Method for constructing spatial proteomics sequencing library from paraffin-embedded tissue sections and sequencing method

Abstract

Provided is a method for constructing a spatial proteomics sequencing library from paraffin-embedded tissue sections. The method comprises: providing a solid-phase carrier and an antibody-nucleic acid conjugate; attaching a paraffin-embedded tissue section sample to the solid-phase carrier and carrying out baking, dewaxing, hydration, de-crosslinking, and blocking treatments to obtain a blocked solid-phase carrier; incubating the antibody-nucleic acid conjugate and the blocked solid-phase carrier to obtain a protein-captured solid-phase carrier; carrying out a permeabilization treatment and DNA synthesis on the protein-captured solid-phase carrier to obtain cDNA; and releasing the cDNA from the solid-phase carrier, collecting the cDNA, and amplifying the cDNA to obtain a sequencing library.

Description

Construction method and sequencing method for spatial proteomics sequencing library of tissue paraffin sections Technical Field

[0001] The present invention relates to the field of biology, and in particular to a method for constructing a spatial proteomics sequencing library for tissue paraffin sections and a sequencing method. Background Art

[0002] Immunohistochemistry (abbreviated as immunohistochemistry) refers to the application of the basic principles of immunology, namely the principle of specific binding between antigens and antibodies, to determine the antigens (peptides and proteins) in tissue cells through chemical reactions to make the color developer (fluorescein, enzyme, metal ion, isotope) labeled with antibodies develop color, and conduct localization, qualitative and relative quantitative research on them.

[0003] Although traditional immunohistochemistry is widely used, the types of antibodies that can be detected by immunohistochemistry technology are very limited, and it can only detect one or several markers on a single slice. Currently, the detection of multiple proteins on a single slice has become a trend, and the current mainstream technologies are mainly divided into two categories: (1) Products based on cyclic hybridization imaging technology, such as Akoya Bioscience's Phenocycler platform, Nanostring's CosMX platform, and 10Xgenomics' Xenium platform. (2) Technologies that use spatial barcode arrays for analyte detection, such as 10Xgenomics' Visium platform and BGI's Stereo-seq technology. All of the above can achieve the simultaneous detection of ultra-high-weight proteins on a single slice.

[0004] However, both of the above two categories of technologies have certain technical defects. For example, for cyclic hybridization imaging technology, since the fluorescent signals that can be detected in one round are limited, multiple rounds of hybridization-photography-elution cycles are required. This cyclic detection will inevitably cause antigen instability on the tissue. And as the number of detection targets increases, the number of cycles required also needs to increase, so the detection time for a sample will increase accordingly. In addition, in some tissues, the presence of autofluorescence will also affect the interpretation of positive staining results. Moreover, the above technology cannot achieve quantitative detection of protein abundance on a single slice. For the spatial barcode array technology Visium platform, although quantitative detection can be achieved, its current resolution can only reach 100 microns. This size means that it contains multiple cell types, and its detection accuracy cannot reach the single-cell level.

[0005] Therefore, the existing spatial proteomics technologies still need to be improved.

[0006] Summary of the Invention

[0007] The present invention aims to solve, at least to a certain extent, the technical problems existing in the prior art. To this end, the present invention proposes a method for constructing a spatial proteomics sequencing library, a spatial proteomics detection method, and a kit for constructing a spatial proteomics sequencing library. The method of the present invention can achieve multiple protein detection on tissue paraffin section samples, with high spatial resolution, no autofluorescence interference, no antigen instability caused by multiple rounds of detection, and an increase in the number of protein detection multiplicities without affecting the detection time. It can detect the expression abundance of different proteins in the sample and has high application value.

[0008] In one aspect of the present invention, the present invention proposes a method for constructing a spatial proteomics sequencing library. According to an embodiment of the present invention, the method includes: providing a solid phase carrier and an antibody-nucleic acid conjugate, wherein a probe is connected to the solid phase carrier, the probe includes a capture sequence and a spatial positioning sequence, and the capture sequence is located at the end of the probe away from the solid phase carrier; the antibody-nucleic acid conjugate includes an antibody and a nucleic acid sequence coupled to the antibody, the nucleic acid sequence includes an antibody sequence and a capture complementary sequence in the direction away from the antibody, and the capture sequence is at least partially complementary to the capture complementary sequence; attaching a tissue paraffin section sample to the solid phase carrier, baking, dewaxing, hydrating, and lysing the sample. Cross-linking and blocking treatment are performed to obtain a closed solid phase carrier; the antibody-nucleic acid conjugate is incubated with the closed solid phase carrier to obtain a protein capture solid phase carrier; the protein capture solid phase carrier is permeabilized so that the capture sequence is combined with the capture complementary sequence to obtain a permeabilized solid phase carrier; DNA synthesis is performed on the permeabilized solid phase carrier to extend the complementary sequence of the nucleic acid sequence on the capture sequence or extend the complementary sequence of the probe on the nucleic acid sequence to obtain cDNA; the cDNA is released from the solid phase carrier, the cDNA is collected and amplified to obtain a sequencing library.

[0009] According to the method of an embodiment of the present invention, the antibody-nucleic acid conjugate can specifically bind to the antigen in the tissue paraffin section (also referred to as "FFPE" in the present invention) sample, and through tissue permeabilization, the capture complementary sequence in the antibody-nucleic acid conjugate is combined with the capture sequence and hybridized, and then DNA synthesis is performed to extend the complementary sequence of the antibody nucleic acid sequence on the capture sequence or extend the complementary sequence in the probe on the nucleic acid sequence to obtain cDNA with spatial information and antibody information, and then the cDNA is amplified to obtain a sequencing library. In this way, multiple protein detection on tissue paraffin section samples can be achieved, with high spatial resolution, no autofluorescence interference, no antigen instability caused by multiple rounds of detection, and the increase in the number of protein detection does not affect the detection time, and the expression abundance of different proteins in the sample can be detected, with high application value.

[0010] In another aspect, the present invention provides a spatial proteomics detection method. According to an embodiment of the present invention, the method comprises: constructing a sequencing library using the aforementioned method for constructing a spatial proteomics sequencing library; and sequencing the sequencing library.

[0011] In another aspect, the present invention provides a kit for constructing a spatial proteomics sequencing library. According to an embodiment of the present invention, the kit includes: a cross-linking decontamination reagent and a permeabilization reagent; the cross-linking decontamination reagent includes collagenase and Tris-EDTA buffer; and the permeabilization reagent includes: phosphate buffer, Tween, nonylphenoxypolyethoxyethanol, and digitonin.

[0012] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0014] FIG1 shows a schematic flow chart of a method for constructing a spatial proteomics sequencing library according to one embodiment of the present invention;

[0015] FIG2 shows a schematic diagram of the structure of a solid phase carrier connected with a probe according to one embodiment of the present invention;

[0016] FIG3 shows a schematic structural diagram of an antibody-nucleic acid conjugate according to one embodiment of the present invention;

[0017] FIG4 shows a schematic diagram of hybridization and extension between an antibody-nucleic acid conjugate and a probe according to one embodiment of the present invention;

[0018] FIG5 shows a schematic flow chart of a method for constructing a spatial proteomics sequencing library according to another embodiment of the present invention;

[0019] FIG6 shows a diagram of tissue section detection under different cross-linking reagent conditions according to one embodiment of the present invention;

[0020] FIG7 shows a diagram of tissue section detection under different de-crosslinking time conditions according to one embodiment of the present invention;

[0021] FIG8 shows a diagram of tissue section detection under different collagenase concentrations according to one embodiment of the present invention;

[0022] FIG9 shows a diagram of tissue section detection under different fixative conditions according to one embodiment of the present invention;

[0023] FIG10 shows a diagram of tissue section detection under different elution reagent conditions according to one embodiment of the present invention;

[0024] FIG. 11 shows a diagram of tissue section detection under different permeabilization reagent conditions according to an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0027] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0028] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0029] The present invention proposes a method for constructing a spatial proteomics sequencing library, a spatial proteomics detection method, and a kit for constructing a spatial proteomics sequencing library, which will be described in detail below.

[0030] Methods for constructing spatial proteomics sequencing libraries

[0031] In one aspect, the present invention provides a method for constructing a spatial proteomics sequencing library. According to an embodiment of the present invention, referring to Figure 1 , the method comprises: S100 providing a solid phase support and an antibody-nucleic acid conjugate; S200 attachment, baking, dewaxing, hydration, cross-linking, and blocking; S300 antibody incubation; S400 permeabilization; S500 DNA synthesis; and S600 release and amplification. Each step is described in detail below.

[0032] S100 provides solid phase carriers and antibody-nucleic acid conjugates

[0033] In this step, a solid support and an antibody-nucleic acid conjugate are provided (see Figure 2). A probe is attached to the solid support. The probe includes a capture sequence and a spatial positioning sequence. The capture sequence is located at the end of the probe away from the solid support and can be used to capture the antibody-nucleic acid conjugate. The spatial positioning sequence can be used to spatially position the probe. In some embodiments, the solid support is a chip.

[0034] According to embodiments of the present invention, the probe further comprises an excision region, a second amplification linker sequence (also referred to herein as "amplification linker B sequence"), and a UMI sequence. The excision region is connected to the solid support. The excision region can be chemically bonded to the solid support, or the nucleic acid can be released from the solid support using chemical reagents or other means. The second amplification linker can be used for subsequent PCR amplification, and the UMI sequence can be used for quantification and removal of repetitive sequences introduced by subsequent PCR.

[0035] It should be noted that the purpose of designing the excision region in the present invention is mainly to achieve the purpose of nucleic acid separation by excising the excision region after the reaction is completed. There is no special limitation on the sequence information of the excision region. Nucleic acid sequence information known in the art can be used, such as uracil-specific excision reagent (Uracil-Specific Excision Reagent, USER) that can produce a single nucleotide gap at the dU position of DNA, or multiple peptide bonds, such as proteases that can hydrolyze multiple peptide bonds.

[0036] Referring to the antibody-nucleic acid conjugate structure in Figure 3, the antibody-nucleic acid conjugate includes an antibody and a nucleic acid sequence coupled to the antibody. The nucleic acid sequence includes an antibody sequence and a capture complementary sequence in the direction away from the antibody, and the capture sequence is at least partially complementary to the capture complementary sequence. The antibody achieves the purpose of capturing the antigen by specifically binding to the antigen. The capture complementary sequence contained in the nucleic acid sequence can hybridize and bind with the capture sequence on the solid phase carrier, thereby facilitating the capture of the antibody-nucleic acid conjugate carrying the antigen onto the solid phase carrier. The antibody sequence records the identity information of the antibody for antibody identification.

[0037] According to an embodiment of the present invention, the nucleic acid sequence further includes a first amplification linker sequence (also referred to herein as "amplification linker A sequence"), which is connected to the antibody sequence, which is in turn connected to the capture complementary sequence. Thus, the nucleic acid sequence can be used for subsequent PCR amplification and can avoid loss of the antibody sequence.

[0038] According to an embodiment of the present invention, the capture sequence is a Poly T sequence, and the complementary sequence of the capture sequence is a Poly A sequence.

[0039] According to an embodiment of the present invention, the probes containing the Poly T sequence are multiple clusters, and the distance between two adjacent clusters is 495 nm to 505 nm; the length of the Poly A sequence is 22 bp to 35 bp.

[0040] It should be noted that the antibody-nucleic acid conjugates in this application are not particularly limited and can be reasonably selected according to the research purpose. For example, they can be selected from any one or more of the following antibodies: CD169 antibody, CD3 antibody, CD8 antibody, CD19 antibody, CD20, CD21, CD45R-B220, CD163, CD38, CD11b, CD140a, CD335, CD371, CD90.2, TER-119, CD274, CD279, CD56, CD14, CD340, CD324, CD38, CD29, CD68, CD44, CD21 / 35, IgD, CD5, CD4, IgM, CD79b, CD11c, F4 / 80, CD27, CD31 and CD8a. It should also be noted that the probe capture sequence in this application is not limited to PolyT and can be any other sequence.

[0041] In the preferred embodiment described above, the spacing between the two Poly T cluster probes on the chip is set at approximately 500 nm, facilitating colocalization of the entire transcriptome and proteome at 0.5 μm resolution. Since the diameter of a single cell is typically around 10 μm, this application enables protein detection at single-cell resolution, approaching that of immunofluorescence (the gold standard, with a resolution of 0.2 μm).

[0042] S200 attachment, baking, dewaxing, hydration, decrosslinking and blocking

[0043] In this step, the tissue paraffin section sample is attached to a solid phase carrier, and is subjected to baking, dewaxing, hydration, cross-linking removal and blocking treatment to obtain a blocked solid phase carrier.

[0044] By baking FFPE samples, the waxed tissue sections are firmly adhered to the solid phase carrier, preventing the sections from falling off during subsequent operations. Baking also preserves the immune reactivity in the tissue sections at room temperature for a longer period of time.

[0045] The main purpose of dewaxing is to restore the tissue sample to a water-soluble state for subsequent antigen repair and antibody staining, to ensure that the antibody can fully bind to and react with the antigen in the tissue, and to avoid deformation and incomplete immersion of the tissue section in subsequent operations through hydration.

[0046] In histological studies, tissue samples are often fixed to maintain their morphological structure and protein stability. This fixation usually uses a cross-linking agent (such as formalin) to fix proteins and nucleic acids to prevent them from degradation and loss of structure during the experiment. However, this fixation may cause protein cross-linking, making its structure more stable, but also limiting the binding between antibodies and proteins and nucleic acids. Therefore, a cross-linking removal step becomes a necessary operation to remove the cross-links between proteins and make them easier for antibodies to bind.

[0047] According to an embodiment of the present invention, the reagents used in the cross-linking deconvolution treatment include a collagenase solution and a Tris-EDTA buffer. The inventors have discovered that the type of reagent used in the cross-linking deconvolution treatment can significantly affect the uniformity and signal intensity of the sequencing analysis, thereby affecting the accuracy of the sequencing analysis. Furthermore, after extensive experiments, the inventors found that treating FFPE samples with a collagenase solution and a Tris-EDTA buffer can effectively deconvolute cross-linked protein molecules.

[0048] It should be noted that the collagenase solution and Tris-EDTA buffer of the present invention are provided separately and not in the form of a mixed solution.

[0049] According to an embodiment of the present invention, the collagenase is selected from type I collagenase, and the concentration of the collagenase solution is 0.2 to 1.0 U / μL. In some embodiments, the concentration of the collagenase solution is 0.2 U / μL, 0.4 U / μL, 0.5 U / μL, 0.6 U / μL, 1.0 U / μL, or a range between any two values ​​as endpoint values, preferably 0.4 to 0.6 U / μL. Thus, the cross-linking between the antigen molecules is further effectively decomposed, which helps the subsequent antibody recognition.

[0050] According to an embodiment of the present invention, the cross-linking treatment includes: incubating the collagenase solution with the hydrated solid phase carrier at 35-39°C for 10-40 minutes, and discarding the liquid; the solid phase carrier treated in the previous step and Tris-EDTA buffer in a hot water bath at 95-100°C for 15-25 minutes, and then lowering the temperature of the solid phase carrier to room temperature in the water bath. In some embodiments, the collagenase incubation temperature is 35°C, 36°C, 37°C, 38°C, 39°C or any two values ​​as a range between the endpoint values, the collagenase incubation time is 10min, 20min, 30min, 40min or any two values ​​as a range between the endpoint values, preferably 20-40min, the hot water bath temperature is 95°C, 96°C, 97°C, 98°C, 99°C, 100°C or any two values ​​as a range between the endpoint values, and the hot water bath time is 15min, 18min, 20min, 22min, 25min or any two values ​​as a range between the endpoint values.

[0051] The solid phase carrier after the cross-linking treatment is blocked to prevent non-specific binding of the antibody to certain sites on the tissue section and the solid phase carrier, which may cause false positive results. According to an embodiment of the present invention, the blocking reagent used in the blocking treatment includes: a sequence at least partially complementary to the capture sequence, salmon sperm DNA, an Fc receptor blocker, serum, and a buffer. In some embodiments, the sequence at least partially complementary to the capture sequence is selected from Poly A. As a result, the sequencing results are accurate and stable, and the background signal is low.

[0052] According to an embodiment of the present invention, the blocking reagent includes: 5-30 μM Poly A; 1-3 mg / mL salmon sperm DNA; 2-10% by volume of an Fc receptor blocker; 10-20% by volume of serum, and a buffer selected from a PBS buffer containing Triton-X100. In some embodiments, the concentration of Poly A is 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, or a range between any two values ​​as endpoints; the concentration of salmon sperm DNA is 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 3 mg / mL, or a range between any two values ​​as endpoints; the concentration of the Fc receptor blocker is 2% by volume, 3% by volume, 5% by volume, 8% by volume, 10% by volume, or a range between any two values ​​as endpoints; and the concentration of serum is 10% by volume, 12% by volume, 15% by volume, 18% by volume, 20% by volume, or a range between any two values ​​as endpoints. Therefore, the accuracy and stability of sequencing results can be further improved by using the blocking reagent of the above composition for treatment.

[0053] According to an embodiment of the present invention, based on the tissue species selection, for human tissue, the Fc receptor blocker is selected from Human TruStain FcX TM (Cat.No.422301), for mouse tissue, Fc receptor blocker selected from TruStain FcX TM PLUS (anti-mouse CD16 / 32) Antibody (Cat. No. 156604), serum was selected from mixed serum, including horse serum and goat serum (1:1).

[0054] S300 antibody incubation

[0055] In this step, the antibody-nucleic acid conjugate is incubated with a blocked solid support to form a protein capture solid support. The antibodies on the antibody-nucleic acid conjugate can specifically bind to the antigens on the FFPE sample, achieving the capture of the target protein.

[0056] For intracellular proteins, the blocked solid phase carrier can be permeabilized before antibody incubation to change the permeability of the cell membrane, allowing the antibody to enter the cell and specifically bind to the intracellular protein, thereby achieving the purpose of efficiently capturing the intracellular protein.

[0057] In order to confirm whether the antibody-nucleic acid conjugate is successfully bound to the probe on the solid phase carrier, secondary antibody incubation and staining can be optionally performed after antibody incubation, and then fluorescence color development or other fluorescent marker detection and photography can be performed.

[0058] S400 permeabilization

[0059] In this step, the protein capture solid support is permeabilized to allow the capture sequence to bind to the capture complementary sequence, resulting in a permeabilized solid support. This permeabilization allows the capture complementary sequence in the antibody-nucleic acid conjugate to hybridize and bind to the capture sequence on the solid support, thereby capturing the antibody-nucleic acid conjugate carrying the antigen.

[0060] According to an embodiment of the present invention, the permeabilization reagent used in the permeabilization treatment includes: a buffer and a surfactant, and the surfactant includes at least one of the following: Triton TM X-100, saponin, methanol, acetone, Tween, nonylphenoxypolyethoxyethanol, and digitonin. Combining these permeabilization agents can achieve enhanced permeabilization, reduce antibody diffusion, improve clarity, and facilitate hybridization between capture sequences and complementary capture sequences. Tween, nonylphenoxypolyethoxyethanol, and digitonin are particularly effective as surfactants.

[0061] According to an embodiment of the present invention, the permeabilization reagent includes: 0.05-0.5% by volume of Tween 20; 0.05-0.5% by volume of nonylphenoxypolyethoxyethanol; 0.00001-0.1% by volume of digitonin; and the balance is 2× sodium citrate buffer. In some embodiments, the concentration of Tween 20 is 0.05% by volume, 0.2% by volume, 0.4% by volume, 0.5% by volume, or a range between any two values; the concentration of nonylphenoxypolyethoxyethanol is 0.05% by volume, 0.1% by volume, 0.2% by volume, 0.5% by volume, or a range between any two values; and the concentration of digitonin is 0.00001% by volume, 0.0001% by volume, 0.001% by volume, 0.01% by volume, 0.1% by volume, or a range between any two values.

[0062] According to an embodiment of the present invention, the protein capture solid phase support is eluted and fixed before permeabilization. The inventors have discovered that elution can improve the signal-to-noise ratio of detection and reduce the signal in blank areas of tissue. Fixation prevents the antibody-nucleic acid conjugate from shifting during the permeabilization process, reduces antibody diffusion, and improves clarity.

[0063] According to an embodiment of the present invention, the elution reagent used in the elution treatment includes at least one of a formamide solution and a dimethyl sulfoxide solution. Thus, the use of such an elution reagent can further improve the signal-to-noise ratio of the detection and reduce nonspecific signals in tissue and blank areas. Among them, a formamide solution is preferred.

[0064] According to an embodiment of the present invention, the concentration of the formamide solution is 40 to 100 volume%, and the concentration of the dimethyl sulfoxide solution is 50 to 100 volume%. In some embodiments, the concentration of the formamide solution is 40 volume%, 50 volume%, 60 volume%, 80 volume%, 100 volume%, or a range value between any two of the values ​​as endpoints, and the concentration of the dimethyl sulfoxide solution is 50 volume%, 60 volume%, 80 volume%, 100 volume%, or a range value between any two of the values ​​as endpoints. Thus, the signal-to-noise ratio of the detection can be further improved, and non-specific signals in tissues and blank areas can be reduced.

[0065] According to an embodiment of the present invention, the temperature of the elution treatment is 20-70°C, and the time is 5-30 minutes. In some embodiments, the temperature of the elution treatment is 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, or a range between any two values ​​as endpoint values, and the time is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or a range between any two values ​​as endpoint values.

[0066] According to an embodiment of the present invention, the fixation treatment includes incubating the eluted solid support with a fixation reagent, wherein the fixation reagent includes at least one of a methanol solution, a formaldehyde solution, and a paraformaldehyde solution. Thus, the use of the above fixation reagent can further reduce the degree of antibody diffusion and improve clarity.

[0067] According to an embodiment of the present invention, the concentration of the methanol solution is 95-100% by volume, the concentration of the formaldehyde solution is 10-20% by volume, and the concentration of the paraformaldehyde solution is 1-6% by volume. In some embodiments, the concentration of the methanol solution is 95% by volume, 96% by volume, 97% by volume, 98% by volume, 99% by volume, 100% by volume, or a range between any two of the values ​​as endpoints, the concentration of the formaldehyde solution is 10% by volume, 12% by volume, 15% by volume, 18% by volume, 20% by volume, or a range between any two of the values ​​as endpoints, and the concentration of the paraformaldehyde solution is 1% by volume, 2% by volume, 4% by volume, 6% by volume, or a range between any two of the values ​​as endpoints.

[0068] According to an embodiment of the present invention, the fixed treatment temperature of the formaldehyde solution or paraformaldehyde solution is 20 to 30°C (for example, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C or any two values ​​as the range value between the endpoint values), and the time is 1 to 10 min (for example, 1 min, 2 min, 4 min, 5 min, 6 min, 8 min, 10 min or any two values ​​as the range value between the endpoint values); the fixed treatment temperature of the methanol solution is -25 to -15°C (for example, -25°C, -22°C, -20°C, -18°C, -15°C or any two values ​​as the range value between the endpoint values), and the time is 1 to 10 min (for example, 1 min, 2 min, 4 min, 5 min, 6 min, 8 min, 10 min or any two values ​​as the range value between the endpoint values).

[0069] S500 DNA synthesis

[0070] In this step, the permeabilized solid phase support is subjected to DNA synthesis to extend the complementary sequence of the nucleic acid sequence on the capture sequence or to extend the complementary sequence of the probe on the nucleic acid sequence to obtain cDNA.

[0071] As shown in FIG4 , through a DNA synthesis reaction such as reverse transcription, the antibody sequence complementary sequence and the first amplification linker complementary sequence are extended on the probe, thereby obtaining a cDNA having spatial information and antibody information.

[0072] S600 release and amplification

[0073] In this step, the cDNA is released from the solid support, collected, and amplified to produce a sequencing library. Specifically, when extending the complementary sequence of the probe on the nucleic acid sequence, the cDNA can be released from the solid support by excising the excision region above the probe. When extending the complementary sequence of a nucleic acid sequence other than the capture complementary sequence on the capture sequence, the generated cDNA can be released from the template strand by melting.

[0074] According to an embodiment of the present invention, the method further comprises: preparing the amplified product obtained by amplification into DNA nanoballs.

[0075] The following, with reference to Figure 5, further details the construction method for a spatial proteomics sequencing library. After attaching the FFPE sample to the chip, the chip is baked to prevent detachment, dewaxed, hydrated, and cross-linked, then blocked to prevent nonspecific binding of the antibody-nucleic acid conjugate to the FFPE and chip. During the antibody incubation step, the antibody-nucleic acid conjugate and a corresponding negative control antibody-nucleic acid conjugate are added. A series of wash steps are then performed to retain the antibody-nucleic acid conjugate bound to the specific antigen. To confirm successful binding of the antibody-nucleic acid conjugate, a secondary antibody incubation and fluorescence development or other fluorescent marker detection and imaging can be performed. Subsequently, tissue permeabilization is performed to allow the antibody-conjugated nucleic acid sequence to bind and hybridize with the probes on the chip. Subsequently, through DNA synthesis reactions such as reverse transcription (Figure 4), the complementary sequence of the antibody sequence and the complementary sequence of the amplification adapter A are extended onto the probe to obtain cDNA containing both spatial and antibody information. These cDNAs are then released from the chip and recovered by magnetic bead purification for sequencing library preparation and sequencing. By sequencing the spatial positioning sequence, antibody sequence and UMI sequence, the type and quantity of antibodies bound at a specific spatial location can be determined.

[0076] Spatial proteomics detection methods

[0077] In another aspect, the present invention provides a spatial proteomics detection method. According to an embodiment of the present invention, the method comprises: constructing a sequencing library using the aforementioned method for constructing a spatial proteomics sequencing library; and sequencing the sequencing library. Thus, the method of the present invention can be used to conduct spatial proteomics research based on paraffin-embedded tissue sections, obtaining information about proteins at specific spatial locations.

[0078] It should be noted that the features and advantages described above for the construction method of spatial proteomics sequencing library are also applicable to this spatial proteomics detection method and will not be repeated here.

[0079] Kit for spatial proteomics sequencing library construction

[0080] In another aspect, the present invention provides a kit for constructing a spatial proteomics sequencing library. According to an embodiment of the present invention, the kit comprises: a cross-linking decontamination reagent and a permeabilization reagent; the cross-linking decontamination reagent comprises collagenase and Tris-EDTA buffer; and the permeabilization reagent comprises a buffer, Tween, nonylphenoxypolyethoxyethanol, and digitonin. The buffer is selected from a phosphate buffer or a sodium citrate buffer.

[0081] According to an embodiment of the present invention, the collagenase is selected from type I collagenase; and the concentration of the collagenase solution is 0.1 to 0.8 U / μL.

[0082] According to an embodiment of the present invention, the permeabilization reagent includes: 0.05-0.5 volume % Tween 20; 0.05-0.5 volume % nonylphenoxypolyethoxyethanol; 0.00001-0.1 volume % digitonin; and the balance 2× sodium citrate buffer.

[0083] According to an embodiment of the present invention, the kit further includes at least one of the following: a blocking reagent, an elution reagent and a fixation reagent; the blocking reagent includes: Poly A, salmon sperm DNA, Fc receptor blocker, serum and buffer; the elution reagent includes at least one of a formamide solution and a dimethyl sulfoxide solution; the fixation treatment includes incubating the solid phase carrier after the elution treatment with the fixation reagent, and the fixation reagent includes at least one of a methanol solution, a formaldehyde solution and a paraformaldehyde solution.

[0084] According to an embodiment of the present invention, the blocking reagent includes: 5-30 μM Poly A; 1-3 mg / mL salmon sperm DNA; 2-10% by volume of Fc receptor blocker; 10-20% by volume of serum; a buffer solution selected from PBS buffer solution containing Triton-X100; and an Fc receptor blocker selected from Human TruStain FcX TM (Cat.No.422301) or TruStain FcX TM PLUS (anti-mouse CD16 / 32) Antibody (Cat. No. 156604); the concentration of the formamide solution is 40-100% by volume, the concentration of the dimethyl sulfoxide solution is 50-100% by volume; the concentration of the methanol solution is 95-100% by volume, the concentration of the formaldehyde solution is 10-20% by volume, and the concentration of the paraformaldehyde solution is 1-6% by volume.

[0085] According to an embodiment of the present invention, the elution reagent is selected from formamide solution.

[0086] It should be noted that the features and advantages of the cross-linking removal reagent, permeabilization reagent, blocking reagent, elution reagent and fixation reagent described above for the construction method of the spatial proteomics sequencing library are also applicable to this kit and will not be repeated here. Beneficial effects

[0087] 1. The spatial proteomics sequencing library construction method and sequencing method of the present invention can be used to achieve spatial proteomics analysis of paraffin section tissues.

[0088] 2. Compared with cyclic imaging technology, the present invention has no autofluorescence interference and no antigen instability caused by multiple rounds of cyclic detection. The increase in the number of protein detection multiplexes does not affect the detection time, and the expression abundance of different proteins in the sample can be detected.

[0089] 3. Compared with the Visium platform, the present invention can achieve protein detection with high spatial resolution (0.5 microns).

[0090] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0091] Example 1

[0092] Experimental tissue: human tonsil FFPE tissue.

[0093] Sources of reagents: as shown in Table 1.

[0094] Table 1 Sources of reagents

[0095] (3) Reagent preparation

[0096] Type I collagenase working solution: Dilute the type I collagenase stock solution to 0.4 U / μL with HBSS.

[0097] Mixed serum: Thaw serum at -20℃, mix 10μL horse serum with 10μL goat serum in a ratio of 1:1, centrifuge at 14000g for 10 min at 4℃, and collect the supernatant.

[0098] Blocking reagent preparation and permeabilization reagent configuration: The blocking reagent composition and permeabilization reagent composition are shown in Table 2 and Table 3, respectively.

[0099] Table 2 Blocking reagent composition

[0100] Table 3 Permeabilization reagent composition

[0101] (IV) Experimental steps

[0102] 1. Baked slices

[0103] Bake the tissue slices at 60°C for 1-2 hours

[0104] 2. Dewaxing and Rehydration

[0105] After baking, the slides were immediately dewaxed and rehydrated. The process is shown in Table 4.

[0106] Table 4 Dewaxing and rehydration procedures

[0107] 3. Decrosslinking

[0108] The decrosslinking step was divided into I and II, and the experimental group was divided into four groups according to different combinations of decrosslinking conditions.

[0109] Table 5 Decrosslinking conditions

[0110] a) Add 100 μL of collagenase type I working solution (diluted to 0.4 U / μL in HBSS) to the chip, ensuring that the liquid evenly covers the entire chip. Incubate at 37°C for 20 min; alternatively, skip steps ae) and proceed directly to step f).

[0111] b) Use a pipette to aspirate the cross-linking reagent from one corner of the chip, removing as much surface liquid as possible while keeping the chip moist.

[0112] c) Add PBS to the chip at a volume of 100 μL / chip;

[0113] d) Tilt the culture dish and use a pipette to add PBS from one corner of the chip to minimize the surface liquid while keeping the chip moist.

[0114] e) Repeat steps c and d once;

[0115] f) Immerse the chip in a box containing Tris-EDTA (TE) or sodium citrate antigen retrieval solution in a 99°C water bath for 20 minutes;

[0116] g) After de-crosslinking is completed, remove the box and place in a cold water bath for 5 minutes;

[0117] h) Remove the chip and add 100 μL of washing buffer per chip;

[0118] i) Use a pipette to aspirate the wash buffer from one corner of the chip, removing as much surface liquid as possible while keeping the chip moist.

[0119] j) Repeat steps h and i once.

[0120] 4. Closed

[0121] Add the prepared blocking reagent to the chip at a volume of 50 μL / chip, ensuring that the blocking reagent evenly covers the entire chip, and incubate at room temperature for 30 minutes.

[0122] 5. Primary Antibody Incubation

[0123] The preparation of primary antibody incubation solution is shown in Table 6.

[0124] Table 6 Composition of primary antibody incubation solution

[0125] a) Use a pipette to aspirate the blocking reagent from one corner of the chip, removing as much surface liquid as possible while keeping the chip moist.

[0126] b) Add the prepared antibody incubation solution to the chip at a rate of 50 μL / chip, ensuring that the solution evenly covers the entire chip, and incubate at room temperature for 45 minutes.

[0127] c) Tilt the culture dish and use a pipette to aspirate the primary antibody incubation solution from one corner of the chip, removing as much surface liquid as possible while keeping the chip moist.

[0128] d) Add 100 μL of washing buffer to the chip per chip;

[0129] e) Tilt the culture dish and use a pipette to remove the wash solution from one corner of the chip, minimizing the amount of liquid on the surface while keeping the chip moist.

[0130] f) repeat steps d and e once;

[0131] g) Add 0.1×SSC to the chip at a volume of 100 μL / chip;

[0132] h) Tilt the culture dish and use a pipette to remove the wash solution from one corner of the chip, minimizing the amount of liquid on the surface while keeping the chip moist.

[0133] i) Repeat steps g and h once.

[0134] 6. Tissue Permeabilization

[0135] Prepare the permeabilization reagent according to Table 7.

[0136] Table 7 Permeabilization reagent composition

[0137] a) Add permeabilization reagent to the chip at a volume of 100 μL / chip;

[0138] b) Perform permeabilization reaction at 37°C (permeabilization time can be adjusted according to actual conditions);

[0139] c) Add 0.1×SSC to the chip at a volume of 100 μL / chip;

[0140] d) Tilt the culture dish and use a pipette to remove the wash solution from one corner of the chip, minimizing the amount of liquid on the surface while keeping the chip moist.

[0141] 7. Reverse transcription, library construction, and sequencing

[0142] a) Add reverse transcription reagent and perform reverse transcription at 42°C for 3 hours.

[0143] b) Add 400 μL of tissue removal solution and digest at 55°C for 30 min. Remove the tissue removal solution and add 400 μL of digestion solution and incubate at 55°C for 3 hours.

[0144] c) Purify cDNA from the digestion solution, amplify the library, prepare DNB, and sequence it.

[0145] d) Restore protein spatial location information through sequencing results.

[0146] (V) Results and Analysis

[0147] As shown in Figure 6, the sequencing effect obtained by collagenase + TE cross-linking decomposition is significantly better than TE cross-linking decomposition, sodium citrate cross-linking decomposition, and collagenase + sodium citrate cross-linking decomposition. Specifically, it is reflected in the increase of the reduction signal of the sequencing of the same antibody, such as Vimentin, CD45RO and other antibodies; the uniformity of antibody recognition is improved, such as Vimentin antibody.

[0148] Example 2

[0149] (I) Experimental tissue: human tonsil FFPE tissue.

[0150] (2) Source of reagents: Same as Example 1.

[0151] (3) Reagent preparation: same as in Example 1.

[0152] (IV) Experimental steps: Except for the cross-linking step, the steps are the same as those in Example 1, with the following specific differences:

[0153] The decrosslinking step was divided into I and II, and the experimental group was divided into four groups according to different decrosslinking conditions.

[0154] Table 8 Decrosslinking Conditions

[0155] (V) Results and Analysis

[0156] As shown in FIG7 , the collagenase treatment time was optimized in this example. Compared with the TE decrosslinking group and the 10-min group, the uniformity and signal of antibody detection were significantly improved after the collagenase treatment for 20 min and 40 min.

[0157] Example 3

[0158] (I) Experimental tissue: human tonsil FFPE tissue.

[0159] (2) Source of reagents: Same as in Example 1.

[0160] (3) Reagent preparation: same as in Example 1.

[0161] (IV) Experimental steps: Except for the cross-linking decomposition step, the other experimental steps are the same as those in Example 1, with the following specific differences:

[0162] According to the different concentrations of type I collagenase in decrosslinking, the experimental group was divided into four groups, namely collagenase 0.2 U / μL, collagenase 0.3 U / μL, collagenase 0.4 U / μL and collagenase 0.6 U / μL.

[0163] (V) Results and Analysis

[0164] As shown in FIG8 , the collagenase concentration was optimized in this example. The antibody uniformity and signal were significantly improved in the 0.4 U / μL collagenase and 0.6 U / μL collagenase treatment groups compared with the 0.2 U / μL collagenase and 0.3 U / μL collagenase treatment groups.

[0165] Example 4

[0166] (I) Experimental tissue: human tonsil FFPE tissue.

[0167] (2) Source of reagents: Same as Example 1.

[0168] (3) Reagent preparation: same as in Example 1.

[0169] (IV) Experimental procedures: TE was used for the cross-linking removal step in a 99°C water bath for 20 min. A fixation step was added before permeabilization. The specific procedures are shown in Table 9.

[0170] Table 9 Fixation treatment conditions

[0171] (V) Results and Analysis

[0172] As shown in Figure 9, fixation prior to permeabilization significantly reduced the diffusion of the Vimentin antibody, improving detection clarity. Similar effects were observed with different fixatives.

[0173] Example 5

[0174] (I) Experimental tissue: human tonsil FFPE tissue.

[0175] (2) Source of reagents: Same as Example 1.

[0176] (3) Reagent preparation: same as in Example 1.

[0177] (IV) Experimental steps: TE was used for the decrosslinking step in a 99°C water bath for 20 min. A pre-permeabilization elution step was added before permeabilization. The specific operation was as follows. Other steps were the same as in Example 1.

[0178] Table 10 Elution treatment conditions

[0179] (V) Results and Analysis

[0180] As shown in Figure 10, compared to the control group, elution with 50% formamide and 100% formamide (FAM) before permeabilization significantly improved the signal-to-noise ratio of antibody detection and reduced the signal in blank areas of tissue. 60% DMSO and 100% DMSO also had similar effects, but the effects were less pronounced than formamide.

[0181] Example 6

[0182] (I) Experimental tissue: human tonsil FFPE tissue.

[0183] (2) Source of reagents: Same as Example 1.

[0184] (3) Reagent preparation: same as in Example 1.

[0185] (IV) Experimental Procedures: TE was used for cross-linking deconvolution in a 99°C water bath for 20 min. Non-ionic permeabilization group: The permeabilization reagent used was the permeabilization reagent of Example 1. Pepsin group: The permeabilization reagent used was pepsin, and the reaction was carried out at 37°C for 20 min.

[0186] (V) Results and Analysis

[0187] As shown in FIG11 , compared with the pepsin group, the nonionic permeabilizing agent of the present invention can significantly reduce the diffusion of antibodies and improve the detection signal and uniformity.

[0188] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a spatial proteomics sequencing library, characterized in that: include: A solid phase support and an antibody-nucleic acid conjugate are provided, wherein a probe is connected to the solid phase support, the probe comprising a capture sequence and a spatial positioning sequence, and the capture sequence is located at the end of the probe away from the solid phase support; the antibody-nucleic acid conjugate comprises an antibody and a nucleic acid sequence coupled to the antibody, the nucleic acid sequence comprising, in order away from the antibody, an antibody sequence and a capture complementary sequence, and the capture sequence and the capture complementary sequence are at least partially complementary; attaching the tissue paraffin section sample to the solid phase carrier, performing baking, dewaxing, hydration, cross-linking removal and blocking treatment to obtain a blocked solid phase carrier; Incubating the antibody-nucleic acid conjugate with the closed solid phase carrier to obtain a protein capture solid phase carrier; Permeabilizing the protein capture solid phase carrier so that the capture sequence binds to the capture complementary sequence to obtain a permeabilized solid phase carrier; Performing DNA synthesis on the permeabilized solid phase support to extend the complementary sequence of the nucleic acid sequence on the capture sequence or to extend the complementary sequence of the probe on the nucleic acid sequence to obtain cDNA; The cDNA is released from the solid phase carrier, collected and amplified to obtain a sequencing library.

2. The method according to claim 1, characterized in that The nucleic acid sequence further includes a first amplification linker sequence, the first amplification linker sequence is connected to the antibody sequence, and the antibody sequence is connected to the capture complementary sequence; The probe further includes: a resection region, a second amplification linker sequence, and a UMI sequence, wherein the resection region is connected to the solid phase support.

3. The method according to claim 1, characterized in that The reagents used in the de-crosslinking treatment include collagenase solution and Tris-EDTA buffer.

4. The method according to claim 3, characterized in that The collagenase is selected from type I collagenase; The concentration of the collagenase solution is 0.2-1.0 U / μL.

5. The method according to claim 1, wherein The de-crosslinking treatment comprises: Incubate the collagenase solution with the hydrated solid support at 35-39°C for 10-40 minutes, and discard the liquid; The solid phase carrier treated in the previous step and Tris-EDTA buffer were placed in a 95-100°C hot water bath for 15-25 minutes, and then the temperature of the solid phase carrier was lowered to room temperature in a cold water bath.

6. The method according to claim 1, characterized in that The permeabilization reagent used in the permeabilization treatment includes: a buffer solution and a surfactant; The surfactant comprises at least one of the following: Triton TM X-100, saponin, methanol, acetone, Tween, nonylphenoxypolyethoxyethanol, and digitonin.

7. The method according to claim 6, characterized in that The permeabilization reagent includes: 0.05-0.5% by volume of Tween 20; 0.05-0.5% by volume of nonylphenoxypolyethoxyethanol; 0.00001-0.1% by volume of digitonin; The balance is 2× sodium citrate buffer.

8. The method according to claim 1, characterized in that Before the permeabilization treatment, the protein capture solid phase carrier is subjected to elution and fixation treatments; The elution reagent used in the elution treatment includes at least one of a formamide solution and a dimethyl sulfoxide solution; The fixation treatment comprises incubating the solid phase carrier after the elution treatment with a fixation reagent, wherein the fixation reagent comprises at least one of a methanol solution, a formaldehyde solution and a paraformaldehyde solution.

9. The method according to claim 8, characterized in that The concentration of the formamide solution is 40 to 100% by volume, and the concentration of the dimethyl sulfoxide solution is 50 to 100% by volume; The concentration of the methanol solution is 95-100% by volume, the concentration of the formaldehyde solution is 10-20% by volume, and the concentration of the paraformaldehyde solution is 1-6% by volume.

10. The method according to claim 8, characterized in that The eluting reagent is selected from formamide solution.

11. The method according to claim 8, characterized in that The elution treatment temperature is 20-70°C and the time is 5-30 minutes; The fixing treatment temperature of the formaldehyde solution or paraformaldehyde solution is 20-30° C., and the time is 1-10 minutes; the fixing treatment temperature of the methanol solution is -25--15° C., and the time is 1-10 minutes.

12. The method according to claim 1, characterized in that The blocking reagent used in the blocking treatment includes: a sequence at least partially complementary to the capture sequence, salmon sperm DNA, an Fc receptor blocker, serum and a buffer.

13. The method according to claim 12, characterized in that The blocking reagent includes: 5-30 μM Poly A; 1-3 mg / mL salmon sperm DNA; 2-10% by volume of an Fc receptor blocker; 10-20% serum by volume; Buffer, wherein the buffer is selected from PBS buffer containing Triton-X100.

14. The method according to claim 1, wherein Further including: The amplified products obtained by the amplification are prepared into DNA nanoballs.

15. The method according to claim 1, wherein The capture sequence is a Poly T sequence, and the complementary sequence of the capture sequence is a Poly A sequence.

16. A spatial proteomics detection method, characterized in that: include: Constructing a sequencing library using the method for constructing a spatial proteomics sequencing library according to any one of claims 1 to 15; The sequencing library is sequenced, and the sequencing results are analyzed to obtain spatial proteomics data.

17. A kit for constructing a spatial proteomics sequencing library, characterized in that: include: Cross-linking and permeabilization reagents; The cross-linking removal reagent includes collagenase and Tris-EDTA buffer; The permeabilization reagent comprises: buffer, Tween, nonylphenoxypolyethoxyethanol and digitonin.

18. The kit according to claim 17, characterized in that The collagenase is selected from type I collagenase; The concentration of the collagenase solution is 0.1-0.8 U / μL.

19. The kit according to claim 17, characterized in that The permeabilization reagent includes: 0.05-0.5% by volume of Tween 20; 0.05-0.5% by volume of nonylphenoxypolyethoxyethanol; 0.00001-0.1% by volume of digitonin; The balance is 2× sodium citrate buffer.

20. The kit according to claim 17, wherein further comprising at least one of the following: a blocking reagent, an elution reagent, and a fixation reagent; The blocking reagent includes: Poly A, salmon sperm DNA, Fc receptor blocker, serum and buffer; The elution reagent includes at least one of a formamide solution and a dimethyl sulfoxide solution; The fixing agent includes at least one of a methanol solution, a formaldehyde solution and a paraformaldehyde solution.

21. The kit according to claim 20, characterized in that The blocking reagent includes: 5-30 μM Poly A; 1-3 mg / mL salmon sperm DNA; 2-10% by volume of an Fc receptor blocker; 10-20% serum by volume; Buffer, the buffer is selected from PBS buffer containing Triton-X100; The concentration of the formamide solution is 40 to 100% by volume, and the concentration of the dimethyl sulfoxide solution is 50 to 100% by volume; The concentration of the methanol solution is 95-100% by volume, the concentration of the formaldehyde solution is 10-20% by volume, and the concentration of the paraformaldehyde solution is 1-6% by volume.

22. The kit according to claim 20, characterized in that The eluting reagent is selected from formamide solution.

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