Method, application, system and device for analyzing cathepsin D cleavage type II collagen site and computer readable storage medium
Through the algorithm based on non-trypsin cleavage fragment scoring, the problem of difficulty in accurately predicting the collagen site of cathepsin D cleavage type 2 in the prior art is solved, and accurate prediction of these sites is achieved and specific inhibition is helpful to develop agent, treat related diseases.
Patent Information
- Application Number
- CN202411500462.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2025-02-18
AI Technical Summary
When the existing polypeptide sequencing technology is looking for cathepsin D to cleave type 2 collagen sites, it is difficult to accurately obtain enzyme cleavage sites and distinguish between true and false positive results, which are greatly affected by background noise and experimental pollution.
Using an algorithm based on non-trypsin-cleaved fragment scoring, the short peptide sequence after trypsin is not spliced, but instead statistically scored the non-trypsin-cleaved fragments to calculate their significance, so as to achieve the cleavage of type 2 collagen for cathepsin D. Accurate prediction of loci.
By calculating the significance of nontrypsin-cleaved fragments, accurate prediction of the collagen site of cathepsin D cleavage type 2 is achieved, helping to design and discover specific inhibitors, and thus having a positive effect on the treatment of related diseases such as osteoarthritis.
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Figure CN119479792A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of protein analysis, and in particular to a method, application, system, device and computer-readable storage medium for analyzing the site of cathepsin D cleaving type II collagen. Background Art
[0002] At present, peptide sequencing technology mainly uses UPLC-MS / MS (ultra-high performance liquid chromatography-tandem mass spectrometry) technology, which measures the short peptide sequences after trypsin cleavage and reassembles these short sequences to obtain a theoretical complete sequence. However, this type of method has the following problems when searching for cleavage products of other proteases (such as cathepsin D): since the cleavage sites of other proteases are unknown, the theoretical cleavage fragments are often not accurately obtained; due to the influence of background noise and contamination during the experiment, the scoring of the beginning and end sequences of the spliced sequence sometimes presents a gradual signal, making it difficult to determine the specific starting and ending sites of the fragment.
[0003] Type II collagen is an important structural protein, mainly found in tissues such as cartilage, intervertebral discs and the cornea of the eye, and plays a key role in various tissues of the human body. Type II collagen plays a key role in various physiological and pathological processes, especially in bone development, joint health, and cartilage damage repair. In addition, abnormal expression and structural variation of type II collagen are associated with a variety of diseases, such as osteoarthritis and rheumatoid arthritis. The detection of type II collagen can be used for the diagnosis of diseases such as osteoarthritis. Drug targets for type II collagen, such as cathepsin D, have also become a key direction of drug research and development. By studying the enzyme cleavage sites of type II collagen, specific inhibitors can be developed for the treatment of related diseases.
[0004] Therefore, it is of great significance to study the site where cathepsin D cleaves type II collagen. Summary of the invention
[0005] Based on the above problems, the present application provides a method, application, system, device and computer-readable storage medium for analyzing the sites where cathepsin D cleaves type II collagen.
[0006] This application adopts the following technical solutions:
[0007] The first aspect of the present application discloses a method for analyzing the sites of cathepsin D cleaving type II collagen, comprising: using cathepsin D to perform enzymatic cleavage on type II collagen to obtain a first enzymatic cleavage product; using trypsin to perform enzymatic cleavage on the first enzymatic cleavage product, and performing polypeptide sequencing by mass spectrometry to obtain sequencing data; inputting the sequencing data into the following formula to calculate the standardized count of each site:
[0008] Among them, count s,i is the standardized count of the i-th site in the s-th sample. Assuming that there are n peptide sequences generated by non-trypsin cleavage of the i-th site, I s,f is the relative intensity of the fth non-trypsin cleavage peptide in the sth sample measured by mass spectrometry, I f is the total relative intensity of the peptide produced by the f-th non-trypsin cleavage in all samples, M s,f is the molecular weight of the peptide produced by the f-th non-trypsin cleavage in the s-th sample, E s,f is the charge number of the polypeptide produced by the f-th non-trypsin cleavage in the s-th sample, parameter Pi is 0 or 1, parameter Pm is 0 or 1, and parameter Pe is 0 or 1; the standardized counts of each site are ranked to obtain a ranking score p for each site, and the site where tissue plasminogen activator D cleaves type II collagen is predicted based on the ranking score p.
[0009] It should be noted that in the present application, an algorithm based on scoring of non-trypsin cleavage fragments is provided. The algorithm of the present application does not splice the short polypeptide sequence after trypsin cleavage, but searches for non-trypsin cleavage fragments (trypsin cleaves the polypeptide sequence at lysine K and leucine R). By statistically scoring these non-trypsin cleavage fragments and calculating the significance of non-trypsin cleavage fragments, accurate prediction of the sites where cathepsin D cleaves type II collagen is achieved. The principle is that if a site appears frequently in a single sample and one end (or both ends) of the starting point or end point is not at a known trypsin cleavage site, it may be a cathepsin D cleavage site. Among them, due to the different ionization degrees of different polypeptide sequences, this application introduces I f The standardization is performed and regulated by the parameter Pi. Since the peptide ionization is also affected by the molecular weight of the peptide, we introduce the parameter M s,f (controlled by parameter Pm) for correction; since the relative intensity after ionization is affected by the charge number, we introduce parameter E s,f (controlled by parameter Pe) for correction.
[0010] It should also be noted that when Pi is equal to 0, must be equal to 1, then count s,i The analysis will not pass I fStandardize or correct; when Pi is equal to 1, Equal to I f , then count s,i Will pass I f Normalize or correct; this is intended to be input by the user to control whether the count score is corrected by the total relative ion intensity. The same applies to the values of 0 or 1 for parameters Pm and Pe.
[0011] In one implementation of the present application, the site with a ranking score of p>0.999 is a potential cleavage site of cathepsin D cleaving type II collagen. It should be noted that ranking refers to sorting the standardized counts from large to small, and p greater than 0.999 refers to items ranked above the top 99.9% percentile.
[0012] In one implementation of the present application, ultra-high performance liquid chromatography-tandem mass spectrometry is used to perform the polypeptide sequencing.
[0013] In one implementation of the present application, after the sequencing data is processed using database search software, the formula is input to calculate the standardized count. It should be noted that database search software refers to a software tool used to process mass spectrometry (MS) data and compare it with a known protein sequence database. Common database search software includes MaxQuant, Mascot, Sequest, PeptideProphet, Percolator, etc.
[0014] The second aspect of the present application discloses an application of the method disclosed in the first aspect in finding the site where cathepsin D cleaves type II collagen.
[0015] The third aspect of the present application discloses a system for analyzing the sites of cathepsin D cleaving type II collagen, comprising: a first enzyme cleavage module, used to use cathepsin D to perform enzyme cleavage on type II collagen to obtain a first enzyme cleavage product; a sequencing module, used to use trypsin to perform enzyme cleavage on the first enzyme cleavage product, and use mass spectrometry to perform polypeptide sequencing to obtain sequencing data; a standardized counting module, used to input the sequencing data into the following formula to calculate the standardized count of each site:
[0016] Among them, count s,i is the standardized count of the i-th site in the s-th sample. Assuming that there are n peptide sequences generated by non-trypsin cleavage of the i-th site, I s,f is the relative intensity of the fth non-trypsin cleavage peptide in the sth sample measured by mass spectrometry, I fis the total relative intensity of the peptide produced by the f-th non-trypsin cleavage in all samples, M s,f is the molecular weight of the peptide produced by the f-th non-trypsin cleavage in the s-th sample, E s,f is the charge number of the polypeptide produced by the f-th non-trypsin cleavage in the s-th sample, the parameter Pi is 0 or 1, the parameter Pm is 0 or 1, and the parameter Pe is 0 or 1; a ranking scoring module is used to rank the standardized counts of each site to obtain a ranking score p for each site, and predict the site where tissue plasminogen activator D cleaves type II collagen according to the ranking score p.
[0017] The fourth aspect of the present application discloses a device for analyzing the sites of cathepsin D cleaving type II collagen, comprising a memory and a processor, wherein the memory is used to store programs, and the processor executes the programs stored in the memory to implement the method for analyzing the sites of cathepsin D cleaving type II collagen as described in the first aspect of the present application.
[0018] The fifth aspect of the present application discloses a computer-readable storage medium, wherein the storage medium stores a program, and the program can be executed by a processor to implement the method for analyzing the site of cathepsin D cleaving type II collagen as described in the first aspect of the present application.
[0019] The beneficial effects of this application are:
[0020] In the present application, an algorithm based on scoring of non-trypsin cleavage fragments is provided. The algorithm of the present application does not splice the short polypeptide segment sequence after trypsin cleavage, but searches for the non-trypsin cleavage fragments therein (trypsin cleaves the polypeptide sequence at lysine K and leucine R). By statistically scoring these non-trypsin cleavage fragments and calculating the significance of the non-trypsin cleavage fragments, accurate prediction of the sites where tissue plasminogen activator D cleaves type II collagen can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a diagram of the immunoblotting results after cathepsin D involved in the present application cleaves type II collagen.
[0022] Figure 2 It is a schematic diagram of the data processed by the database search software involved in this application.
[0023] Figure 3 It is a schematic diagram of the standardized counting results and ranking results of each site involved in this application.
[0024] Figure 4 It is a visualization result diagram of the standardized counts of each site involved in this application.
[0025] Figure 5 This is a result diagram of the molecular docking experiment involved in this application. DETAILED DESCRIPTION
[0026] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and the related operations can be fully understood according to the description in the specification and the general technical knowledge in the art.
[0027] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.
[0028] At present, peptide sequencing technology mainly uses UPLC-MS / MS technology, which measures the short peptide sequences after trypsin cleavage and reassembles these short sequences to obtain complete sequences. However, this type of method has the following problems when searching for other protease cleavage products: (1) It is impossible to determine the exact cleavage site; (2) There is no suitable statistical indicator to distinguish between true positive and false positive results. First, this method often requires pre-calculated theoretical cleavage fragments as an aid. However, since the cleavage site is unknown, this theoretical cleavage fragment is often not accurately obtained. Secondly, due to the influence of background noise and contamination during the experiment, the scoring of the beginning and end sequences of the spliced sequence sometimes presents a gradual signal, making it difficult to determine the specific fragment start and end sites. This results in the inability to accurately determine the specific cleavage site when searching for the protease cleavage site.
[0029] To this end, we invented an algorithm based on scoring of non-trypsin cleavage fragments. Our algorithm does not splice the short polypeptide sequence after trypsin cleavage, but looks for the non-trypsin cleavage fragments therein - trypsin cleaves the polypeptide sequence at lysine K and leucine R. By statistically scoring these non-trypsin cleavage fragments, it is possible to accurately predict the sites where cathepsin D cleaves type II collagen. This application also uses the short polypeptide sequence data obtained by UPLC-MS / MS to accurately predict the sites where cathepsin D cleaves type II collagen by calculating the significance of non-trypsin cleavage fragments. This application contributes to the design and discovery of specific cathepsin D inhibitors, and also has a positive effect on the treatment of type II collagen-related diseases such as osteoarthritis.
[0030] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.
[0031] It should be noted that, in this example, cathepsin D and type II collagen were mainly analyzed, and the method can also be applied to other non-trypsin and other proteins.
[0032] Example:
[0033] (1) Cathepsin D metabolizes type II collagen
[0034] First, 2 mg / mL type II collagen (chondrex, USA) was dissolved in 0.025 M acetic acid. The type II collagen solution was heat treated at 45°C for 1 h to obtain enough type II collagen α1 chains. Thereafter, 0.5 U / mL of cathepsin D (McLean, Shanghai) was added to the type II collagen solution and incubated at 37°C for 24 h. After the incubation, SDS-PAGE loading buffer was added to the incubation mixture and heated at 100°C for 5 min. The treated samples were electrophoresed using 10% acrylamide gel. The protein bands after electrophoresis were developed using Coomassie Brilliant Blue, as shown below. Figure 1 The results shown, Figure 1 This is a diagram of the immunoblotting results after cathepsin D involved in the present application cleaves type II collagen.
[0035] (2) Sequencing
[0036] It was found that cathepsin D cleaves type II collagen, of which p32 is a type II collagen atom fragment produced after cleavage. Taking p32 as an example, peptide sequencing of p32 was performed to explore the site where cathepsin D cleaves type II collagen. p32 was cut from the acrylamide gel and processed as follows:
[0037] ①Proteolysis:
[0038] Cutting rubber: Cut the sample into rubber particles of about 1mm;
[0039] Decolorization (staining gel): add 1 mL of decolorizing solution, vortex for 10 seconds, decolorize at 37°C for 30 minutes, centrifuge briefly, dry, and repeat the decolorization several times until the blue color is completely removed (add a quality control sample BSA for each project);
[0040] Washing gel particles (silver-stained gel): wash the gel 3 times, adding 1 mL of ultrapure water each time, and vortexing for 1 min;
[0041] Dehydration: add 500 μL acetonitrile to dehydrate until the particles turn completely white, repeat once;
[0042] Thiol reduction: remove acetonitrile, open the centrifuge tube cover and place in a clean bench for 10 min to air dry the acetonitrile, add 10 mM DTT until the liquid covers the pellet (twice the volume of the pellet), and place in a 56°C water bath for 1 h;
[0043] Thiol blocking: After cooling to room temperature, blot dry, quickly add 55 mM IAM until the liquid covers the particles (twice the volume of the particles), and place in a dark room for 45 min;
[0044] Washing: Aspirate the liquid, wash twice with 500 μL decolorizing solution, and wash once with pure water;
[0045] Dehydration: remove the liquid, add 500 μL acetonitrile, vortex and shake for 5 minutes, remove the acetonitrile, open the centrifuge tube cover and place it on the clean bench for 10 minutes to air dry thoroughly;
[0046] Add enzyme: dilute the enzyme solution (pre-packaged 1μg / μL Trypsin) to 0.01μg / μL with 25mM NH4HCO3, and use the enzyme solution to completely cover the micelles;
[0047] Particles swelling: 4℃ or on ice for 30min;
[0048] Add buffer: After the colloid particles swell, add corresponding buffer until the colloid particles are covered;
[0049] Incubate at 37°C overnight;
[0050] Gradient dissolution of peptides: add 5 times the volume of 50% ACN, vortex and shake for 5 minutes, centrifuge at 5,000g for 1 minute, and transfer the supernatant to a new centrifuge tube with the same number; then add 5 times the volume of 100% ACN, vortex and shake for 5 minutes, centrifuge at 5,000g for 1 minute, transfer the supernatant to a new centrifuge tube with the same number, and finally centrifuge the obtained supernatant at 25,000g for 5 minutes and take the supernatant;
[0051] Drainage: Freeze and drain the extracted peptide solution.
[0052] ②High performance liquid chromatography detection
[0053] The dried peptide samples were re-dissolved with mobile phase A (2% ACN, 0.1% FA), centrifuged at 20,000 g for 10 min, and the supernatant was injected.
[0054] The samples were separated by Thermo UltiMate 3000UHPLC. The samples were first enriched and desalted in a trap column, and then connected in series with a self-assembled C18 column (75 μm inner diameter, 3 μm column particle size, 25 cm column length), and separated at a flow rate of 300 nL / min through the following effective gradient: 0-5 min, 5% mobile phase B (98% ACN, 0.1% FA); 5-45 min, mobile phase B linearly increased from 5% to 25%; 45-50 min, mobile phase B increased from 25% to 35%; 50-52 min, mobile phase B increased from 35% to 80%; 52-54 min, 80% mobile phase B; 54-60 min, 5% mobile phase B. The end of the nanoliter liquid separation was directly connected to the mass spectrometer.
[0055] ③Mass spectrometry detection
[0056] The peptides separated by liquid phase were ionized by nanoESI source and then entered into the tandem mass spectrometer Q-Exactive HFX (Thermo Fisher Scientific, San Jose, CA) for DDA (Data Dependent Acquisition) mode detection. The main parameter settings were: ion source voltage was set to 1.9 kV; the primary mass spectrometry scanning range was 350-1,500 m / z; the resolution was set to 60,000; the secondary mass spectrometry starting m / z was fixed at 100; the resolution was 15,000. The screening conditions for the parent ions of the secondary fragmentation were: the parent ions with charges of 2+ to 6+ and peak intensities exceeding 10,000 ranked in the top 30. The ion fragmentation mode was HCD, and the fragment ions were detected in the Orbitrap. The dynamic exclusion time was set to 30 s. The AGC settings were: primary 3E6, secondary 1E5.
[0057] (3) Data analysis:
[0058] After UPLC-MS / MS sequencing of p32, the next step is to perform a maxquant library search. The library search uses the P02458 protein as a template and considers proline and lysine hydroxylation modifications. After searching the library, the following is obtained: Figure 2 txt file shown, Figure 2 This is a schematic diagram of the data processed by the database search software involved in this application. In R language, it is input into the following algorithm through the file1 and file2 parameters in the find_clevagesite() function:
[0059]
[0060] In this embodiment, the parameter Pm is set to 1, and the parameters Pi and Pe are both set to 0. After entering the algorithm, it will automatically run and calculate the standardized count count of each site. Among them, the fragments that begin or end with lysine or leucine (K / R) are not counted, that is, only the fragments that do not begin or end with lysine or leucine are counted, thereby excluding the fragments cut by trypsin. After the calculation is completed, the algorithm will automatically output the result to the site indicated by the output parameter. The output result is as follows Figure 3 As shown, Figure 3 It is a schematic diagram of the standardized counting and ranking results of each site involved in this application, in which the first column is the amino acid sequence site pointer, the second column is the calculated non-trypsin cleavage fragment standardized count, and the third column is its ranking score. It should be noted that Figure 3 The output results of some sites are only schematically listed. You can also use the figure parameter to indicate whether to visualize the results. The output results are as follows Figure 4 As shown, Figure 4 It is a visualization result diagram of the standardized counts of each site involved in this application, where the black line is the statistical significance threshold set by the p.threshold parameter. When the standardized count count of a site is greater than the threshold, the corresponding ranking score p is statistically significant.
[0061] As shown in the above results, there is a potential cleavage site at the 1028th amino acid position, and the cleavage site is found to be GPPGL|TGPAG by reading the sequence.
[0062] (4) Molecular docking experimental verification
[0063] Molecular docking was used to verify the selected fragments. The macromolecular receptor used for docking was cathepsin D (1YLB), and the small molecule ligand used was a decapeptide consisting of the cleavage site and the first four and last four amino acids. The small molecule structure was generated by alphafold using the peptide sequence, and the peptide skeleton was set to be non-rotatable to simulate the real structure of type II collagen. Molecular docking simulation was performed using AutoDockVina, and all parameters were set to default values. The search grid box set included the active site of cathepsin D (33-D and 231-D). The simulation docking results used the root mean square deviation between the Cartesian coordinates of the ligand atoms to cluster the binding modes, and the simulation results were sorted according to the binding free energy. The binding mode with the lowest binding free energy and the largest cluster member was considered to be the best docking conformation. Figure 5 This is a result diagram of the molecular docking experiment involved in the present application, showing that the best conformation verification ligand completely matches the cleavage mode of cathepsin D. The algorithm of the present application can accurately find the site where cathepsin D cleaves type II collagen.
[0064] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.
Claims
1. A method for analyzing the site of cathepsin D cleavage of type II collagen, characterized in that: include: Using cathepsin D to digest type II collagen to obtain a first digestion product; The first enzyme digestion product is digested with trypsin, and peptide sequencing is performed by mass spectrometry to obtain sequencing data; The sequencing data was input into the following formula to calculate the standardized counts of each site: Among them, count s,i is the standardized count of the i-th site in the s-th sample. Assuming that there are n peptide sequences generated by non-trypsin cleavage of the i-th site, I s,f is the relative intensity of the f-th non-trypsin cleavage peptide in the s-th sample measured by mass spectrometry, I f is the total relative intensity of the peptide produced by the f-th non-trypsin cleavage in all samples, M s,f is the molecular weight of the peptide produced by the f-th non-trypsin cleavage in the s-th sample, E s,f is the charge number of the peptide produced by the f-th non-trypsin cleavage in the s-th sample, parameter Pi is 0 or 1, parameter Pm is 0 or 1, and parameter Pe is 0 or 1; The standardized counts of each site are ranked to obtain a ranking score p of each site, and the site where cathepsin D cleaves type II collagen is predicted based on the ranking score p.
2. The method according to claim 1, characterized in that The sites with a ranking score of p>0.999 are potential cleavage sites for cathepsin D to cleave type II collagen.
3. The method according to claim 1, characterized in that The peptide was sequenced using ultra-high performance liquid chromatography-tandem mass spectrometry.
4. The method according to claim 1 or 3, characterized in that: After the sequencing data is processed using the library search software, the formula is input to calculate the standardized count.
5. Use of the method according to any one of claims 1 to 4 in finding the site where cathepsin D cleaves type II collagen.
6. A system for analyzing the site of cathepsin D cleavage of type II collagen, characterized in that: include: The first enzyme cleavage module is used to perform enzyme cleavage on type II collagen using cathepsin D to obtain a first enzyme cleavage product; A sequencing module, used to perform enzyme digestion on the first enzyme digestion product using trypsin, and perform polypeptide sequencing using mass spectrometry to obtain sequencing data; The standardized counting module is used to input the sequencing data into the following formula to calculate the standardized count of each site: Among them, count s,i is the standardized count of the i-th site in the s-th sample. Assuming that there are n peptide sequences generated by non-trypsin cleavage of the i-th site, I s,f is the relative intensity of the f-th non-trypsin cleavage peptide in the s-th sample measured by mass spectrometry, I f is the total relative intensity of the peptide produced by the f-th non-trypsin cleavage in all samples, M s,f is the molecular weight of the peptide produced by the f-th non-trypsin cleavage in the s-th sample, E s,f is the charge number of the peptide produced by the f-th non-trypsin cleavage in the s-th sample, parameter Pi is 0 or 1, parameter Pm is 0 or 1, and parameter Pe is 0 or 1; The ranking scoring module is used to rank the standardized counts of each site to obtain a ranking score p for each site, and predict the site where cathepsin D cleaves type II collagen based on the ranking score p.
7. The system according to claim 6, characterized in that The sites with a ranking score of p>0.999 are potential cleavage sites for cathepsin D to cleave type II collagen.
8. The system according to claim 6, characterized in that The polypeptide sequencing is performed using ultra-high performance liquid chromatography-tandem mass spectrometry; the sequencing data is processed using library search software, and then the formula is input to calculate the standardized count.
9. A device for analyzing the site of cathepsin D cleaving type II collagen, characterized in that: The method comprises a memory and a processor, wherein the memory is used to store a program, and the processor implements the method for analyzing the site of cathepsin D cleaving type II collagen as claimed in any one of claims 1 to 4 by executing the program stored in the memory.
10. A computer-readable storage medium, characterized in that: The storage medium stores a program, and the program can be executed by a processor to implement the method for analyzing the site of cathepsin D cleaving type II collagen as claimed in any one of claims 1 to 4.