Weak acid buffer solution and application thereof in immune peptide group separation reagent

Abstract

The invention relates to the field of biology, in particular to a weak acid buffer solution and application thereof in an immune peptide group separation reagent, the weak acid buffer solution comprises amino acid and / or polypeptide, the mass-to-charge ratio of the amino acid and / or polypeptide is theoretically predictable and experimentally reproducible, or the mass-to-charge ratio exceeds a preset acquisition range of a mass spectrometer; the weak acid buffer solution further comprises volatile acid, and the volume concentration of the volatile acid is 0.01%-10%; the pH value of the weak acid buffer solution is 3.0 to 3.5. The weak acid buffer solution disclosed by the invention has high compatibility, and pollution and signal interference to an analysis system can be avoided to the greatest extent; the experimental process is simple and convenient, and the whole operation can be completed within tens of minutes, so that the technical threshold is low, and the acceptability is high; the identification number of the immune peptide group can be increased, so that the experiment efficiency is improved; the reagent is low in price and easy to obtain, so that the method is low in cost, suitable for large-scale use and even industrial-grade use, and relatively high in universality.

Description

Technical Field

[0001] The present invention relates to the biological field, in particular to a weak acid buffer and application thereof in an immune peptide group separation reagent. Background Art

[0002] 1. Definition and Application of Immunopeptidome

[0003] The immunopeptidome refers to the collection of peptides presented by major histocompatibility complex (MHC) molecules on the cell surface. These peptides, derived from degradation products of endogenous cellular proteins, are a core mechanism by which the immune system distinguishes between "self" and "non-self." In virology, immunopeptidome results provide crucial information on virus-specific antigenic epitopes, helping to unravel the mechanisms of host-virus interactions and providing key targets for vaccine design. Furthermore, immunopeptidomics has revealed viral escape mechanisms, such as how influenza and hepatitis C viruses evade immune recognition through mutations in their MHC binding sites, providing theoretical support for the development of broad-spectrum vaccines. In cancer immunotherapy, immunopeptidomics helps identify tumor-specific neoantigens for the development of personalized vaccines and T cell therapies, which have demonstrated significant efficacy in diseases such as melanoma and lung cancer. Furthermore, immunopeptidomics has important applications in the diagnosis of infectious diseases and the research of autoimmune diseases, such as early diagnosis through the detection of virus-specific MHC peptides and the analysis of abnormally presented autoantigenic peptides to reveal disease mechanisms.

[0004] 2. Existing immunopeptidome experimental methods and their limitations

[0005] Currently, immunopeptidome sample collection primarily relies on immunoaffinity methods: peptide enrichment using MHC-specific antibodies. However, this method is costly, complex, and can lead to nonspecific binding due to antibody cross-reactivity. As an alternative, mild acid elution (MAE) dissociates MHC-peptide complexes using a weakly acidic buffer, directly releasing peptides. This method is simple to use, low-cost, and economical, suitable for large-scale experiments, and has been used in high-quality publications in recent years.

[0006] Existing MAE methods generally use a citric acid-sodium phosphate (Na2HPO4) buffer system as a weak acid. Citric acid is a commonly used buffer in biochemical experiments, but it has several unavoidable drawbacks when used in immunopeptidase experiments, as follows:

[0007] 1) Difficulty in purification: Citric acid is a non-volatile acid and cannot be removed by spin drying or other methods. The C18 desalting columns commonly used in omics experiments also require multiple rounds of washing to gradually dilute the solution, and residual citric acid will still interfere with subsequent experiments.

[0008] 2) Poor compatibility of analytical instruments:

[0009] 2.1) In immunopeptidomic experiments, peptide samples often require high-performance liquid chromatography (HPLC) fractionation and peptide concentration determination using a NanoDrop instrument. Citric acid and its complexes with peptides and other salt components in the sample have strong UV absorption at 214 nm and 280 nm, which can mask the signal of the target peptide, leading to baseline drift and abnormal peak shape.

[0010] 2.2) During mass spectrometry analysis, residual buffer components and the resulting complexes can inhibit peptide ionization, generating unpredictable mass spectrometric interference signals that degrade the quality of acquired mass spectrometric data. After multiple consecutive analyses, the accumulation of unionized complexes at the needle can even damage the ion source.

[0011] Therefore, current MAE experiments require multiple washes during the cleaning step to reduce background interference. However, multiple washes may also cause the loss of low-abundance peptides, resulting in a decrease in the number of peptide identifications and reduced 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 been able to fundamentally solve the problem of buffer components interfering with downstream analysis.

[0013] In summary, traditional immunopeptidomic technologies such as weak acid elution based on citric acid buffer have bottlenecks such as loss of low-abundance peptides, mass spectrometry signal interference and low experimental efficiency, which limit their application in translation and clinical practice. Summary of the Invention

[0014] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a weak acid buffer and its use in an immune peptide group separation reagent to solve the problems in the prior art.

[0015] The core innovation of the present invention is to propose a new weak acid buffer system based on amino acids or short peptides, which can improve the overall experimental efficiency of the immune peptide group.

[0016] To achieve the above-mentioned and other related purposes, the present invention first provides a weak acid buffer, which 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 certain embodiments of the present invention, the amino acid or polypeptide has no ultraviolet absorption at 280 nm.

[0018] In certain embodiments of the present invention, the amino acids are selected from amino acids such as histidine, arginine, and alanine, which have stable hydrophilicity and hydrophobicity, a defined molecular weight, and generally do not complex with salts such as metal ions. The number of amino acids in the polypeptide is 3 or less, for example, 3 amino acids constitute a tripeptide, or 2 amino acids constitute a dipeptide.

[0019] The volatile acid is selected from formic acid, acetic acid, hydrochloric acid and the like.

[0020] The weak acid buffer may further comprise any 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 the chloride ions or sodium ions is, for example, 100 to 300 mM.

[0021] The present invention also provides use of the weak acid buffer in preparing a protein separation reagent.

[0022] The protein is selected from the group consisting of immunological peptides, antigens or antibodies.

[0023] The present invention also provides a method for separating immune peptide group samples, which comprises the following experimental steps:

[0024] 1) mixing the weak acid buffer with the sample and pipetting to elute the crude product of the immune peptide group from the sample;

[0025] 2) removing amino acids and / or polypeptides in the weak acid buffer in the product obtained in step 1);

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

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

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

[0029] As described above, the weak acid buffer of the present invention and its use in an immune peptide group separation reagent have the following beneficial effects:

[0030] 1. High compatibility. The main components of this buffer are amino acids or short peptides, which are combined with volatile acids to form a weak acid buffer system. The types of amino acids and short peptides can be flexibly configured according to the buffering capacity and other requirements of the experiment. Amino acid components such as histidine have no ultraviolet absorption contamination at 280nm, which can ensure the accuracy of HPLC fractionation and concentration detection of immune peptide group samples. In addition, these amino acids and short peptides are either not within the acquisition range of the mass spectrometer or have a stable and predictable mass-to-charge ratio (m / z), which can minimize contamination and signal interference of the analysis 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 signal of the analysis system.

[0031] 2. The experimental process is simple. Formic acid and acetic acid are volatile acids and can be easily removed by spin drying or other procedures. Amino acids and short peptides can be quickly removed using a C18 desalting column, reducing wash times and streamlining the process. Even if a small amount of residue remains, as mentioned above, it will not affect the analytical system. The entire process can be completed in tens of minutes, making the technical threshold low and the acceptance high.

[0032] 3. Increased identifications. Fewer washes reduce the risk of sample loss, ensuring that low-abundance peptides are retained and enter the analysis system. Furthermore, amino acids and peptides can act as carrier proteins, stabilizing hydrophobic MHC peptides and improving detection sensitivity. Overall, this results in an increased number of peptide identifications in the results, thereby improving experimental efficiency.

[0033] 4. Highly economical. Amino acids, short peptides, formic acid, and acetic acid are all commonly used industrial products, inexpensive and readily available. Therefore, this method is low-cost and suitable for large-scale, even industrial-scale, use. Furthermore, these components pose little chemical risk to the analytical system, causing no corrosion or clogging, thereby extending the life of the analytical system.

[0034] 5. Applicable to elution of products from other immunoassays. The fundamental 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 antigenic products. By disrupting hydrogen bonds between complexes, it achieves efficient elution of antigens. This is particularly true for target products such as IgA, which are difficult to obtain through immunoaffinity precipitation. Therefore, this method also has high universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shown is the UV absorption pollution evaluation chart of Example 1 of the present invention.

[0036] Figure 2 Shown is a mass spectrometry signal interference evaluation diagram of Example 1 of the present invention.

[0037] Figure 3 Shown is a graph showing the results of the elution evaluation of a single immune peptide according to Example 2 of the present invention.

[0038] Figure 4 Shown is a graph showing the evaluation results of the polypeptide identification quantity in Example 3 of the present invention.

[0039] Figure 5 Shown is a graph evaluating the elution effect of the immune peptide according to Example 3 of the present invention. DETAILED DESCRIPTION

[0040] This invention intends to improve the efficiency and reliability of immunopeptidase experiments 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, which includes amino acids and / or polypeptides. The mass-to-charge ratio (m / z) 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 a mass spectrometer. The weak acid buffer also includes a volatile acid, and the volume concentration of the volatile acid is 0.01%-10%. The pH of the weak acid buffer is 3.0 to 3.5.

[0042] The amino acid has a buffering effect and is used to adjust the pH of the weak acid buffer. The concentration of the amino acid is not specifically limited, and the pH of the weak acid buffer reaches 3.0 to 3.5 after the amino acid and the volatile acid are mixed. For example, under general experimental requirements, 5mM of neutral amino acid dissolved in 0.1% volatile acid can achieve the target pH range; in some cases, the buffer may need to have a stronger pH stabilizing ability, and in this case, the concentration of amino acids or acids needs to be increased to meet the requirements. In some cases, the buffer system may also need to have a stronger ability to act as a "carrier protein", and in this case, the concentration of hydrophobic amino acids in the buffer system needs to be increased to meet the requirements.

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

[0044] In certain embodiments of the present invention, the amino acid or polypeptide has no ultraviolet absorption at 280 nm, which can ensure the accuracy of HPLC fractionation and concentration detection of the immune peptide group sample.

[0045] The types of amino acids and peptides can be flexibly configured based on experimental requirements such as buffering capacity. For example, any amino acid or derivative thereof compatible with the experimental requirements can be selected, including but not limited to its isomers and the isomers of its derivatives. Any dipeptide or tripeptide compatible with the experimental requirements can be selected, including but not limited to its isomers. Amino acids and short peptides can also be mixed.

[0046] In certain embodiments of the present invention, the amino acids are selected from amino acids such as histidine, arginine, and alanine, which have stable hydrophilicity and hydrophobicity, a defined molecular weight, and do not complex with salts such as metal ions. The number of amino acids in the polypeptide is 3 or less, for example, 3 for a tripeptide, or 2 for a dipeptide.

[0047] The volatile acid is selected from formic acid, acetic acid, hydrochloric acid and the like.

[0048] In certain experiments, it may be desirable for the buffer system to maintain cell viability for a longer period of time. In such cases, the weak acid buffer may further include a variety of supplementary components. For example, in certain embodiments of the present invention, the weak acid buffer further includes any one or more of the following: protease inhibitors, chloride ions, sodium ions, magnesium ions, potassium ions, and the like. Based on the total volume of the weak acid buffer, the final concentration of the chloride ions or sodium ions is, for example, 100 to 300 mM.

[0049] Unless otherwise specified, the reagents of the present invention are all analytically pure or higher purity reagents.

[0050] The present invention also provides use of the weak acid buffer in preparing a protein separation reagent.

[0051] The protein separation reagent may also be referred to as a protein enrichment reagent or a protein collection reagent.

[0052] The protein is selected from the group consisting of immunogenic peptides, antigens or antibodies.

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

[0054] The present invention also provides a method for separating immune peptide group samples, which comprises the following experimental steps:

[0055] 1) mixing the weak acid buffer with the sample and pipetting to elute the crude product of the immune peptide group from the sample;

[0056] 2) removing amino acids and / or polypeptides in the weak acid buffer in the product obtained in step 1);

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

[0058] In certain 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 an isolated tissue or cell.

[0059] In certain embodiments of the present invention, in step 1), a freshly prepared weak acid buffer is mixed with the sample and slowly pipetted to achieve elution of the immune peptide group. Alternatively, this can be achieved using other operating methods such as microfluidic chips and robotic arm operating systems, which are also within the scope of protection of this patent.

[0060] Under typical experimental requirements, elution of the immune peptide panel can be completed within 1 minute at 4°C with 20 pipettings. In some cases, extending or reducing the number and duration of pipetting may meet additional experimental needs. Researchers can choose the appropriate treatment method based on their experimental requirements, and all of them are protected by this patent.

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

[0062] In step 2), amino acids and / or polypeptides in the weak acid buffer can be removed by using C18 column desalting, online desalting or low molecular weight retentate column, and non-polypeptide parts in the solution can also be removed.

[0063] The desalting step for the C18 column can be performed according to the manufacturer's instructions, but only one to two washes are needed to effectively remove buffer components. In some cases, if a high level of cell debris is produced during the experiment or a purer peptide product is desired, additional or prolonged centrifugation or molecular weight retention column purification, or increased C18 column wash times may be necessary. Researchers can choose the appropriate treatment method based on their experimental requirements, all of which are covered by this patent.

[0064] In step 3), the product can be concentrated using a rotary evaporator or enriched by other methods such as pre-column enrichment.

[0065] For the immune peptide group obtained in step 3), corresponding subsequent experiments can be carried out according to the specific experimental purpose. For example, after fractionation by HPLC, the absorption at 280 nm is measured to estimate the concentration. After gradient separation and elution of the mixed polypeptides by liquid chromatography, signal data is collected by mass spectrometry. Liquid chromatography can adopt a pre-column-analytical column tandem mode or an analytical column mode, and any gradient, mobile phase, flow rate and filling material required by the experiment can be used. After elution, the charged ions of the polypeptides that enter the mass spectrometer through the ion source are usually collected by tandem mass spectrometry, and any collection mode, resolution, etc. required by the experiment can be used.

[0066] The above steps are exemplified by immune peptides, but this scheme 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 describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0068] Before further describing the 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 specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0069] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical 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 those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0070] Example 1

[0071] Experimental purpose: To investigate the effects of several novel buffer systems on the interference of the analytical system and to compare them with the control group (the existing immune peptide group experimental scheme).

[0072] Experimental steps:

[0073] 1) Preparation of eluent.

[0074] For the formic acid-histidine buffer, prepare a 0.1% formic acid solution using HPLC-grade water. Add protease inhibitors (Pierce, Cat. No. A32955) to a final concentration of 1x and NaCl to a final concentration of 150 mM. Then, add histidine powder in small amounts to the solution, dissolve and mix thoroughly, and measure the pH of the solution using a pH meter until it reaches between 3.30 and 3.35 at 23°C. Filter the prepared eluate through a 0.22 μm filter and pre-cool on ice.

[0075] For the acetate-arginine buffer group, prepare a 1.5% acetic acid solution using HPLC-grade water. Add the same protease inhibitors as for the formate-histidine buffer group to a final concentration of 1x, and add NaCl to a final concentration of 150 mM. Then, add arginine powder to the solution in small amounts and multiple times. Dissolve and mix thoroughly, then measure the pH of the solution using a pH meter until it reaches between 3.30 and 3.35 at 23°C. Filter the prepared eluate through a 0.22 μm filter and pre-cool on ice.

[0076] For the control group, citric acid powder and sodium monohydrogen phosphate (Na2HPO4) powder were dissolved in HPLC-grade water. The same protease inhibitors as in the experimental group were added to a final concentration of 1x, and NaCl was added to a final concentration of 150 mM. The pH was then adjusted by adding NaOH or HCl in small amounts until the solution reached approximately 3.3 at 23°C. The prepared eluate was filtered through a 0.22 μm filter and pre-chilled on ice.

[0077] 2) Ultraviolet absorption pollution assessment.

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

[0079] 3) Liquid chromatography-tandem mass spectrometry system signal interference assessment

[0080] 1 μL of each of the two experimental buffers, control buffer, and mass spectrometry buffer was injected into a Bruker nanoElue2 nanoLC system coupled with a Bruker timsTOF Pro2 system for mass spectrometry signal interference assessment. The system's default mass spectrometry method used ddaPASEF, with a 30-minute gradient in dual-column mode and a flow rate of 500 nL / min. The resulting mass spectrometry ion chromatograms were analyzed using Bruker Data Analysis software.

[0081] result

[0082] 1) Ultraviolet absorption pollution assessment.

[0083] like Figure 1As shown, the control group exhibited strong A280 absorption between 1 and 4 minutes, with a peak absorption of 50 mAU. In contrast, the A280 absorption of both experimental groups was essentially consistent with the blank, with a peak absorption of only 1 mAU. This is due to systematic error caused by background noise. This result demonstrates that the buffer system of the present invention does not cause additional contamination of UV absorption.

[0084] 2) Mass spectrometry signal interference assessment.

[0085] like Figure 2 As shown, the control group exhibited a distinct mass spectrometry signal with a signal intensity of 4E6 during the 30-minute analytical gradient. In contrast, the experimental group exhibited very low mass spectrometry signals, with the exception of some characteristic mass spectrometry signals, along the rest of the gradient, essentially consistent with the blank sample. This result demonstrates that the buffer system of the present invention does not interfere with mass spectrometry signals.

[0086] Example 2

[0087] Experimental purpose: To evaluate the effect of weak acid buffer systems with different amino acid types on the elution of single immune peptides

[0088] Experimental steps:

[0089] 1) Preparation of eluent.

[0090] Four formic acid-amino acid buffer systems were prepared as experimental groups according to the method of Example 1: formic acid-histidine, formic acid-serine, formic acid-arginine, and formic acid-alanine. A control group was also prepared according to the method of Example 1. For the blank control group, phosphate-buffered saline (PBS) was used, and the same protease inhibitors as used in the experimental and control groups were added to a final concentration of 1x, and the cells were pre-chilled on ice.

[0091] 2) Elution of single immune peptide.

[0092] A single OVA peptide-MHC protein complex sample prepared and purified in the laboratory was divided into six groups, each containing 200 ng of protein in a volume of approximately 5 μL, with three biological replicates. The sample was transferred to a 0.6 mL protein low-binding centrifuge tube. Slowly add 50 μL of buffer to each tube and pipette gently 20 times.

[0093] 3) C18 column desalting.

[0094] The supernatant was desalted using a Thermo Fisher Scientific C18 Spin Tip desalting column, following the product instructions. To ensure experimental consistency, in this example, both the experimental and control groups underwent five washes to obtain purified single OVA polypeptides.

[0095] 4) Incorporation of quantitative peptides.

[0096] Lysine-labeled OVA polypeptide with 98% purity was ordered from Jeptide Biotechnology Co., Ltd., dissolved in ultrapure water, and spiked into the above groups at a rate of 200 pg per group for subsequent quantification by mass spectrometry analysis.

[0097] 5) Single immune peptide data collection.

[0098] The purified peptide obtained was concentrated in a rotary evaporator and re-dissolved in mass spectrometry buffer, and 1 / 2 was taken out for data acquisition. First, the peptide sample was separated by a Bruker nanoElue2 nanoLC system using a 15-minute gradient with a flow rate of 500nL / min. The peptide sample was enriched on a pre-column before entering the mass spectrometer and then separated on the analytical column. The prmPASEF data was collected using a TimsTOF Pro2 mass spectrometer system. The parameters were set to positive ion mode, a mass scan range of fixed m / z 482.2791 and 486.2861, a mass window of 0.5Da, and an ion mobility range of 1 / K0 0.75-1.3Vs / cm 2 A dynamic collision energy (20–59 eV) was used.

[0099] 6) Immune peptidome data analysis.

[0100] The acquired data were analyzed using Skyline spectral analysis software in PRM mode, with the mass-to-charge ratio of the OVA immune peptide set to 482.2791, the mass-to-charge ratio of the heavy-labeled standard peptide set to 486.2861, and a mass tolerance of 10 ppm. Quantitative statistical analysis of the mass spectrometry results was performed using Excel software. Relative quantification ratios were calculated by dividing the quantitative results of the OVA immune peptide in each group by those of the heavy-labeled standard peptide. Visualization was performed using the R programming language.

[0101] result

[0102] like Figure 3 As shown, the blank control group had a quantitative ratio of 0, while the control group and each experimental group produced relatively high relative quantitative ratios, ranging from 6 to 8.7. This indicates that both the control buffer system and the weak acid buffer systems of each experimental group can effectively elute the immune peptide. Weak acid buffer systems containing a variety of amino acids can elute the immune peptide, demonstrating the method's high flexibility in amino acid selection.

[0103] Example 3

[0104] Experimental purpose: To evaluate the effect of a new buffer system on the elution of immune peptide samples.

[0105] Experimental steps:

[0106] 1) Preparation of eluent.

[0107] A formic acid-histidine buffer system was prepared as the experimental group according to the method of Example 1. A control group was also prepared according to the method of Example 1. The solution was pre-chilled on ice.

[0108] 2) Cell culture and collection.

[0109] For the DC2.4 (murine dendritic cell) cell line, culture in a shake flask using standard culture 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 a 15 ml centrifuge tube and centrifuge at 200g and 4°C for 5 minutes. After aspirating the supernatant, resuspend the cell pellet in PBS buffer. Repeat the centrifugation and aspiration of the supernatant three times. Before the final wash, estimate the total number of cells using a fluorescent dye and a cell counter, and based on the results, divide the cells into six new 15 ml centrifuge tubes, with a total number of cells in each tube of approximately 1E7.

[0110] 4) Elution of immune peptide group or target product.

[0111] Slowly add 1 mL of buffer to each centrifuge tube containing the cell pellet. Three experimental and three control tubes were added. Slowly pipette up and down 20 times, with the total time controlled within 1 minute.

[0112] 5) Neutralization of eluent, gradient washing and purification by molecular retention column.

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

[0114] 6) C18 column desalting.

[0115] The supernatant was desalted using a Sep-Pak C18 desalting column manufactured by Waters, following the product instructions. To ensure experimental consistency, in this example, both the experimental and control groups were washed five times. Purified polypeptides were obtained.

[0116] 7) Immunopeptidome data collection.

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

[0118] 8) Immune peptidome data analysis.

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

[0120] result

[0121] 1) Peptide identification quantity evaluation.

[0122] like Figure 4 As shown, the number of peptide identifications in the three biological replicates of the control group ranged from 330 to 346, while the number of peptide identifications in the three biological replicates of the experimental group all reached over 400, more than double that of the control group. This result indicates that the buffer system of the present invention can significantly increase the number of peptide identifications.

[0123] 2) Evaluation of immune peptide elution effect.

[0124] Among the identified peptides, the present invention evaluated the motif distribution and additionally analyzed their MHC (H2-Kb type) affinity using the affinity prediction software MHC Flurry, and performed a Venn diagram analysis on high-affinity peptides. Figure 5 As shown in A, the experimental group and the control group showed very consistent motif distribution, indicating that the buffer system of the present invention will not bias the results of the immune peptide group. Figure 5 As shown in Figure B, the experimental group identified 55 more high-affinity peptides than the control group. This result shows that the buffer system of the present invention can achieve higher efficiency in immunopeptidase experiments.

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

Claims

1. A weak acid buffer, characterized in that The weak acid buffer comprises amino acids and / or polypeptides, 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, the volume concentration of the volatile acid is 0.01%-10%; the pH of the weak acid buffer is 3.0 to 3.

5.

2. The weak acid buffer according to claim 1, wherein The amino acid or polypeptide has no ultraviolet absorption at 280 nm.

3. The weak acid buffer according to claim 1, wherein The amino acid has stable hydrophilicity and hydrophobicity and does not complex with metal ions; preferably, the amino acid is selected from histidine, arginine or alanine; and / or the number of amino acids in the polypeptide is 3 or less.

4. The weak acid buffer according to claim 1, wherein The volatile acid is selected from formic acid, acetic acid or hydrochloric acid.

5. The weak acid buffer according to claim 1, wherein The weak acid buffer further comprises any one or more of the following: protease inhibitors, chloride ions, sodium ions, magnesium ions, and potassium ions; preferably, based on the total volume of the weak acid buffer, the final concentration of the chloride ions or sodium ions is 100 to 300 mM.

6. Use of the weak acid buffer according to any one of claims 1 to 5 in the preparation of a protein separation reagent.

7. The use according to claim 6, characterized in that The protein is selected from the group consisting of immunogenic peptides, antigens or antibodies.

8. A method for separating immune peptide samples, characterized in that: The method comprises the following experimental steps: 1) mixing the weak acid buffer according to any one of claims 1 to 5 with the sample and pipetting to elute the crude immunopeptide product from the sample; 2) removing amino acids and / or polypeptides in the weak acid buffer in the product obtained in step 1); 3) Concentrating or enriching the product obtained in step 2) to obtain the immune peptide group sample.

9. The method according to claim 8, characterized in that In step 1), the weak acid buffer is a newly prepared weak acid buffer; and / or the sample is selected from tissues or cells.

10. The method according to claim 8, characterized in that After step 1), centrifugation is further performed to remove cell or tissue precipitates and to collect the supernatant. Preferably, the supernatant is passed through a molecular retention column to remove protein fragments.

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