Polypeptide sequencing technology based on single-molecule immune array

Abstract

The invention relates to a polypeptide sequencing technology based on a single molecule immune array, the technology can analyze a polypeptide sample with extremely low concentration, the concentration and sequence information of each polypeptide in the sample can be obtained only in one experiment, the analysis cost is low, and the technology is suitable for large-scale application.

Description

Technical Field

[0001] The invention belongs to the technical field of proteomics, and particularly relates to a polypeptide sequencing technology based on a single molecule immune array. Background Art

[0002] Proteomics is a science that studies the composition, distribution, changes and interaction patterns of proteins in cells, tissues and organisms. It involves in-depth analysis of protein expression patterns and functional characteristics of the proteome. Proteomics research has promoted the development of basic science and clinical applications by revealing biological mechanisms and identifying disease-related biomarkers, and has provided important support for personalized medicine and drug development. Peptide sequencing is one of the key technical means of studying proteomics. This technology can infer and identify protein types by analyzing amino acid sequences.

[0003] Traditional peptide sequencing is mainly based on the Edman degradation cycle. This method analyzes the N-terminal amino acids produced in each cycle to gradually identify the amino acid sequence of the peptide. This method was widely used in the early days of the era, but each cycle can only obtain information on one site in the sample, resulting in a slow overall analysis speed and the inability to handle situations where the sample contains multiple peptides. The new generation of peptide sequencing solutions is based on mass spectrometry. By collecting the fragment ion information of the peptide in the mass spectrometry data and combining it with certain bioinformatics algorithms to infer the sequence of the original peptide, this method has made significant progress in improving sequencing speed and accuracy, but it is often accompanied by high costs and has strict requirements on the amount and purity of the sample. Therefore, there is an urgent need for a new type of peptide sequencing technology that is high-throughput, fast, economical, has low sample requirements, and can simultaneously determine multiple peptide sequences in a sample. Summary of the invention

[0004] In view of the defects of the prior art, the technical problem to be solved by the present invention is to provide a polypeptide sequencing technology based on single molecule immune array.

[0005] The present invention provides a polypeptide sequencing method, comprising:

[0006] S1: Provide analysis platform;

[0007] S2: Probe preparation; wherein the probe includes two groups of DNA chains, A and B, wherein the group A DNA chain is a specific DNA chain for coupling with amino acids, and the group B DNA chain is a complementary chain of the group A DNA chain, and the complementary chain is connected to the enzyme;

[0008] S3: sample processing;

[0009] S4: adding the processed sample to the analysis platform;

[0010] S5: Perform detection.

[0011] Preferably, the analysis platform in step S1 comprises a substrate, wherein the substrate is provided with sample cavities distributed in an array; wherein the sample cavities are microporous structures or microwell structures.

[0012] Furthermore, the sample chamber is a well structure with a volume of fL level. For the fluorescent luminescence system, such a volume can ensure that the concentration of the fluorescent product produced by the catalysis of a single enzyme molecule reaches a level that can be observed by an ordinary fluorescent microscope. For the chemiluminescent system, the low background noise corresponding to the micro-well structure limitation can also make the chemiluminescence reach a level that can be observed by a microscope.

[0013] Furthermore, the method for preparing the sample cavity includes: at least one of a SIMOA method, a soft lithography method, and a silicon-based process.

[0014] The preparation method of SIMOA is to put the entire optical fiber into hydrochloric acid for etching, and control the time to obtain a microporous structure of appropriate depth. Due to the different materials of the optical fiber core and cladding, the etching rates in hydrochloric acid are different, and the etching rate of the core is faster.

[0015] A soft lithography process is used: a cylindrical structure is made on the wafer, and multiple structures are arranged to form a dense array. Then a PDMS mold is coated, and after peeling it off, a microwell structure can be obtained on the PDMS, and multiple microwells are arranged to form a dense array.

[0016] Using silicon-based technology, RIE (reactive ion etching) / DRIE (deep reactive ion etching) is performed on the oxide layer silicon wafer / silicon wafer after masking to directly form a microporous structure.

[0017] Furthermore, the size of the sample cavity is maintained so that at most one microbead can fall into each hole. At the same time, the fL volume is not fixed and can be expanded, but the expansion of the volume will make the concentration of the fluorescent product too low / the background noise too high, making it difficult to observe. At this time, it is necessary to use molecular brushes / polymerases and other solutions to increase the concentration of the marker enzyme to enhance the signal. In addition, after the array is prepared, the array surface needs to be sealed to prevent the subsequent non-specific binding of the array surface and the enzyme from affecting the observation.

[0018] In step S2, the DNA strands of group A are labeled A1, A2, A3, ..., An, respectively; the complementary strands of group B are labeled B1, B2, B3, ..., Bn, respectively; wherein n is a positive integer greater than 1.

[0019] The ends of the complementary strands are ligated to an enzyme;

[0020] The enzyme is one or more of an enzyme that catalyzes the production of fluorescent products, a chemiluminescent enzyme, and a color reaction enzyme.

[0021] The enzymes include, but are not limited to, one or more of HRP enzyme, alkaline phosphatase, and β-galactosidase.

[0022] Regarding the probe preparation in step S2:

[0023] In order to achieve specific labeling of amino acids, two groups of DNA chains with different sequences need to be prepared in advance. The end of group A is modified for coupling with specific amino acids (the specific modification is determined by the chemical properties of the amino acid functional group). The DNA chains of group B and group A are equal in number and correspond one to one in sequence (complementary). The enzymes (such as HRP enzyme, etc.) are connected to the ends in advance. Each DNA chain here is a single strand.

[0024] Methods for connecting enzymes (such as HRP enzymes) include but are not limited to:

[0025] Add biotin to the DNA end (5' or 3' end) and incubate with avidin-HRP enzyme conjugate;

[0026] HRP was oxidized with p-benzoquinone to generate active sites, which were then condensed with polyethyleneimine to form a complex, and then linked to DNA with glutaraldehyde.

[0027] Notice:

[0028] The DNA chain here is generally selected to be 5-40 nt in length. The following must be ensured during design: (1) The Hamming distance between different DNA chains is as large as possible to prevent mismatching; (2) No secondary structure should appear to ensure stability in a solution environment; the Hamming distance refers to the number of different characters at the same position between two strings of equal length.

[0029] In addition, the HRP enzyme is only used as an example, and any enzyme that can catalyze the production of fluorescent products / chemiluminescence / color development reactions can meet the requirements, and other enzymes include alkaline phosphatase and β-galactosidase.

[0030] The sample processing in step S3 includes: performing Boc tert-butyloxycarbonyl protection treatment on the sample, then connecting with the microsphere, then using the A group DNA chain for specific amino acid labeling, and finally removing the Boc tert-butyloxycarbonyl protecting group;

[0031] The microspheres are amino-modified microspheres.

[0032] The Boc tert-butyloxycarbonyl protection treatment is performed: the polypeptide is treated with di-tert-butyl dicarbonate, di-tert-butyl pyrocarbonate, triethylamine, and tetrahydrofuran to convert the two hydrogen atoms of the free amino group at the N-terminus into tert-butyloxycarbonyl groups. This process is called Boc protection, and the purpose is to avoid the interconnection of amino groups and carboxyl groups between polypeptides during the subsequent coupling process, and the modification of primary amines, which makes it impossible to perform Edman degradation cycles;

[0033] The same microsphere connection: using amino, carboxyl coupling protocol: under the action of carbodiimide, the surface amino modified microspheres and polypeptide samples are treated, and the two are connected by a stable amide bond. In this step, it should be noted that the number of microspheres should be much larger than the number of polypeptides to ensure that most of the microspheres have only one or zero polypeptide chains on their surfaces. Generally, the ratio of microspheres to polypeptides is 10:1.

[0034] The specific amino acid labeling using group A DNA chains: the labeling method is to chemically couple different sequence DNA probes with amino acids according to the properties of different amino acids, for example: (the DNA chains mentioned here are group A chains, and each DNA chain in group A is only responsible for connecting to a specific amino acid functional group);

[0035] For example, the amino-modified DNA is treated with SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, a cross-linking agent), and the resulting product is incubated with the polypeptide in PBS buffer, so that the DNA is linked to the -SH thiol group on the polypeptide.

[0036] Use carbodiimide or dicyclohexylcarbodiimide to connect the amino DNA to the -COOH carboxyl group on the peptide;

[0037] Use carbodiimide or dicyclohexylcarbodiimide to link the carboxylated DNA to the -NH2 amino group on the peptide.

[0038] Note: The modification schemes mentioned here are only examples. In actual operation, different amino acid functional groups can be labeled through various schemes. At the same time, more functional groups besides the three functional groups mentioned above can also be labeled. As long as the various labeling schemes do not affect each other, for example, the solution environment required for the next labeling step will destroy the labeling completed in the previous step, it can be regarded as a qualified scheme.

[0039] The purpose of this step is to allow different amino acids to connect to different DNA chains, converting the original detection of amino acids into detection of DNA chains.

[0040] The removal of the Boc tert-butyloxycarbonyl protecting group: using trifluoroacetic acid and dichloromethane to treat and remove the Boc (tert-butyloxycarbonyl) protecting group, exposing the free amino group at the N-terminus of each polypeptide, so as to facilitate the smooth Edman degradation cycle in subsequent sequencing.

[0041] Alternatively, the sample processing in step S3 includes: performing Boc tert-butyloxycarbonyl protection treatment on the sample, then using group A DNA chains for specific amino acid labeling, separating the labeling substances and connecting them to the microspheres; wherein the separation of the labeling substances is to use HPLC (liquid chromatography) or other methods to separate the DNA chains labeled with specific amino acids.

[0042] In the step S4: adding the processed sample to the analysis platform (sample loading).

[0043] The sample mixture is a microsphere, each of which has one or zero polypeptides bound to its surface. The specific amino acid functional groups (such as -SH, -COOH, -NH2) on each polypeptide have been labeled with DNA chains of different sequences.

[0044] The mixture is passed into the resulting array in the analysis platform for drop-in.

[0045] Note: The microspheres in this step include but are not limited to: magnetic beads, polystyrene beads, and silica beads, as long as the surface is amino-modified. Different types of microspheres have different methods of dropping into the hole. For example, magnetic beads can be assisted by magnets, while others can be assisted by vacuuming / centrifugation and other methods. The situation inside the hole after dropping is as follows Figure 2 shown.

[0046] The detection in step S5 includes: DNA binding, substrate introduction, incubation observation, DNA dissociation and washing, and Edman degradation cycle.

[0047] Furthermore, the detection in step S5 includes: (1) first adding the complementary chain B1 of group B to the analysis platform for DNA binding, incubating and observing after the substrate is introduced, and then performing DNA dissociation and washing; (2) replacing B1 in step (1) with the complementary chain B2 and repeating step (1) ... and so on, adding the complementary chain Bn to replace Bn-1 for DNA binding, incubating and observing after the substrate is introduced, and then performing DNA dissociation and washing; and then performing Edman degradation cycle;

[0048] Repeat the above process until there is no obvious fluorescence at all sites.

[0049] Note: The cycle from step (1) to Edman degradation is a large cycle, which is repeated until no obvious fluorescence is observed at all sites. The enzymes used to connect the complementary chains B1 to Bn in the above method can be the same or different.

[0050] Furthermore, the detection in step S5 includes: firstly adding all complementary chains B1 to Bn to the analysis platform for DNA binding, wherein B1 to Bn are respectively connected to different enzymes; each time a substrate corresponding to an enzyme is introduced, incubated and observed, and washed, until a substrate corresponding to the enzyme connected to Bn is added for incubation and observation, washed, and then an Edman degradation cycle is performed;

[0051] Repeat the above process until there is no obvious fluorescence at all sites.

[0052] Note: Adding the first substrate to the Edman degradation cycle is a large cycle, and the process is repeated until there is no obvious fluorescence at all sites.

[0053] For example, different enzymes are connected to the DNA chains of group B, and they are marked as enzyme 1, enzyme 2, enzyme 3... enzyme n respectively; then all the DNA chains of group B are added for DNA binding, and the substrate corresponding to enzyme 1 is introduced first, incubated, observed, and washed; then the substrate corresponding to enzyme 2 is introduced, incubated, observed, and washed; the substrate corresponding to enzyme 3 is introduced, incubated, observed, and washed, and so on, until the substrate corresponding to the enzyme connected to Bn is added, incubated, observed, washed, and then Edman degradation cycle is performed;

[0054] Repeat the above steps until there is no obvious fluorescence at all sites.

[0055] The introduction of the substrate (the substrate corresponding to the enzyme) generates fluorescence, color development or chemiluminescence; the incubation observes and records the luminescence site and intensity information.

[0056] After the detection in step S5 is completed, data processing is performed to determine the type and / or concentration of the polypeptide.

[0057] The polypeptide type determination: mapping the sequence information obtained in the experiment to the proteome, and searching for polypeptides that match the sequence information, so that high coverage measurement of the proteome can be achieved in a very short cycle;

[0058] Determination of peptide concentration: The analysis can obtain the information of peptide types at each site, record the number of each peptide, and then observe the array under bright field to count the number of all microspheres at this time. At this time, the ratio of the number of each peptide to the total number of microspheres represents the concentration of the peptide in the original sample, and the standard curve method is used to determine the concentration of the original peptide in the sample.

[0059] Note: First, use different concentrations of peptides to incubate the reaction, and observe the proportion of peptides in the microspheres in the corresponding situation to make a calibration curve. Then, the concentration of the original peptide in the sample is determined based on the calibration curve.

[0060] The present invention provides a polypeptide sequencing kit, which comprises a probe, wherein the probe comprises two groups of DNA chains, A and B, wherein the group A DNA chains are specific DNA chains for coupling with amino acids, the group B DNA chains are complementary chains of the group A DNA chains, and the complementary chains are connected to an enzyme, an analysis platform (single molecule array), and a substrate corresponding to the enzyme.

[0061] Beneficial Effects

[0062] The present invention proposes a polypeptide sequencing technology based on a single-molecule immune array, which can analyze polypeptide samples with extremely low concentrations. The concentration and sequence information of each polypeptide in the sample can be obtained in only one experiment, and the analysis cost is low, which is suitable for large-scale applications.

[0063] The present invention proposes a polypeptide sequencing technology based on a single-molecule immune array as a detection platform, combined with DNA probe technology, using Edman degradation to successively cleave terminal amino acids, so that the concentration and sequence information of each polypeptide in the sample can be obtained in one experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram of a single molecule array;

[0065] Figure 2 Schematic diagram of microsphere falling into the hole;

[0066] Figure 3 This is the labeling status of the polypeptide ACBCCAC after it is put into the complementary strand of DNA strand A;

[0067] Figure 4 The labeling status of the polypeptide ACBCCAC after it is put into the complementary strand of DNA strand B;

[0068] Figure 5 This is the experimental procedure for detecting the terminal amino acid of the ACBCCAC polypeptide in a single well. DETAILED DESCRIPTION

[0069] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0070] Peptide sequencing technology based on single molecule immune array as the detection platform, the analysis steps include:

[0071] 1. Preparation of analytical platform;

[0072] 2. Probe preparation;

[0073] 3.Preparation before sample loading;

[0074] 4. Sample loading;

[0075] 5. Detection after loading;

[0076] 6. Data analysis.

[0077] 1. Preparation of analytical platform:

[0078] The analytical platform constructs well structure arrays with volumes at the fL level.

[0079] In some specific embodiments, the traditional SIMOA preparation method is used: the entire optical fiber is placed in hydrochloric acid for etching, and the etching time is controlled to obtain a microporous structure of appropriate depth.

[0080] In some specific embodiments, a soft lithography process is used to prepare: a cylindrical structure with a diameter of about 4.5um and a height of about 3um is produced on a wafer, and multiple structures are arranged to form a dense array. Then a PDMS mold is coated, and after peeling off, a micro-well structure with a pore diameter of 4.5um and a height of about 3um can be obtained on the PDMS, and multiple micro-wells are arranged to form a dense array. Figure 1 shown.

[0081] In some specific implementations, silicon-based processes are used for preparation: RIE (reactive ion etching) / DRIE (deep reactive ion etching) is performed on the oxide layer silicon wafer / silicon wafer after masking to directly form a microporous structure.

[0082] 2. Probe Preparation

[0083] Two groups of DNA chains with different sequences are prepared. The end of group A is modified for coupling with specific amino acids (the specific modification is determined by the chemical properties of the amino acid functional group). The DNA chains of group B and group A are equal in number and correspond one to one in sequence (complementary). The enzymes are connected to the ends in advance. Each DNA chain here is a single strand.

[0084] In some embodiments, the ligase is HRP enzyme.

[0085] Method for connecting HRP enzyme: In some embodiments, biotin is added to the end of DNA (5' or 3' end) and incubated with avidin-HRP enzyme conjugate; in some embodiments, HRP is oxidized with p-benzoquinone to generate active sites, condensed with polyethyleneimine to form a complex, and then connected to DNA with glutaraldehyde.

[0086] In some embodiments the ligase is alkaline phosphatase or β-galactosidase.

[0087] 3. Sample preparation

[0088] The steps are as follows: the sample is treated with Boc tert-butyloxycarbonyl protection, then connected with the microsphere, then the A group DNA chain is used for specific amino acid labeling, and finally the Boc tert-butyloxycarbonyl protection group is removed.

[0089] The sample was subjected to Boc tert-butyloxycarbonyl protection treatment: the polypeptide was treated with di-tert-butyl dicarbonate, di-tert-butyl pyrocarbonate, triethylamine, and tetrahydrofuran to convert the two hydrogen atoms of the free amino group at its N-terminus into tert-butyloxycarbonyl groups.

[0090] Connecting with microspheres: Using amino and carboxyl coupling protocols: Under the action of carbodiimide, the surface amino-modified microspheres and the polypeptide samples are treated and connected by a stable amide bond. In some embodiments, the ratio of the number of microspheres to the number of polypeptides is 10:1.

[0091] Using group A DNA chains for specific amino acid labeling: In some embodiments, SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester, a cross-linking agent) is used to treat the aminated DNA, and the resulting product is incubated with the polypeptide in PBS buffer, so that the DNA is linked to the -SH thiol group on the polypeptide;

[0092] In some embodiments, carbodiimide or dicyclohexylcarbodiimide is used to link the amino DNA to the -COOH carboxyl group on the polypeptide.

[0093] In some embodiments, carbodiimide or dicyclohexylcarbodiimide is used to link the carboxylated DNA to the -NH2 amino group on the polypeptide.

[0094] Removal of Boc tert-butyloxycarbonyl protecting group: Use trifluoroacetic acid and dichloromethane to treat and remove the Boc (tert-butyloxycarbonyl) protecting group, exposing the free amino group at the N-terminus of each polypeptide, so as to facilitate the smooth progress of the Edman degradation cycle during subsequent sequencing.

[0095] 4. Sample loading

[0096] The prepared samples are introduced into the obtained array in the analysis platform for falling into the wells.

[0097] In some specific embodiments, the microspheres are at least one of magnetic beads, polystyrene beads, and silica beads; they can be modified with amino groups on the surface. Different types of microspheres have different methods of dropping into holes. In some specific embodiments, for example, magnetic beads can be assisted by magnets to drop into holes, while others can be assisted by vacuuming / centrifugation and other methods to drop into holes. The situation inside the hole after dropping into the hole is as follows Figure 2 .

[0098] 5. Post-sampling detection

[0099] DNA binding, introduction of fluorescent substrate, incubation observation, DNA dissociation and washing and / or Edman degradation cycles.

[0100] In some specific implementations, taking the detection process of labeling three specific functional groups (-SH, -COOH, -NH2) as an example, there are three types of DNA chains in group A, which are labeled with the above three functional groups, denoted as A1, A2, and A3, and have been connected to the corresponding amino acids in (III). There are three types of DNA chains in group B, denoted as B1, B2, and B3, which are complementary to A1, A2, and A3, respectively.

[0101] (1) DNA binding: Add the B1 chain to the array. At this time, the B1 chain will complement the A1 chain connected to the polypeptide. Due to the presence of HRP enzyme at the end of the B1 chain, the detection of all -SH in the polypeptide sample is converted into the detection of HRP enzyme. Subsequently, the array is thoroughly washed with PBS solution to completely remove the unbound B1 chain in the system.

[0102] (2) Introduction of fluorescent substrate: A mixed solution of Amplex Red and H2O2 is introduced. The substrate will produce the fluorescent product resorufin under the catalysis of HRP enzyme.

[0103] (3) Incubation observation: Inject mineral oil or fluorinated oil, or use glass slides for physical sealing, so that the solutions in all wells in the array are each sealed into fL-level isolation chambers. Due to the extremely small size, the fluorescent product produced by the catalysis of a single enzyme molecule can quickly reach a level that can be observed under an ordinary fluorescence microscope.

[0104] A CCD camera is used to take pictures and record the luminescent sites and intensity information. All luminescent sites contain two pieces of information: there are polypeptides on these sites, and there are sulfhydryl groups on the polypeptides (i.e., there is cysteine).

[0105] (4) DNA dissociation and cleaning: The temperature is raised to allow the bound DNA chains to dissociate (generally, the dissociation temperature of a 15 nt long DNA chain is around 50°C), and then the flow channel is thoroughly cleaned to remove the substrate added in the above process and the dissociated DNA chains.

[0106] (5) Replace the DNA chain with B2, repeat steps 1-4, and record the luminescent sites and intensity information. All luminescent sites contain two pieces of information: there are polypeptides at these sites, and there are side chain carboxyl groups on the polypeptides (i.e., there are aspartic acid and glutamic acid).

[0107] (6) Replace the DNA chain with B3, repeat steps 1-4, and record the luminescent sites and intensity information. All luminescent sites contain two pieces of information: there are polypeptides at these sites, and there are side chain amino groups on the polypeptides (i.e., there are arginine and lysine).

[0108] (7) Edman degradation cycle: A mixed solution of phenyl isothiocyanate and trimethylamine is introduced into the array to couple with the primary amine end of the protein. Trifluoroacetic acid is then introduced to remove the N-terminal conjugate, resulting in the remaining polypeptide chain after removing the first amino acid at the N-terminus. At this time, the primary amine at the N-terminus is still exposed and can be used for the next Edman degradation.

[0109] (8) After the Edman degradation cycle, the above process is repeated to observe the luminescence intensity information under each different group B DNA binding before and after the cycle. If the luminescence intensity of a certain site drops sharply or even disappears under a certain group B DNA binding condition, it means that the polypeptide at that site contains the amino acid labeled by the DNA chain and the amino acid is located at the N-terminus of the polypeptide.

[0110] For example, when B1 binds, obvious fluorescence is observed at a certain site. After one cycle of Edman degradation, it is combined with B1 again and incubated with substrate. It is observed that the fluorescence at the corresponding site disappears. This indicates that the first amino acid at the N-terminus of the polypeptide at this site must have a thiol group (i.e., cysteine).

[0111] Repeat the above process until there is no obvious fluorescence at all sites.

[0112] The HRP enzyme + Amplex Red system used in the above specific implementation is only an example, and any system can be used as long as it can catalyze the generation of fluorescent substrate / chemiluminescence phenomenon / color development reaction, or even a detectable current signal.

[0113] The above process uses only one fluorescent system. Some specific implementations use a combination of different enzymes and substrates to label the Group B DNA probes, thereby simplifying the steps involved in 4-6 and greatly improving the processing efficiency.

[0114] Specifically, DNA chain B1 is labeled with HRP enzyme, B2 is labeled with AP enzyme, and B3 is labeled with galactosidase. Each time DNA binds, a mixture of three DNAs is introduced simultaneously to allow them to bind simultaneously. In subsequent observations, one luminescent substrate is introduced each time (if there is no acid-base or other environmental conflict between the substrates catalyzed by multiple enzymes, multiple substrates can also be introduced) and the image is recorded. Similarly, the type of amino acid at the N-terminus can be determined by analyzing the image changes before and after Edman degradation.

[0115] In the above process, only three amino acids -SH, -COOH, and -NH2 are used as examples. The actual labeling is not limited to these three amino acids. Any amino acid that is chemically active and can be specifically labeled can be added to the scheme. The specific number is not limited to three. In theory, the more labeled amino acids, the more accurate the subsequent test results.

[0116] The Edman degradation method used above aims to cut off one amino acid at the N-terminus of the polypeptide each time. In this case, aminopeptidase is used to gradually cleave the N-terminal amino acid, which can also achieve the same effect. In addition, the polypeptide can be anchored to the solid surface with sulfhydryl or even amino groups, and then the C-terminal amino acid can be cleaved with carboxypeptidase. That is, the anchoring method and the cleavage method are not fixed, as long as they are not contradictory.

[0117] In some specific embodiments, as follows Figure 3 ,4,5 show the sequencing process of a peptide (the sequence from N-terminal to C-terminal is ACBCCAC). A, B, and C are only references and do not refer to specific amino acids. A and B can be modified, but C cannot be modified. Figure 3 , 4 shows the binding effect after the complementary DNA chain is released. Figure 5 The specific steps for detecting the terminal amino acid of a single-site polypeptide are shown.

[0118] 6. Data Processing

[0119] According to the detection steps given in (V) (assuming that the labeling group is still -SH, -COOH, -NH2), the position distribution of all 1. cysteine ​​2. aspartic acid or glutamic acid 3. arginine or lysine in the polypeptide chain can be obtained, as well as the specific position of the polypeptide chain in the array. The method for determining the type and concentration of the polypeptide from the experimental results is as follows:

[0120] (1) Determination of peptide types: The above experimental results only obtained the position information of some amino acids, and could not obtain the type information of all amino acids in the peptide. However, since the human proteome information is already very complete, it is only necessary to map the partial sequence information obtained from the experiment to the proteome and search for peptides that match the sequence information. In this way, high coverage measurement of the proteome can be achieved within a very short cycle.

[0121] Studies have shown that the peptides produced by cyanobromide are anchored to the solid phase support through the C-terminus, labeling the four amino acids on the polypeptide, and then processed through the Edman degradation cycle, ultimately achieving a proteome coverage of about 98%.

[0122] (2) Determination of polypeptide concentration. According to the analysis in (1), the polypeptide type information of each site can be obtained, the number of each polypeptide can be recorded, and then the array can be observed under bright field, and the number of all microspheres at this time can be counted. At this time, the ratio of the number of each polypeptide to the total number of microspheres represents the concentration of the polypeptide in the original sample. The concentration calculation principle is based on SIMOA (single molecule array), which can achieve ultra-low concentration detection that is several orders of magnitude lower than traditional ELISA. It should be noted that the concentration of each polypeptide here cannot be directly calculated by calculating the ratio of each polypeptide to the total number of microspheres. It is necessary to first use different concentrations of polypeptides for reaction incubation, and observe the ratio of polypeptides to microspheres in the corresponding situation, make a calibration curve, and then judge the concentration of the original polypeptide in the sample based on the calibration curve.

[0123] Example 2

[0124] like Figure 5 Shown are the steps of detecting the first amino acid at the N-terminus of the peptide ACBCCAC after labeling, connecting to microspheres, and dropping into the well, wherein amino acids A and B are labeled amino acids, and amino acid C is not labeled.

[0125] The specific detection steps and the phenomenon analysis of each step are as follows:

[0126] 1. Pass the complementary chain of DNA chain A (with HRP enzyme connected to the end)

[0127] 2. Pass HRP enzyme fluorescent substrate and incubate for observation;

[0128] 3. Obvious fluorescence was observed under a microscope, indicating that the polypeptide at this site contained at least one A amino acid;

[0129] 4. Heat to dissociate the complementary strand of the DNA strand A just put in, and then wash;

[0130] 5. Pass the complementary strand of DNA strand B (with HRP enzyme attached to the end);

[0131] 6. Pass HRP enzyme fluorescent substrate and incubate for observation;

[0132] 7. Obvious fluorescence was observed under the microscope, indicating that the polypeptide at this site contained at least one B amino acid;

[0133] 8. Heat to dissociate the complementary strand of the DNA strand B just added, and then wash;

[0134] 9. Perform edman degradation

[0135] The above completes a basic cycle. Next, continue

[0136] 10. Pass the complementary strand of DNA strand A (with HRP enzyme attached to the end);

[0137] 11. Add HRP enzyme fluorescent substrate and incubate for observation;

[0138] 12. Obvious fluorescence was observed under a microscope, and the fluorescence intensity was reduced compared to the fluorescence intensity observed when the complementary chain of DNA chain A was incubated with the substrate before Edman degradation.

[0139] This indicates that the terminal amino acid of the Edman degradation cut just now is amino acid A.

[0140] 13. Heat to dissociate the complementary strand of the DNA strand A just added, and then wash;

[0141] 14. Pass the complementary strand of DNA strand B (with HRP enzyme attached to the end);

[0142] 15. Pass HRP enzyme fluorescent substrate;

[0143] 16. Obvious fluorescence was observed under a microscope, and the fluorescence intensity was unchanged compared to the fluorescence intensity observed when the complementary strand of DNA strand B and the substrate were incubated before Edman degradation;

[0144] This indicates that the terminal amino acid of the Edman degradation is not the B amino acid.

[0145] In summary, the first amino acid at the N-terminus of the polypeptide is amino acid A;

[0146] (If you want to continue detecting the peptide, then do another Edman degradation after step 16, and then repeat steps 1-9).

Claims

1. A polypeptide sequencing method, comprising: S1: Provide analysis platform; S2: Probe preparation; wherein the probe includes two groups of DNA chains, A and B, wherein the group A DNA chain is a specific DNA chain for coupling with amino acids, and the group B DNA chain is a complementary chain of the group A DNA chain, and the complementary chain is connected to the enzyme; S3: sample processing; S4: adding the processed sample to the analysis platform; S5: Perform detection.

2. The sequencing method according to claim 1, characterized in that: In step S1, the analysis platform includes a substrate, wherein the substrate is provided with sample cavities distributed in an array; wherein the sample cavities are microporous structures or microwell structures.

3. The sequencing method according to claim 1, characterized in that: In step S2, the DNA strands of group A are labeled A1, A2, A3...An respectively; the complementary strands of group B are labeled B1, B2, B3...Bn respectively; The ends of the complementary strands are ligated to an enzyme; The enzyme is one or more of an enzyme that catalyzes the production of fluorescent products, a chemiluminescent enzyme, and a color reaction enzyme.

4. The sequencing method according to claim 1, characterized in that The sample processing in step S3 includes: performing Boc tert-butyloxycarbonyl protection treatment on the sample, then connecting with the microsphere, then using the A group DNA chain for specific amino acid labeling, and finally removing the Boc tert-butyloxycarbonyl protecting group; or performing Boc tert-butyloxycarbonyl protection treatment on the sample, then using the A group DNA chain for specific amino acid labeling, separating the label and connecting with the microsphere; The microspheres are amino-modified microspheres.

5. The sequencing method according to claim 1, characterized in that: The detection in step S5 includes: DNA binding, substrate introduction, incubation observation, DNA dissociation and washing, and Edman degradation cycle.

6. The sequencing method according to claim 5, characterized in that: The detection in step S5 includes: (1) first adding the complementary chain B1 of group B to the analysis platform for DNA binding, passing the substrate, incubating and observing, and then performing DNA dissociation and washing; (2) replacing B1 in step (1) with the complementary chain B2 and repeating step (1) ... and so on, adding the complementary chain Bn to replace Bn-1 for DNA binding, passing the substrate, incubating and observing, and then performing DNA dissociation and washing; and then performing Edman degradation cycle; Repeat the above process until there is no obvious fluorescence at all sites.

7. The sequencing method according to claim 5, characterized in that: The detection in step S5 includes: firstly adding all complementary chains B1 to Bn to the analysis platform for DNA binding, wherein B1 to Bn are respectively connected to different enzymes; each time introducing a substrate corresponding to an enzyme, incubating and observing, washing, until adding a substrate corresponding to the enzyme connected to Bn, incubating and observing, washing, and then performing Edman degradation cycle; Repeat the above process until there is no obvious fluorescence at all sites.

8. The sequencing method according to claim 6 or 7, characterized in that: After the substrate is introduced, fluorescence, color development or chemiluminescence is generated; during the incubation, the luminescence site and intensity information are observed and recorded.

9. The sequencing method according to claim 1, characterized in that: After the detection in step S5 is completed, data processing is performed to determine the type and / or concentration of the polypeptide.

10. A polypeptide sequencing kit, characterized in that: The kit includes a probe; wherein the probe includes two groups of DNA chains, A and B, wherein the group A DNA chains are specific DNA chains used for coupling with amino acids, and the group B DNA chains are complementary chains of the group A DNA chains, and the complementary chains are connected to the enzyme, the analysis platform, and the substrate corresponding to the enzyme.

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