Method for quantitatively detecting triple-helix structure of collagen based on mass spectrum

Abstract

The invention relates to a method for quantitatively detecting a triple helix structure of collagen based on mass spectrometry, which comprises the following steps: acquiring a to-be-detected solution containing target collagen, combining a specific antibody of the target collagen with an alpha y chain in the target collagen to form an antigen-antibody compound, and performing precipitation separation; carrying out enzymolysis on the obtained antigen-antibody compound to obtain a polypeptide mixed solution containing an alpha y chain and a corresponding specific peptide fragment generated by carrying out enzymolysis on another alpha x chain in the target collagen; and carrying out mass spectrometric detection after desalination, and calculating the amount and the proportion of the triple-helical structure of the target collagen according to the number of specific peptide fragments corresponding to alpha x and alpha y chains obtained through detection and a formula that the number of the triple-helical structure of the target collagen is equal to alpha x / n and the proportion (%) of the triple-helical structure of the target collagen is equal to m * alpha x / (n * alpha y) * 100%. According to the method, different types of collagen triple helix structures can be distinguished, and the detection result is more accurate.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more specifically to a method for quantitatively detecting the triple helix structure of collagen based on mass spectrometry. Background Art

[0002] Collagen is a type of structural protein widely found in animals. It is mainly synthesized and secreted by fibroblasts and constitutes the main component of connective tissues such as skin, bones, cartilage and tendons. Its typical secondary structure is a triple helix structure formed by three left-handed helical α chains coiled in a right-handed helix through interactions such as interchain hydrogen bonds. This unique triple helix protein is widely used in biomedical materials, drug delivery carriers, tissue engineering, cosmetics and food because it can promote cell growth and tissue repair, and has physical and chemical properties such as high biocompatibility and biodegradability. However, the triple helix structure, which is an important indicator of the biological activity of collagen, is easily affected by the processing environment, resulting in the destruction of the structure and loss of its biological activity. Existing studies have found that mutations or degradation of collagen are closely related to a variety of diseases such as osteogenesis imperfecta, arthritis, cancer, and thrombosis. Therefore, detecting these collagens can help us better understand their roles in physiological and pathological processes, and also provide possibilities for disease diagnosis and treatment.

[0003] At present, the detection of collagen is mainly divided into the detection of collagen and the detection of the triple helix structure of collagen. For example, the immunohistochemistry method uses a specific collagen antibody to incubate with an antigen, and then adds a secondary antibody with a marker for color development, which is used to qualitatively detect the distribution and location of collagen in the tissue; the enzyme-linked immunosorbent assay method uses a double antibody sandwich, and uses an enzyme marker to measure the absorbance value according to the color depth of the substrate, and then quantitatively analyzes the content of collagen. However, these methods actually detect the total amount of collagen, and cannot distinguish between non-triple helix structure collagen and triple helix structure collagen.

[0004] The immunoblotting method uses SDS-PAGE to separate the extracted proteins according to their molecular weight, uses specific collagen antibodies to incubate and bind with the proteins, adds labeled secondary antibodies, and semi-quantitatively analyzes the content of collagen by detecting the labeled signal. However, this method can only detect collagens with a known molecular weight range, and may not be able to accurately detect some new collagens with unknown molecular weights. The existing commonly used methods for detecting the triple helix structure of collagen include circular dichroism and infrared spectroscopy, but these methods can only indirectly and qualitatively determine the degree of damage to the triple helix structure in collagen by comparing with reference materials, and cannot quantitatively detect the content of the triple helix structure.

[0005] Patent CN108659117B discloses a method for quantitatively detecting the content of collagen triple helix structure, which utilizes collagen with a complete triple helix structure that cannot be hydrolyzed by trypsin, while collagen with a completely or partially destroyed triple helix structure can be hydrolyzed by trypsin, and by detecting the change in the content of hydroxyproline, a characteristic amino acid of collagen before and after hydrolysis, the content of collagen with a complete triple helix structure is calculated, and then the quantitative detection of collagen triple helix structure is realized. However, collagen has a variety of different types, and they differ in molecular structure, tissue distribution, biological function, etc. However, this method cannot distinguish different types of collagen with a complete triple helix structure, and there is no conclusion on how to define whether the triple helix structure of collagen is complete, and whether the collagen with a complete triple helix structure can resist trypsin is also controversial. Therefore, it is of great significance to study a method for quantitatively detecting the content of its triple helix structure for different types of collagen in biological samples. Summary of the invention

[0006] In response to the above defects or improvement needs of the prior art, the present invention provides a method for quantitatively detecting the triple helix structure of collagen based on mass spectrometry, the purpose of which is to use collagen-specific antibodies to extract the target collagen in the sample, then enzymatically hydrolyze the target collagen to obtain specific peptides produced by enzymatic hydrolysis of the target collagen, detect the number of specific peptides by affinity purification mass spectrometry, and calculate the amount and proportion of the collagen triple helix structure based on the composition of the target collagen α chain, thereby solving the technical problem that the existing quantitative detection method of the collagen triple helix structure is difficult to distinguish the triple helix structures of different types of collagen and the detection is not accurate enough.

[0007] To achieve the above object, according to one aspect of the present invention, a method for quantitatively detecting the triple helix structure of collagen based on mass spectrometry is provided, which comprises the following steps:

[0008] (1) obtaining a test solution containing a target collagen, wherein the target collagen has a trimeric helical structure encoded by a heterologous gene and has at least two different α chains; using a specific antibody for the target collagen to bind to the αγ chain in the target collagen to form an antigen-antibody complex, and precipitating and separating the complex;

[0009] (2) Enzymatic hydrolysis: enzymatically hydrolyzing the antigen-antibody complex obtained in step (1) to obtain a polypeptide mixture containing the αy chain and another αx chain in the target collagen protein, which is hydrolyzed to produce the corresponding specific peptide segment;

[0010] (3) Quantitative detection of the target collagen triple helix structure: The peptide mixture is desalted and then subjected to mass spectrometry. Based on the number of specific peptide segments corresponding to the αx and αy chains obtained by the detection, the amount and proportion of the target collagen triple helix structure are calculated according to the following formula:

[0011] The number of target collagen triple helical structures = α x / n;

[0012] The proportion of target collagen triple helix structure (%) = m*α x / (n*α y )*100%;

[0013] Where m represents the number of αy chains in a target collagen triple helix structure, n represents the number of αx chains in a target collagen triple helix structure, m≤n;

[0014] α x It represents the total number of specific peptides produced by enzymatic hydrolysis of all αx chains in the antigen-antibody complex;

[0015] α y It represents the total number of specific peptides produced by enzymatic hydrolysis of all αγ chains bound to specific antibodies in the antigen-antibody complex.

[0016] Preferably, in the method, the αy chain is an α chain with 1 chain number in the target collagen.

[0017] Preferably, in the method, the target collagen is type I collagen composed of one α2 chain and two α1 chains, the specific antibody is a COL1A2 antibody that binds to the α2 chain, and the amount and proportion of the triple helix structure of type I collagen are calculated according to the following formula:

[0018]

[0019] In the formula, α1 represents the number of specific peptides produced by enzymatic hydrolysis of all α1 chains in the antigen-antibody complex, and α2 represents the number of specific peptides produced by enzymatic hydrolysis of all α2 chains in the antigen-antibody complex.

[0020] Preferably, in the method, the amino acid sequence of the specific peptide corresponding to the α1 chain of type I collagen is as shown in SEQ ID No.1, TTGVEGPKGDTGPRGPRGP, and the amino acid sequence of the specific peptide corresponding to the α2 chain is as shown in SEQ ID No.2, GIPGPVGAAGATGAR.

[0021] Preferably, in the method, the enzymatic hydrolysis in step (2) is performed by using trypsin, a mixed enzyme of Trypsin and Lys-C, and trypsin without Lys-C.

[0022] Preferably, in the method, in the enzymatic hydrolysis of step (2), trypsin is added at a protein to enzyme mass ratio of 25:1-5 for the first enzymatic hydrolysis, and the enzymatic hydrolysis is carried out at 35-40°C for 16-20 hours; a mixed enzyme of Trypsin and Lys-C is added at a protein to enzyme mass ratio of 25:1-5 for the second enzymatic hydrolysis, and the enzymatic hydrolysis is carried out at 35-40°C for 4-6 hours; and trypsin without Lys-C is added at a protein to enzyme mass ratio of 25:1-5 for the third enzymatic hydrolysis, and the enzymatic hydrolysis is carried out at 35-40°C for 12-15 hours.

[0023] Preferably, in the method, in the first enzymatic hydrolysis and / or the third enzymatic hydrolysis, CaCl2 is further added to make its final concentration 2-4 mM.

[0024] Preferably, in the method, the test solution containing the target collagen in step (1) is prepared according to the following method:

[0025] Biological tissues or cells are firstly cross-linked and fixed with 1-4% paraformaldehyde to collagen with a triple helix structure; then the cells are lysed under non-denaturing conditions and centrifuged to obtain a supernatant containing the target collagen.

[0026] Preferably, the method, wherein the cleavage is performed at a rate of (1 to 10) × 10 7 Add 5 mL IP lysis buffer to each cell for lysis.

[0027] Preferably, in the method, the precipitation separation is performed by using Protein A / G agarose beads to specifically bind to the antigen-antibody complex to precipitate it, followed by centrifugal separation.

[0028] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0029] The present invention fully considers that different types of collagen have similar triple helical structures, and existing methods for quantitative detection of collagen triple helical structures are difficult to distinguish the triple helical structures of different types of collagen. The method for quantitative detection of collagen triple helical structure provided by the present invention can select its specific antibody according to the type of target collagen to be tested in the biological sample, bind to the αy chain in the target collagen to form an antigen-antibody complex, separate by precipitation, obtain a polypeptide mixture containing the αy chain in the target collagen and another αx chain enzymatically hydrolyzed to produce the corresponding specific peptide segment, and then combine affinity purification mass spectrometry for quantitative detection, according to the number of specific peptide segments corresponding to the αx and αy chains obtained by the detection, according to the formula, the number of target collagen triple helical structures = α x / n; The proportion of target collagen triple helix structure (%) = m*α x / (n*α y)*100%, calculate the amount and proportion of collagen triple helix structure. Compared with the existing methods, this method can not only quantitatively detect the collagen triple helix structure in biological samples, but also distinguish different types of collagen triple helix structures, and the detection results are more accurate. In addition, it can also be used for quantitative detection of recombinant collagen triple helix structure, and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the detection principle of this method;

[0031] Figure 2 It is the affinity purification mass spectrometry result of different treatment groups in Example 2;

[0032] Figure 3 It is the proportion of collagen triple helix structure in different treatment groups of Example 2. DETAILED DESCRIPTION

[0033] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0034] Collagen is a family of proteins. At least 30 kinds of collagen chain coding genes have been found, which can form more than 16 kinds of collagen molecules. Some collagens have a trimer helical structure encoded by heterologous genes:

[0035] Table 1 Composition of different types of collagen α chains

[0036]

[0037]

[0038] The present invention is based on the composition characteristics of different types of collagen, utilizes a target collagen-specific antibody to specifically bind to a specific αy chain in the target collagen to form an antigen-antibody complex, wherein the antigen-antibody complex includes a complex formed by the target collagen with a triple helix structure and the antibody, and a complex formed by the specific binding of a single αy chain and the antibody, separates the antibody-bound target collagen from a sample to be tested by precipitation separation, enzymatically hydrolyzes the antigen-antibody complex to obtain a polypeptide mixture of all αy chains (including the αy chain and a single αy chain in the target collagen) and corresponding specific peptide segments generated by enzymatic hydrolysis of another αx chain in the target collagen, detects using mass spectrometry, calculates the amount of the target collagen triple helix structure and its proportion based on the number of specific peptide segments corresponding to the αx and αy chains obtained by the detection and the ratio of the αx and αy chains in the target collagen, and can analyze and detect the triple helix structures of different types of fibroblast collagen.

[0039] Based on this, the present invention provides a method for quantitatively detecting the triple helix structure of collagen based on mass spectrometry, which comprises the following steps:

[0040] (1) obtaining a test solution containing a target collagen, wherein the target collagen has a trimeric helical structure encoded by a heterologous gene and has at least two different α chains; using a specific antibody for the target collagen to bind to the αγ chain in the target collagen to form an antigen-antibody complex, and precipitating and separating the complex;

[0041] (2) Enzymatic hydrolysis: enzymatically hydrolyzing the antigen-antibody complex obtained in step (1) to obtain a polypeptide mixture containing the αy chain and another αx chain in the target collagen protein, which is hydrolyzed to produce the corresponding specific peptide segment;

[0042] (3) Quantitative detection of the target collagen triple helix structure: The peptide mixture is desalted and then subjected to mass spectrometry. Based on the number of specific peptide segments corresponding to the αx and αy chains obtained by the detection, the amount and proportion of the target collagen triple helix structure are calculated according to the following formula:

[0043] The number of target collagen triple helical structures = α x / n;

[0044] The proportion of target collagen triple helix structure (%) = m*α x / (n*α y )*100%;

[0045] Wherein m represents the number of αy chains in a target collagen triple helix structure, n represents the number of αx chains in a target collagen triple helix structure, m≤n;

[0046] α x It indicates the number of specific peptides produced by enzymatic hydrolysis of all αx chains in the antigen-antibody complex;

[0047] α y It indicates the number of specific peptides produced by enzymatic hydrolysis of all αy chains bound to specific antibodies in the antigen-antibody complex.

[0048] Preferably, the αy chain is an α chain with 1 in the target collagen. The α chains of collagen have similarities in some aspects, but their amino acid sequences are different. Each type of collagen chain has its own specific amino acid sequence, which determines the stability and function of the collagen. For example, in the present invention, the target collagen is type I collagen composed of one α2 chain and two α1 chains. The specific antibody for the target collagen in step (1) is selected from the COL1A1 antibody that specifically binds to the α1 chain of type I collagen or the COL1A2 antibody that specifically binds to the α2 chain of type I collagen.

[0049] In some embodiments, the target collagen is type I collagen, and preferably the specific antibody is COL1A2 antibody. The amount and proportion of the triple helix structure of type I collagen are calculated according to the following formula:

[0050]

[0051] In the formula, α1 represents the number of specific peptide segments corresponding to the α1 chain of type I collagen, and α2 represents the number of specific peptide segments corresponding to the α2 chain of type I collagen; the amino acid sequence of the specific peptide segment corresponding to the α1 chain of type I collagen is as shown in SEQ ID No.1, TTGVEGPKGDTGPRGPRGP, and the amino acid sequence of the specific peptide segment corresponding to the α2 chain of type I collagen is as shown in SEQ ID No.2, GIPGPVGAAGATGAR, wherein the specific peptide segment refers to a corresponding characteristic peptide segment produced after enzymatic hydrolysis of an α chain of collagen.

[0052] In some embodiments, the target collagen is composed of an α1 chain, an α2 chain, and an α3 chain (type V collagen), and the specific antibody used may be any one of the specific antibodies corresponding to the α1 chain, the α2 chain, and the α3 chain. Taking the specific antibody as COL1A2 antibody as an example, the amount and proportion of the triple helix structure of the target collagen are calculated according to the following formula:

[0053]

[0054] In the formula, α1 represents the number of specific peptides corresponding to the target collagen α1 chain, and α2 represents the number of specific peptides corresponding to the target collagen α2 chain.

[0055] Furthermore, the test solution containing the target collagen in step (1) is prepared according to the following method:

[0056] Biological tissues or cells are firstly cross-linked and fixed with 1-4% paraformaldehyde to collagen with a triple helix structure; then the cells are lysed under non-denaturing conditions and centrifuged to obtain a supernatant containing the target collagen.

[0057] In some embodiments, in step (1), the 7 Add 5 mL IP lysis buffer to each cell for lysis.

[0058] The IP lysis solution includes 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and any one or more of the inhibitors sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4 and leupeptin.

[0059] The crosslinking agent is a formaldehyde solution with a formaldehyde volume ratio of 1% to 4%. The specific concentration can be adjusted appropriately according to experimental requirements and cell types. For example, formaldehyde powder can be dissolved in a corresponding buffer (such as PBS buffer) to prepare a formaldehyde solution of the required concentration.

[0060] The precipitation separation is to use Protein A / G agarose beads to specifically bind to the antigen-antibody complex to precipitate it, and then centrifuge it.

[0061] The enzymatic hydrolysis in step (2) is performed by using trypsin, a mixed enzyme of trypsin and Lys-C, and trypsin without Lys-C. In some embodiments, the enzymatic hydrolysis in step (2) is specifically as follows:

[0062] The precipitate obtained in (1) was resuspended in UA buffer (100 mM Tris-HCl (pH 8.5), 8 M Urea), DTT was added to make the final concentration 10 mM, and IAA and Tris-HCl (pH 8.0) were added to dilute the Urea in the solution to 1 M;

[0063] According to the mass ratio (protein: enzyme = 25:1), Trypsin (Promega catalog number: V528A) was added for the first enzymatic hydrolysis. CaCl2 was preferably added to increase the enzyme activity, so that the final concentration of CaCl2 was 2mM, and the enzymatic hydrolysis was carried out in a 37°C water bath overnight (16-20h);

[0064] Then, Tris-HCl (pH 8.0) was added to adjust the Urea in the solution to 4 M, and a mixed enzyme of Trypsin and Lys-C was added at a protein to enzyme mass ratio of 20:1 for a second enzymatic hydrolysis, and the enzymatic hydrolysis was carried out in a water bath at 37°C for 4 h.

[0065] Then add Tris-HCl (pH 8.0) to dilute the Urea in the solution to 1M, add trypsin without Lys-C at a protein to enzyme mass ratio of 20:1 for the third enzymatic hydrolysis, preferably add CaCl2 to increase the enzyme activity, so that the final concentration of CaCl2 is 2mM, and perform enzymatic hydrolysis in a 37°C water bath overnight (12-15h); finally, add TFA to make the pH of the enzymatic hydrolyzate <4.

[0066] The peptides were injected and loaded onto the precolumn at a flow rate of 4 μL / min. The peptides were eluted into the mass spectrometer using an HPLC gradient eluent. Mobile phase A was 0.2 M acetic acid, and mobile phase B was 70% acetonitrile / 0.2 M acetic acid. The mobile phase B gradient was: 0-100%, within 50 min, linear gradient. Mass spectrometry data were collected using a hybrid linear ion trap Orbitrap under the following conditions:

[0067] MS scan: 300≤m / z≤2000; MS / MS exclusion list, 2Da isolation window and 25% relative collision energy: 8 most abundant ions (5>z>1); ESI voltage: 1.5 kV; capillary temperature: 150°C.

[0068] The following are examples

[0069] Example 1 Quantitative detection of collagen triple helix structure

[0070] Fibroblasts can synthesize and secrete various types of collagen, such as type I and type III, which participate in the formation of the extracellular matrix. In addition to collagen, fibroblasts also synthesize and secrete components such as fibronectin, laminin, and elastic fibers. This example uses fibroblasts (HFF-1 cells) to simulate the real environment in which the body synthesizes and secretes collagen to quantitatively detect the triple helix structure of collagen. The experimental process is as follows: Figure 1 As shown, the details are as follows:

[0071] 1.1 HFF-1 cell culture

[0072] (1) Aspirate or pour out the old culture medium in the culture bottle.

[0073] (2) Add a small amount of trypsin solution and EDTA mixture into the bottle.

[0074] (3) Place the culture flask in an incubator at 37°C or at room temperature (25°C) for digestion. After 2 to 5 minutes, place the culture flask under an inverted microscope for observation. If the cytoplasm shrinks and the intercellular space increases, the digestion should be stopped immediately.

[0075] (4) Aspirate the digestion solution, add a small amount of Hanks solution to the bottle, gently rotate the culture bottle to wash away the residual digestion solution, and then add culture solution. If only trypsin is used for digestion, after aspirating the trypsin solution, a small amount of serum-containing culture solution can be directly added to terminate the digestion.

[0076] (5) Use an elbow pipette to absorb the culture medium in the bottle, and gently blow the cells on the bottle wall repeatedly in sequence to separate them from the bottle wall to form a cell suspension. Be gentle when blowing to avoid damaging the cells.

[0077] (6) After counting with a counting plate, inoculate the cells into new culture bottles and culture them in a CO2 incubator for 48 hours.

[0078] 1.2 Extraction and separation of target collagen

[0079] (1) Cell treatment: Add 200ul of crosslinking agent (4% formaldehyde solution) to the HFF-1 cells that have been cultured in 1.1, and crosslink and incubate for 5min to crosslink and fix the collagen that has formed a triple helix structure in the cells to avoid damage to the triple helix structure by organic solvents. Then add 5ml of glycine and incubate for 15min to terminate the crosslinking. Remove the cells, discard the supernatant, rinse twice with pre-cooled PBS, add 500ul / 6mm IP lysis buffer (containing the diluted cocktail) to lyse the cells under non-denaturing conditions to release the proteins therein: lyse on ice for 30min, shake every 10min to allow the lysis buffer to fully rinse the cells. After sufficient lysis, scrape the cells with a cell scraper, collect them in a 1.5ml EP tube, centrifuge at 12000rpm, 4℃ for 10min, and take the supernatant to obtain the cell lysate containing the target collagen. IP lysis buffer usually includes 20mM Tris (pH7.5), 150mM NaCl, 1% Triton X-100, and a variety of inhibitors such as sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin, etc. These inhibitor components can effectively inhibit protein degradation and maintain the original protein-protein interactions to avoid destroying the protein-protein interactions.

[0080] (2) Add antibodies to specifically bind to target collagen

[0081] In the present invention, corresponding specific antibodies can be selected according to the type of target collagen in the sample. For example, type I collagen is the most abundant type of collagen, which is composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. Anti-COL1A1 antibody or anti-COL1A2 antibody can be selected, wherein COL1A1 antibody specifically binds to the α1 chain of type I collagen, and COL1A2 antibody specifically binds to the α2 chain of type I collagen, and COL1A2 antibody is preferred.

[0082] For collagen composed of three different subunits, such as α1 chain (encoded by COL2A1 gene), α2 chain and α3 chain (encoded by COL2A2 and COL2A3 genes), an anti-COL2A1 antibody can be selected, which specifically binds to the α1 chain of the collagen.

[0083] Although the proportions of different types of collagen vary in different tissues, for example, in normal skin, the collagen secreted by fibroblasts is mainly type I, accounting for about 80% to 90% of the total collagen secreted, while type III collagen accounts for about 10% to 20%; in perivascular tissue, the secretion of type I collagen accounts for about 60%, and type III collagen accounts for about 40%; in connective tissue, type I collagen accounts for 70% to 80%, and type III collagen accounts for 20% to 30%. However, in the body, collagen is mainly type I collagen and type III collagen, of which type I collagen is the main one.

[0084] This embodiment takes the target collagen as type I collagen as an example, and the details are as follows:

[0085] The cell lysate was divided into three parts, and appropriate amounts of antibodies were added. 10% of the cell lysate was used as input, 45% of the cell lysate was added with COL1A2 antibody to specifically bind to the antigen (α2 chain of type I collagen), and 45% of the cell lysate was added with IgG antibody as background. The protein lysate with the antibody was incubated overnight at 4°C with shaking.

[0086] (3) Protein A / G agarose bead treatment: After fully mixing the Protein A / G agarose beads, cut off the front end of the pipette tip and aspirate 100ul. Wash twice with TBS-T, centrifuge at 1000rpm for 1min, and discard the supernatant. Prepare blocking solution, dissolve 5% BSA in TBS-T. Add 500ul of blocking solution to the washed Protein A / G agarose beads and shake at 4℃ for blocking overnight. Protein A / G agarose beads are made by covalently coupling two proteins, Protein A and Protein G, to agarose beads, and are used to specifically bind to the COL1A2 antibody-antigen complex.

[0087] (4) Purification and separation of antigen-antibody complexes: Centrifuge the blocked Protein A / G agarose beads at 1000 rpm for 1 min, discard the supernatant, wash twice with TBS-T, centrifuge at 1000 rpm for 1 min, discard the supernatant, wash once with IP lysis buffer, evenly divide into antibody-protein lysis buffer tubes, and incubate at 4°C with shaking for 4-6 hours to allow it to bind to the antigen-antibody complex. After the incubation, centrifuge at 4°C, 1000 rpm for 1 min, carefully aspirate and discard the supernatant, and collect the precipitate. Wash the precipitate 2 to 4 times with TBS-T, repeat the centrifugation step after each wash, and collect the precipitate. Half of the sample can be used for silver staining as needed.

[0088] 1.3 Quantitative detection of collagen triple helix structure

[0089] The protein in 1.2 was detected based on mass spectrometry technology, and the content of the triple helix structure of type I collagen was calculated based on the obtained mass spectrometry data, as follows:

[0090] Solution and reagent preparation:

[0091] Table 2 Preparation of UA Buffer

[0092]

[0093] Table 3 Preparation of 0.5M IAA (iodoacetamide)

[0094]

[0095] Table 4 Preparation of 0.1% TFA (trifluoroacetic acid) / / 2.5% ACN

[0096]

[0097] Table 5 Preparation of 0.1% TFA / / 80% ACN

[0098]

[0099] Table 6 Preparation of 0.1% TFA / / 2.5% ACN

[0100]

[0101] Table 7 Preparation of 50 mM acetic acid

[0102]

[0103] Sample preparation before mass spectrometry

[0104] (1) Prepare the required solutions and reagents.

[0105] (2) Acetone precipitation (the following steps are all pure acetone unless otherwise specified)

[0106] ① Take 294 μL of washing solution, add 6 μL of 5M NaCl, make up with 10mM Tris-cl 8.0, and then add NaCl to maintain a basic salt concentration of not less than 100mM;

[0107] ② Add 4 times the volume of acetone (1200 μL) of the previous step, vortex thoroughly to mix (vortex for at least 30 seconds), and place at -20°C overnight to precipitate; centrifuge at 4°C, 21000g for 20 minutes; carefully aspirate and discard the supernatant;

[0108] Add 500 μL of pre-cooled acetone, mix by inversion, and wash the precipitate; centrifuge at 4°C, 21,000 g for 20 min; discard the supernatant and dry for 5-10 min until the acetone is completely evaporated.

[0109] (3) Enzymatic hydrolysis of target collagen

[0110] ④ Use 20 μL UA buffer (100 mM Tris-cl 8.5, 8 M Urea) to resuspend the precipitate obtained in step ③, vortex thoroughly to mix, and leave to dissolve at room temperature for 30 minutes;

[0111] ⑤ Add 2.2 μL of 100 mM DTT to a final concentration of 10 mM, mix well, and incubate at 37°C for 30 min; then add 1 μL of freshly prepared 0.5 M IAA, mix well, and incubate at 37°C for 30 min in a dark place;

[0112] ⑥ Add 140 μL of 50 mM Tris-cl 8.0 to dilute the Urea in the solution to 1 M; enzymatic hydrolysis.

[0113] ⑦ Add 10 μL of 0.25 μg / μL Trypsin (Promega catalog number V528A) at a theoretical mass ratio of protein: enzyme = 25:1, and add 3.4 μL of 100 mM CaCl2 to increase enzyme activity, making the final concentration of CaCl2 2 mM, and perform enzymatic hydrolysis in a 37°C water bath overnight (16-20 h).

[0114] ⑧ Add an equal volume of 50mM Tris-cl 8.0 to dilute the Urea in the solution to 4M; enzymatic hydrolysis. Add 3μL of Trypsin / Lys-C mixed enzyme solution, mix thoroughly, and perform enzymatic hydrolysis for 4 to 6 hours in a 37℃ water bath; the combined use of Trypsin and Lys-C proteases can reduce the missed cleavage sites generated by trypsin when incubated overnight under non-denaturing conditions, thereby improving the ability of trypsin to digest proteins.

[0115] ⑨ Add 50mM Tris-cl 8.0 to dilute the Urea in the solution to 1M; add 5μL of Trypsin (without Lys-C) enzyme, and add CaCl2 to increase enzyme activity. The final concentration of CaCl2 is 2mM. Perform enzymatic hydrolysis in a 37℃ water bath overnight (12-15h); add 10% TFA to make its pH <4 (if it is not less than 4, continue to add 10% TFA until it is less than 4).

[0116] (4) Desalting and recovery of target peptides

[0117] ⑩C18 Tips selection: >20μg protein, select 100μL Tips; <20μg protein, select 10μL Tips; 50% ACN rinse;

[0118] 0.1% TFA / / 2.5% ACN, 50μL in total; 0.1% TFA / / 80% ACN, 50μL, first repeatedly pipette and pump, then soak the tips for about 2 minutes, then repeatedly pipette and pump to complete the elution, and collect the eluate;

[0119] Use a vacuum concentrator to concentrate the collected eluate at room temperature for 20 to 30 minutes; re-dissolve 12 μL of 480 mass spectrometry mobile phase A solution, add A solution, oscillate and mix thoroughly, incubate on a shaker at room temperature for 1 hour (mix frequently during the process), and after complete dissolution, detect with a NanoDrop instrument, select proteins, and measure the concentration using A280 (tryptophan, tyrosine, and other aromatic amino acids with benzene rings, which need to be divided by 0.6 to obtain the final concentration) or A205 (peptide bond) (solution A is blank);

[0120] The sample was centrifuged at 14000 g for 10 min, and the supernatant was transferred to a mass spectrometer loading tube and marked.

[0121] The peptides were injected and loaded onto the precolumn at a flow rate of 4 μL / min. The peptides were eluted into the mass spectrometer using an HPLC gradient eluent. Mobile phase A was 0.2 M acetic acid, and mobile phase B was 70% acetonitrile / 0.2 M acetic acid. The mobile phase B gradient was: 0-100%, within 50 min, linear gradient. Mass spectrometry data were collected using a hybrid linear ion trap Orbitrap under the following conditions:

[0122] MS scan: 300≤m / z≤2000; MS / MS exclusion list, 2Da isolation window and 25% relative collision energy: 8 most abundant ions (5>z>1); ESI voltage: 1.5 kV; capillary temperature: 150°C.

[0123] (5) Results analysis

[0124] All collected raw data were processed using pFind (V3.1.6) software. The peak list was searched through the UniProt human protein database (release version 20190308). Trypsin has four missing cleavages. The mass tolerances for precursor ions and fragment ions were 20ppm and 20ppm, respectively. Using the open search algorithm in pFind, acetylation (Lys) was set as a variable modification. The minimum peptide length was set to 6, and the estimated false discovery rate (FDR) threshold for peptides and proteins was set to a maximum of 1%. For other parameters in pFind, the algorithm default values ​​were used in this example.

[0125] Affinity purification mass spectrometry was used to detect peptides in the sample after enzymatic hydrolysis of the target collagen. Based on the number of specific peptides corresponding to the α1 chain and α2 chain of type I collagen, the amount and proportion of the triple helix structure of type I collagen were calculated according to the following formula:

[0126]

[0127] In the formula: α1 represents the number of specific peptides corresponding to the α1 chain, α2 represents the number of specific peptides corresponding to the α2 chain, the amino acid sequence of the specific peptide corresponding to the type I collagen α1 chain is TTGVEGPKGDTGPRGPRGP, and the amino acid sequence of the specific peptide corresponding to the type I collagen α2 chain is GIPGPVGAAGATGAR.

[0128] In this method, silver staining can also be used to enrich the sample after step 1.2 and before mass spectrometry detection, as follows:

[0129] Silver staining was performed using Pierce Silver Stain Kit (Thermo)

[0130] (1) Prepare the gel, add 10% of the above loading buffer to the sample, and spot the sample into the gel. Set the voltage of the electrophoresis instrument to 70V, and run the electrophoresis in the separation gel for half an hour; after the band runs to the concentrated gel, set the voltage to 120V, continue the electrophoresis for 1-1.5 hours, and observe further. Stop the electrophoresis when the bromophenol blue band approaches the bottom of the glass plate.

[0131] (2) Silver staining was performed using a silver staining kit (Thermo Fisher Scientific). The gel was first washed with ultrapure water for 2 × 5 minutes. The gel was fixed in a 30% ethanol:10% acetic acid solution for 2 × 15 minutes. The gel was washed with 10% ethanol for 2 × 5 minutes, and then washed with ultrapure water for 2 × 5 minutes.

[0132] (3) Prepare the sensitizer working solution (50 μL sensitizer plus 25 mL water). Sensitize the gel for 1 minute, then wash with water 2×1 minute. Prepare the stain working solution (0.5 mL sensitizer and 25 mL stain). Stain the gel for 30 minutes. Prepare the developer working solution (0.5 mL enhancer and 25 mL developer). Wash the gel with ultrapure water 2×20 seconds, then develop for 2-3 minutes until bands appear. Stop with 5% acetic acid for 10 minutes.

[0133] (4) Place the developed gel on a white light source plate and take a picture using a gel imager.

[0134] Example 2 Verification of the method

[0135] UVA (long-wave ultraviolet) irradiation can cause the destruction of type I collagen in the skin, and our previous studies have shown that sialic acid (SA) can promote fibroblasts to synthesize collagen. In this example, the fibroblasts (blank) in Example 1 were used as the control group, and the fibroblasts were treated with SA, UVA and SA+UVA respectively to detect the proportion of triple helical type I collagen, as follows:

[0136] Blank control group: normal human skin fibroblasts (HFF-1 cells) were pre-incubated in 1% FBS+DMEM with or 0.2% for 24 h and further incubated for 48 h;

[0137] SA group: SA was added after preincubation of HFF-1 cells to a final concentration of 0.2%, and the cells were incubated for 48 h;

[0138] UVA group: HFF-1 cells pre-incubated, 6J / cm 2 After UVA irradiation, incubation was continued for 48 h;

[0139] SA+UVA group: SA was added after pre-incubation of HFF-1 cells to a final concentration of 0.2% and 6 J / cm 2 After UVA irradiation, incubation was continued for 48 h.

[0140] The cells were treated in the same manner as in Example 1 to extract, separate and purify type I collagen, and then subjected to mass spectrometry detection after proteolysis. Figure 2 shown.

[0141] The results of affinity purification mass spectrometry showed that compared with the control group, the peptide spectrum matching score (Peptide Spectrum Matches, PSMs, showing the number of times the protein was detected) increased significantly after SA treatment, indicating that the content of type I collagen with a triple helix structure bound by COL1A1 antibody in the SA group increased significantly, mainly because SA enhanced the binding force between the α1 and α2 chains of type I collagen and improved the stability of the triple helix structure of collagen; while the peptide matching score decreased significantly after UVA treatment, mainly because UVA treatment would destroy the triple helix structure of type I collagen, indicating that the results of detecting the triple helix structure of type I collagen using this method are accurate and reliable.

[0142] Based on the number of specific peptide segments of type I collagen α1 and α2 chains, the proportion of type I collagen with a triple helix structure can be calculated according to the formula. The results are as follows: Figure 3 As shown, the details are as follows:

[0143]

[0144] The detection results of this method show that compared with the blank control group, the proportion of collagen triple helix structure increased significantly after SA treatment, while the proportion of collagen triple helix structure decreased significantly after UVA treatment, which is consistent with the phenomenon that sialic acid can promote fibroblasts to synthesize and secrete collagen, and UVA can destroy the triple helix structure of collagen. This shows that the detection results of this detection method are accurate and reliable, and can be used to quantitatively detect collagen with a triple helix structure.

[0145] In addition, this method can also be used to evaluate the structural stability of collagen under different processing conditions or between different samples, wherein the higher the proportion of the collagen triple helix structure, the better its structural stability.

[0146] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for quantitatively detecting the triple helix structure of collagen based on mass spectrometry, characterized in that: The following steps are involved: (1) obtaining a test solution containing a target collagen, wherein the target collagen has a trimeric helical structure encoded by a heterologous gene and has at least two different α chains; using a specific antibody for the target collagen to bind to the αγ chain in the target collagen to form an antigen-antibody complex, and precipitating and separating the complex; (2) Enzymatic hydrolysis: enzymatically hydrolyze the antigen-antibody complex obtained in step (1) to obtain a polypeptide mixture containing the αy chain and another αx chain in the target collagen protein, which is hydrolyzed to produce the corresponding specific peptide segment; (3) Quantitative detection of the target collagen triple helix structure: The peptide mixture is desalted and then subjected to mass spectrometry. Based on the number of specific peptide segments corresponding to the αx and αy chains obtained by the detection, the amount and proportion of the target collagen triple helix structure are calculated according to the following formula: The number of target collagen triple helical structures = α x / n; The proportion of target collagen triple helix structure (%) = m*α x / (n*α y )*100%; Wherein m represents the number of αy chains in a target collagen triple helix structure, n represents the number of αx chains in a target collagen triple helix structure, m≤n; α x It indicates the number of specific peptides produced by enzymatic hydrolysis of all αx chains in the antigen-antibody complex; α y It indicates the number of specific peptides produced by enzymatic hydrolysis of all αy chains bound to specific antibodies in the antigen-antibody complex.

2. The method according to claim 1, characterized in that The αy chain is the α chain with 1 strand in the target collagen.

3. The method according to claim 2, characterized in that The target collagen is type I collagen composed of one α2 chain and two α1 chains, and the specific antibody is the COL1A2 antibody that binds to the α2 chain. The amount and proportion of the triple helix structure of type I collagen are calculated according to the following formula: In the formula, α1 represents the number of specific peptides produced by enzymatic hydrolysis of all α1 chains in the antigen-antibody complex, and α2 represents the number of specific peptides produced by enzymatic hydrolysis of all α2 chains in the antigen-antibody complex.

4. The method according to claim 3, characterized in that The amino acid sequence of the specific peptide segment corresponding to the α1 chain of type I collagen is shown in SEQ ID No.1, and the amino acid sequence of the specific peptide segment corresponding to the α2 chain is shown in SEQ ID No.

2.

5. The method according to any one of claims 1 to 4, characterized in that: The enzymatic hydrolysis in step (2) is performed by using trypsin, a mixed enzyme of Trypsin and Lys-C, and trypsin without Lys-C.

6. The method according to claim 5, characterized in that In the enzymatic hydrolysis of step (2), trypsin is added at a protein to enzyme mass ratio of 25:1-5 for the first enzymatic hydrolysis, and the enzymatic hydrolysis is carried out at 35-40° C. for 16-20 hours; a mixed enzyme of Trypsin and Lys-C is added at a protein to enzyme mass ratio of 25:1-5 for the second enzymatic hydrolysis, and the enzymatic hydrolysis is carried out at 35-40° C. for 4-6 hours; and trypsin without Lys-C is added at a protein to enzyme mass ratio of 25:1-5 for the third enzymatic hydrolysis, and the enzymatic hydrolysis is carried out at 35-40° C. for 12-15 hours.

7. The method according to claim 6, characterized in that In the first enzymolysis and / or the third enzymolysis, CaCl2 is further added to make its final concentration 2-4 mM.

8. The method according to claim 5, characterized in that The test solution containing the target collagen in step (1) is prepared according to the following method: Biological tissues or cells are firstly cross-linked and fixed with 1-4% paraformaldehyde to collagen with a triple helix structure; then the cells are lysed under non-denaturing conditions and centrifuged to obtain a supernatant containing the target collagen.

9. The method according to claim 8, characterized in that The cleavage is carried out at a rate of (1-10)×10 7 Add 5 mL IP lysis buffer to each cell for lysis.

10. The method according to claim 9, characterized in that The precipitation separation is to use Protein A / G agarose beads to specifically bind to the antigen-antibody complex to precipitate it, and then centrifuge it.

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