₄) powder were sequentially dissolved in HPLC-grade water, and the same protease inhibitor as in the experimental group was added to a final concentration of 1, followed by the addition of NaCl to a final concentration of 150 mM. The pH was then adjusted by adding small amounts of NaOH or HCl multiple times until the solution reached approximately 3.3 at 23°C. After filtering the prepared eluent through a 0.22 μm filter membrane, the solution was pre-cooled on ice.

[0077] 2) Assessment of ultraviolet absorption pollution.

[0078] For the two experimental group buffers, the control group buffer, and the mass spectrometry buffer (blank, composed of 0.1% formic acid), 80 μL was injected into the Thermo Vanquish HPLC system for UV absorption contamination assessment, and separation was performed using a 13-minute gradient. The obtained chromatograms were analyzed using Thermo Chameleon software.

[0079] 3) Signal interference assessment of liquid chromatography-tandem mass spectrometry system

[0080] For the two experimental group buffers, control group buffer, and mass spectrometry buffer, 1 μL was injected into the Bruker TimsTOF Pro2 LC system (Bruker Elue 2 nanoLC system) for mass spectrometry signal interference assessment. The mass spectrometry method used was the system's default ddaPASEF, employing a 30-minute gradient in dual-column mode at a flow rate of 500 nL / min. The obtained mass ion chromatograms were analyzed using Bruker Data Analysis software.

[0081] result

[0082] 1) Assessment of ultraviolet absorption pollution.

[0083] like Figure 1As shown, the control group exhibited strong A280 absorption within the 1-4 minute range, with the highest absorption peak reaching 50 mAU; while the A280 absorption of the two experimental groups was essentially consistent with the blank, with the highest absorption peak only 1 mAU. This is attributed to systematic errors caused by background noise. This result demonstrates that the buffer system of this invention does not cause additional contamination in ultraviolet absorption.

[0084] 2) Mass spectrometry signal interference assessment.

[0085] like Figure 2 As shown, the control group exhibited a significant mass spectrometry signal with an intensity of 4E6 during the 30-minute analytical gradient. In contrast, the experimental group showed only a few characteristic mass spectrometry signals, with extremely low mass spectrometry signals at other gradients, essentially consistent with the blank sample. This result demonstrates that the buffer system of this invention does not cause interference with the mass spectrometry signal.

[0086] Example 2

[0087] Experimental Objective: To evaluate the effectiveness of weak acid buffer systems with different amino acid compositions in eluting single immunopeptides.

[0088] Experimental steps:

[0089] 1) Preparation of eluent.

[0090] Following the method in Example 1, four formic acid-amino acid buffer systems were prepared as experimental groups: formic acid-histidine, formic acid-serine, formic acid-arginine, and formic acid-alanine. A control group was also prepared simultaneously following the method in Example 1. For the blank control group, phosphate-buffered saline (PBS) was used, and the same protease inhibitor as in the experimental and control groups was added to a final concentration equal to 1, and the mixture was pre-cooled on ice.

[0091] 2) Elution of single immune peptides.

[0092] Single OVA peptide-MHC protein complex samples, prepared and purified in the laboratory, were divided into six groups, each containing 200 ng (approximately 5 μL), with three biological replicates for each group. The samples were transferred to 0.6 mL protein-lowering centrifuge tubes. 50 μL of buffer was slowly added to each tube, and the tubes were gently pipetted 20 times.

[0093] 3) Desalination using C18 column.

[0094] The supernatant was desalted using a Thermo C18 Spin Tip desalting column, following the product instructions. To ensure consistency, both the experimental and control groups underwent five washes in this embodiment. Purified single OVA peptides were obtained.

[0095] 4) Incorporation of quantitative polypeptides.

[0096] We ordered 98% pure lysine-labeled OVA peptides from Jepeptide Biotechnology Co., Ltd., dissolved them in ultrapure water, and incorporated them into the above groups at a rate of 200 pg per group for subsequent quantification by mass spectrometry analysis.

[0097] 5) Data collection of single immune peptides.

[0098] The purified peptides were concentrated in a rotary evaporator, reconstituted with mass spectrometry buffer, and half of the concentrate was used for data acquisition. First, the peptide samples were separated using a Bruker nanoElue2 nanoLC system with a 15-minute gradient at a flow rate of 500 nL / min. The peptide samples were pre-enriched before entering the mass spectrometer, followed by separation on the analytical column. prmPASEF data acquisition was performed using a TimsTOF Pro2 mass spectrometer system. Parameters were set to positive ion mode, fixed mass scan ranges of m / z at 482.2791 and 486.2861, a mass window of 0.5 Da, and an ion mobility range of 1 / K0 0.75–1.3 Vs / cm. 2 Dynamic collision energy (20-59 eV) is used.

[0099] 6) Analysis of immune peptide data.

[0100] The acquired data were analyzed using Skyline mass spectrometry software in PRM mode, with the mass-to-charge ratio of OVA immunopeptides set to 482.2791, the mass-to-charge ratio of re-labeled standard peptides set to 486.2861, and the mass deviation set to 10 ppm. The obtained mass spectrometry results were then used for quantitative statistical analysis using Excel software. The relative quantitative ratios were obtained by dividing the quantitative results of OVA immunopeptides in each group by the quantitative results of re-labeled standard peptides. Visualization was performed using R language.

[0101] result

[0102] like Figure 3 As shown, the quantification ratio in the blank control group was 0, while both the control group and each experimental group produced relatively high quantification ratios, ranging from 6 to 8.7. This indicates that both the buffer system in the control group and the weak acid buffer systems in each experimental group could effectively elute immunopeptides. The ability of weak acid buffer systems with various amino acid types to elute immunopeptides demonstrates the high flexibility of this method in terms of amino acid selection.

[0103] Example 3

[0104] Experimental objective: To evaluate the effect of a novel buffer system on improving the elution of immunopeptide samples.

[0105] Experimental steps:

[0106] 1) Preparation of eluent.

[0107] A formate-histidine buffer solution was prepared as the experimental group according to the method in Example 1. A control group was also prepared simultaneously according to the method in Example 1. All solutions were pre-cooled on ice.

[0108] 2) Cell culture and collection.

[0109] For the DC2.4 (mouse dendritic cell) cell line, culture in shake flasks using standard medium (DMEM medium mixed with 10% DMEM) until the cell density reaches 80%, with a concentration of approximately 3E6 per mL. Transfer the suspended cell culture medium to 15 mL centrifuge tubes and centrifuge at 200 g, 4°C for 5 minutes. After aspirating the supernatant, resuspend the cell pellet in PBS buffer. Repeat the centrifugation and supernatant aspiration process three times. Before the final wash, estimate the total cell count using a fluorescent dye and cell counter, and based on the result, divide the cells into six new 15 mL centrifuge tubes, with each tube containing approximately 1E7 cells.

[0110] 4) Elution of immune peptide groups or target products.

[0111] Slowly add 1 mL of buffer solution to each centrifuge tube containing cell pellet, with three groups for the experimental group and three groups for the control group. Gently pipette 20 times, keeping the total time under 1 minute.

[0112] 5) Eluent neutralization, gradient washing, and molecular cutoff column purification.

[0113] Add 4 mL of PBS buffer to each centrifuge tube and gently pipette five times. Use pH paper to confirm the solution is neutral. Centrifuge at 200 g, 4°C for 5 minutes, then carefully transfer the supernatant to a new 15 mL centrifuge tube; centrifuge at 350 g, 4°C for 5 minutes, then carefully transfer the supernatant to a new 15 mL centrifuge tube; centrifuge at 4000 g, 4°C for 15 minutes, then carefully transfer the supernatant to a new 2 mL centrifuge tube; centrifuge at 16000 g, 4°C for 15 minutes. Carefully transfer the supernatant to a 5 mL 10 kDa molecular cutoff column and centrifuge at 4000 g, 4°C for at least 30 minutes until the volume of solution on the filter membrane inside the column is less than 500 μL.

[0114] 6) Desalination using C18 column.

[0115] The supernatant was desalted using a Waters Sep-Pak C18 desalting column, following the product instructions. To ensure consistency, both the experimental and control groups underwent five washes in this embodiment. The final purified peptides were obtained.

[0116] 7) Immune peptide data collection.

[0117] The purified peptides were concentrated in a rotary evaporator, reconstituted with mass spectrometry buffer, and 1 / 10 was used for data acquisition. First, the peptide samples were separated using a Bruker nanoElue2 nanoLC system with a 30-minute gradient at a flow rate of 500 nL / min. The peptide samples were pre-enriched before entering the mass spectrometer, followed by separation on the analytical column. ddaPASEF data acquisition was performed using a TimsTOF Pro2 mass spectrometer system. Parameters were set to positive ion mode, mass scan range m / z 100–1700, and ion mobility range 1 / K0 0.6–1.6 Vs / cm. 2 Dynamic collision energy (20-59 eV) is used to adapt to different mobility windows.

[0118] 8) Analysis of immune peptide data.

[0119] The acquired data were analyzed in non-specific mode using FragPipe v22.0 and pFind data search software, with the reference protein file set to the mouse proteome, including 17,184 proteins; the false discovery rate was set to 5%. The results were used for peptide count and visualization in R, and affinity prediction analysis was performed using MHCFlurry software.

[0120] result

[0121] 1) Evaluation of peptide identification quantity.

[0122] like Figure 4 As shown, the number of peptides identified in the three biological replicates of the control group was between 330 and 346, while the number of peptides identified in the three biological replicates of the experimental group all exceeded 400, more than twice that of the control group. This result demonstrates that the buffer system of the present invention can significantly improve the number of peptides identified.

[0123] 2) Evaluation of the elution effect of immune peptides.

[0124] Among the identified peptides, this invention evaluated the motif distribution and additionally analyzed their MHC (H2-Kb type) affinity using the affinity prediction software MHCFlurry. Venn diagram analysis was also performed on peptides with high affinity. Figure 5 As shown in Figure A, the experimental and control groups exhibited very consistent motif distributions, indicating that the buffer system of this invention does not bias the immunopeptidome results. Figure 5 As shown in Figure B, the experimental group identified 55 additional high-affinity peptides compared to the control group. This result demonstrates that the buffer system of this invention can achieve higher efficiency in immunopeptidomics experiments.

[0125] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The use of a weak acid buffer in the preparation of an immunopeptide separation reagent, wherein the weak acid buffer comprises amino acids, the mass-to-charge ratio of which is theoretically predictable and experimentally reproducible, or the mass-to-charge ratio exceeds the preset acquisition range of the mass spectrometer; the weak acid buffer further comprises a volatile acid, the volume concentration of which is 0.01%-10%; the pH of which is 3.0 to 3.5; the volatile acid is selected from formic acid; the amino acid is selected from histidine; the immunopeptide separation reagent refers to a reagent used to separate immunopeptides from MHC-peptide complexes.

2. Use according to claim 1, characterized in that, The weak acid buffer also includes any one or more of the following: protease inhibitors, chloride ions, sodium ions, magnesium ions, and potassium ions.

3. Use according to claim 2, characterized in that, Based on the total volume of the weak acid buffer solution, the final concentration of chloride or sodium ions is 100-300 mM.

4. An immunopeptidomics sample isolation method, characterized by, The method includes the following experimental steps: 1) Mix the weak acid buffer solution as described in any of claims 1 to 3 with the sample and pipette to elute the crude immunopeptide product from the sample; 2) Remove the amino acids from the weak acid buffer solution in the product obtained in step 1); 3) Concentrate or enrich the product obtained in step 2) to obtain the immune peptide group sample; The separation refers to the separation of the immune peptide group from the MHC-peptide complex; The sample is selected from tissues or cells; Step 1) includes centrifugation to remove cell or tissue precipitates, collection of the supernatant, and passing the supernatant through a molecular trapping column to remove protein debris.

5. The method of claim 4, wherein, In step 1), the weak acid buffer solution is a freshly prepared weak acid buffer solution.

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