Method for analyzing disulfide bonds in polypeptides based on nmr technology

Abstract

The present application relates to the field of biological macromolecular structure detection, and particularly relates to a method for analyzing disulfide bonds in polypeptides based on nuclear magnetic resonance technology, which acquires and analyzes 1D 1 H spectrum and 2D spectrum of a polypeptide sample to obtain information such as the number, position and pairing mode of disulfide bonds in the polypeptide sample, wherein the 2D spectrum includes 1 H- 1 H COSY, TOCSY, NOESY and 1 H- 13 CHSQC spectrum. Compared with the prior art, the method for analyzing disulfide bonds in polypeptides provided by the present application is simple, efficient, does not damage the molecular structure, and can also solve the problem of serious signal stacking of polypeptide molecules rich in cysteine to a certain extent.

Description

Technical Field

[0001] This invention relates to the field of biomolecular structure detection, specifically, to a method for analyzing disulfide bonds in peptides based on nuclear magnetic resonance (NMR) technology. Background Technology

[0002] Disulfide bonds (-SS-) are a crucial post-translational modification formed by the covalent cross-linking of two cysteine ​​side chain thiol groups (-SH) within or between polypeptide chains. Disulfide bonds themselves are not entirely stable; oxidized disulfide bonds can be reversibly reduced to free thiol groups, making them typically dynamic chemical bonds. Most natural hormones, growth factors, and monoclonal antibodies contain disulfide bonds. Their presence plays a vital role in stabilizing the spatial structure of these proteins, maintaining correct folding conformation, preserving and regulating biological activity, and performing normal physiological functions. Mismatched disulfide bonds can lead to misfolding of the spatial structure and loss of protein function. Furthermore, many bioactive polypeptides in nature (such as spider, scorpion, and cone snail venom peptides, and plant-derived cyclic peptides) are also rich in disulfide bonds, with approximately 70% or more containing two or more pairs. By forming disulfide bonds, the conformation of peptide molecules is further restricted or locked. Peptides containing multiple disulfide bonds exhibit higher complexity, stronger target binding ability, and higher selectivity, along with increased thermal and protease stability. Given the often superior biological activity and pharmacokinetic stability of disulfide-bonded peptides, drug developers are increasingly focusing on them. The design of drugs containing multiple disulfide bonds is now commonly applied in molecular recognition, supramolecular self-assembly, and biological ion channels. Some disulfide-bonded peptides have been successfully marketed and are used in the treatment and diagnosis of diseases such as diabetes and cancer. Many more disulfide-bonded peptides have become lead compounds in the research and development of analgesia and autoimmune diseases.

[0003] In summary, the linkage mode of intramolecular disulfide bonds directly affects the biological activity of peptides. Determining this linkage mode will help reveal the higher-order structure of peptides or proteins and guide the evaluation of chemically or biologically directed synthesis. Therefore, the precise and rapid localization of disulfide bonds in peptides and proteins is one of the important aspects of studying their structure and function. Given the rapid development of peptide and protein drugs globally, the characterization of disulfide bonds has become an indispensable and crucial step in the safety and efficacy quality evaluation of biopharmaceuticals.

[0004] Characterization of disulfide bonds mainly involves determining the number, position, pairing mode, and conformation of intramolecular disulfide bonds. Determining the number of disulfide bonds is relatively simple; for example, mass spectrometry (MS) can be used to compare the difference in relative molecular mass of the peptide or protein before and after complete reduction with a reducing agent. However, the localization of disulfide bonds (i.e., determining their position and pairing mode) remains challenging. A common method is to combine protease digestion with mass spectrometry peptide analysis. For instance, using TECP reagent under low pH conditions, folded peptides are partially reduced, followed by high-performance liquid chromatography (HPLC) purification of the unfolded intermediates and subsequent alkylation products. These alkylation intermediates are then subjected to complete reduction and a second round of alkylation, followed by Edman assays. This process is exceptionally cumbersome and time-consuming, and may involve rearrangement of the intramolecular disulfide bond bridging ring structure during partial reduction and partial alkylation. Later, researchers improved the method using cyanation, which partially solved the problem. However, the enzyme digestion mass spectrometry method is not efficient for many active peptides, especially for short peptides (such as those with less than 15 amino acids). Finding suitable protease cleavage sites is not easy, and the method will destroy the chemical structure of the sample.

[0005] Currently, high-resolution methods for determining disulfide bond linkages include X-ray single-crystal diffraction and nuclear magnetic resonance (NMR). X-ray crystallography is theoretically ideal for determining the disulfide backbone structure of peptides because the sulfur atoms in the cysteine ​​side chain readily undergo electron diffraction and can be clearly seen in electron density maps. However, most disulfide-rich peptides are difficult to crystallize due to the inherent flexibility of their structures. Nuclear magnetic resonance (NMR), as a non-destructive method, can observe molecular structure and dynamics in solution under physiological conditions (such as buffer, ionic strength, pH, and temperature), offering unique advantages in resolving high-resolution peptide solutions. However, de novo NMR analysis of disulfide-containing peptides often requires obtaining the solution structure of the peptide, and the extraction of NOE distance constraints and iterative structural calculations are complex and time-consuming. Therefore, there is an urgent need for a simple, efficient, and non-destructive method for analyzing disulfide bonds in peptides. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems in related technologies. To this end, this invention provides a method for analyzing disulfide bonds in peptides based on nuclear magnetic resonance (NMR) technology. This method involves collecting 1D samples of peptides... 1 H spectrum and including 1 H- 1 H COSY, TOCSY, NOESY and 1 H -13The 2D spectrum of C HSQC is obtained, and analysis of the spectrum yields information such as the number, position, and pairing mode of disulfide bonds in the polypeptide sample. Compared with existing disulfide bond analysis methods, the method provided by this invention is simple, efficient, and non-destructive to molecular structure.

[0007] Therefore, the first aspect of the present invention provides a method for analyzing disulfide bonds in peptides based on nuclear magnetic resonance technology, the method comprising:

[0008] (1) Pre-process the polypeptide sample to obtain the NMR sample;

[0009] (2) Acquire 1D NMR samples of the NMR sample 1 Spectral analysis was performed using H-spectrum and 2D spectrum.

[0010] in,

[0011] The 2D spectrum includes 1 H- 1 H COSY, TOCSY, NOESY and 1 H- 13 C HSQC spectrum;

[0012] The spectral analysis includes: utilizing the... 1 H- 1 H COSY, TOCSY, and NOESY spectra were used to assign amino acid signals from peptide samples; the above methods were employed to... 1 H- 13 C HSQC spectral pair 13 Assignment based on C chemical shift, according to C β Chemical shift is used to determine the presence and number of disulfide bonds in a peptide sample; the pairing mode of the disulfide bonds is determined based on the NOE signal in the NOESY spectrum.

[0013] This invention utilizes liquid nuclear magnetic resonance to acquire 1D spectra and a series of 2D spectra, including 1 H- 1 HCOSY, TOCSY, NOESY and 1 H- 13 C HSQC spectroscopy is used to assign amino acid signals of peptides based on cysteine ​​C β Chemical shift values ​​determine the state of cysteine, i.e., whether it is in a reduced state (free -SH) or an oxidized state (disulfide bond -SS), based on the β-methylene proton (H) of cysteine. β The NOE-related information between the two molecules is used to locate disulfide bonds. This method provides a simple, efficient, and non-destructive way to obtain information about disulfide bonds in peptides.

[0014] According to an embodiment of the present invention, the NMR sample includes a field-locking reagent.

[0015] According to an embodiment of the present invention, the pH value of the NMR sample is 5.0-6.0. This ensures that the NH signal and peak linewidth signal in the polypeptide backbone region remain normal and unaffected.

[0016] According to an embodiment of the invention, the NMR sample further comprises a phosphate buffer solution. This provides a testing environment consistent with physiological or drug administration conditions.

[0017] According to an embodiment of the present invention, the field-locking reagent comprises deuterated heavy water (D2O).

[0018] According to an embodiment of the present invention, the NMR sample further includes an internal standard, which can serve as a reference for spectral chemical shifts.

[0019] According to embodiments of the present invention, the internal standard includes 4,4-dimethyl-4-silylpentane-1-sulfonic acid (DSS) or 3-trimethylsilylpropionic acid (TMSP).

[0020] According to an embodiment of the present invention, the purity of the polypeptide sample is above 95%.

[0021] According to an embodiment of the present invention, the final concentration of the polypeptide sample is greater than 1 mM.

[0022] According to an embodiment of the present invention, the concentration of the field-locking reagent is not less than 5%.

[0023] According to an embodiment of the present invention, step (2) further includes: acquiring 1D NMR data of the NMR sample by NMR testing. 1 H-spectrum and 2D spectrum.

[0024] According to an embodiment of the present invention, the nuclear magnetic resonance (NMR) test includes: adding the NMR sample into an NMR tube, performing preparation steps, and calling a pulse sequence to suppress the water peak.

[0025] The preparation steps include field locking, field homogenization, and tuning.

[0026] According to embodiments of the present invention, the pulse sequence includes at least one selected from pre-saturation pulse sequences, watergate suppression pulse sequences, and excitation sculpting pulse sequences. A suitable pulse sequence is selected for watergate suppression, and the process is continuously optimized and adjusted. 1 The center frequency of the H-spectrum window overlaps with the frequency of the water peak signal, thereby minimizing interference from the water peak signal.

[0027] According to an embodiment of the present invention, the nuclear magnetic resonance test further includes: testing the 90° pulse width after performing the preparation steps. The net magnetization transfer is maximized by testing the 90° pulse width.

[0028] According to an embodiment of the present invention, the nuclear magnetic resonance test is performed at 280K-298K.

[0029] According to a specific embodiment of the present invention, the sample testing temperature can be selected at room temperature (298K), or low temperature conditions can be selected for spectral acquisition according to the properties of the sample to reduce the tumbling time of the peptide sample in the molecular solution, which is beneficial to acquiring more NOE signals.

[0030] According to an embodiment of the present invention, in step (2), the scanning spectral width during the acquisition of the 2D spectrum is set to be able to cover the 1D spectrum. 1 The frequencies of all signals in the H-spectrum.

[0031] According to an embodiment of the present invention, the scanning spectral width is 14-18 ppm.

[0032] According to an embodiment of the present invention, the mixing time for acquiring the TOCSY spectrum is set to 80-120 msec.

[0033] According to an embodiment of the present invention, the mixing time for acquiring the NOESY spectrum is set to 200-500 msec.

[0034] According to an embodiment of the present invention, the collection of the 1 H- 13 Prior to C HSQC spectroscopy, peptide samples were freeze-dried and reconstituted with D2O. This minimizes the interference of water peak signals on the amino acid HA region signals, facilitating accurate determination of the HA region signal. 1 H- 13 C HSQC spectroscopy analysis of information related to non-exchangeable protons in peptide sample structures.

[0035] According to an embodiment of the present invention, the 1 H- 13 When acquiring C HSQC spectral data, the indirect dimension (F1) spectral width was set to 5-80 ppm.

[0036] According to an embodiment of the present invention, utilizing the 1 H- 1 Assigning amino acid signals from peptide samples using H COSY, TOCSY, and NOESY spectra includes: utilizing the aforementioned 1 H- 1 H COSY and TOCSY spectra are used to assign signals within the amino acid spin system, and the NOESY spectra are used to identify the connection sequence between amino acids.

[0037] According to an embodiment of the present invention, the 1 H- 13 C HSQC spectrum 13 Methods for assigning C chemical shifts include those based on assignment. 1 H chemical shift 1 H- 13 C HSQC spectrum 13 Assignment of C chemical shift.

[0038] According to an embodiment of the present invention, the step according to C β Chemical shift analysis for determining the presence and number of disulfide bonds in a peptide sample further includes: if C β If the chemical shift value is less than 32.0 ppm, cysteine ​​exists in its reduced form, indicating that no disulfide bond has been formed; if C β If the chemical shift value is greater than 35.0 ppm, cysteine ​​exists in the oxidized form, indicating the formation of a disulfide bond; if it is in the range of 32.0-35.0 ppm, the oxidation / reduction state of cysteine ​​needs further determination.

[0039] According to an embodiment of the present invention, determining the pairing mode of disulfide bonds based on the NOE signal in the NOESY spectrum further includes: determining the pairing mode of disulfide bonds based on the NOE signal between the β-methylene protons of the two cysteine ​​residues.

[0040] According to an embodiment of the present invention, step (2) further includes collecting peptide samples ROESY and 2D. 13 C-edited NOESY, 3D 13 At least one of the following in the C-edited HSQC-NOESY spectrum. The ROESY spectrum can be used to replace the NOESY spectrum for assigning amino acid signals in peptides, thus avoiding weak or sparse related peak signals in the NOESY spectrum. 2D 13 C-edited NOESY and 3D 13 C-edited HSQC-NOESY spectroscopy can be used to resolve issues in cysteine-rich peptide samples where severe chemical shift stacking prevents the determination of H+. β The issue of assigning displacements.

[0041] According to an embodiment of the present invention, the method further includes: replacing the disulfide bonds in the polypeptide sample structure with diselenylene bonds, acquiring 2D spectra after sample preparation, and determining the connection information of the disulfide bonds in the original structure. This can solve the problem of "false" NOEs caused by local structural crowding in some cysteine-rich polypeptides, such as toxic polypeptides, resulting in excessively close spatial distances.

[0042] According to an embodiment of the present invention, the 2D spectrum includes selections from 2D... 1 H, 77 Se-HMQC, 1 H, 77 Se-HMBC, 77 Se- 77 At least one of the following in the Se COSY spectrum.

[0043] According to an embodiment of the present invention, determining the connection information of disulfide bonds in the original structure includes: analyzing the 2D... 1 H, 77 Se-HMQC and / or 1 H, 77 Relevant peaks in Se-HMBC spectrum 3 J Hβ-Seγ The information determines the connection information of disulfide bonds in the original structure;

[0044] and / or extract the 77 Se- 77 Se COSY spectrum 1 J Seγ-Seγ Information determines the connection information of disulfide bonds.

[0045] The present invention has the following advantages over the prior art:

[0046] (1) Simple and efficient operation. Compared with commonly used enzyme digestion mass spectrometry analysis methods, this invention uses liquid nuclear magnetic resonance to obtain disulfide bond information in peptides. Disulfide bond analysis can be performed under physiological solution conditions, avoiding cumbersome pretreatment processes, as well as the processing and determination of alkylation intermediates. It also avoids the rearrangement reaction of intramolecular disulfide bond bridged ring structures that may occur during this process. Furthermore, compared with conventional nuclear magnetic resonance analysis methods, it eliminates the need for de novo analysis and calculation of solution structures, saving time.

[0047] (2) No damage to molecular structure. Unlike enzyme digestion mass spectrometry analysis, the method for analyzing disulfide bonds in peptides based on nuclear magnetic resonance technology provided by this invention does not require an enzyme digestion step. Instead, it observes the structure and dynamics of peptides in solution in a non-destructive manner, thereby obtaining relevant information about disulfide bonds in the peptide structure.

[0048] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0049] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0050] Figure 1 The structural formula of the polypeptide drug linaclotide in Example 1 of the present invention is shown;

[0051] Figure 2 The experimental flowchart for analyzing disulfide bond information of peptide samples based on nuclear magnetic resonance technology in Example 1 of the present invention is shown.

[0052] Figure 3 The 1D of the polypeptide drug in Example 1 of the present invention is shown. 1 H spectrum;

[0053] Figure 4-7 The polypeptide drug in Example 1 of this invention is shown respectively. 1 H- 1 H COSY, TOCSY, NOESY 1 H- 13 CHSQC spectral signal attribution. Detailed Implementation

[0054] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0055] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0056] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0057] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0059] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0060] According to embodiments of the present invention, the present invention provides a method for analyzing disulfide bonds in peptides based on nuclear magnetic resonance (NMR) technology, the method comprising:

[0061] (1) Pre-process the polypeptide sample to obtain the NMR sample;

[0062] (2) Acquire 1D NMR samples of the NMR sample 1 Spectral analysis was performed using H-spectrum and 2D spectrum.

[0063] in,

[0064] The 2D spectrum includes 1 H- 1 H COSY, TOCSY, NOESY and 1 H- 13 C HSQC spectrum;

[0065] The spectral analysis includes: utilizing the... 1 H- 1 H COSY, TOCSY, and NOESY spectra were used to assign signals to the amino acids in the peptide samples; for the... 1 H- 13 C HSQC spectrum 13 Assignment based on C chemical shift, according to C β Chemical shift is used to determine the presence and number of disulfide bonds in a peptide sample; the pairing mode of the disulfide bonds is determined based on the NOE signal in the NOESY spectrum.

[0066] According to a specific embodiment of the present invention, a high-purity peptide sample (≥95%) is prepared and dissolved in an aqueous solution (containing ≥5% D₂O lock field). The final concentration of the peptide sample should be greater than 1 mM. The test solution for the sample should be selected to be consistent with physiological or drug administration conditions as much as possible. The solution conditions generally use buffer solutions, preferably phosphate-containing buffer systems. An appropriate amount of internal standard, such as DSS or TMSP, can be added to the test solution as a reference for spectral chemical shift.

[0067] According to a specific embodiment of the present invention, the NMR sample is added to an NMR tube, injected, and preparatory steps such as field locking, shimming, and tuning are performed. The 90° pulse width is measured to maximize net magnetization transfer, and a pulse sequence for suppressing the water peak is invoked. Appropriate experiments are selected for water peak suppression to obtain the best results, such as pre-saturation methods, water gate methods (3919), gradient excitation etching, and other pulse sequence methods.

[0068] According to a specific embodiment of the present invention, during testing, a high-field NMR spectrometer (≥600MHz) equipped with a liquid helium high-sensitivity probe should be selected. The sample testing temperature can be room temperature (298K), or low-temperature conditions can be selected according to the properties of the sample to reduce the tumbling time of the peptide in the molecular solution, which is beneficial to the acquisition of more NOE signals.

[0069] According to a specific embodiment of the present invention, the collection of polypeptide samples... 1 For H-spectroscopy, the number of scans should be selected based on the actual concentration of the sample, for example, 16-64 scans. After acquisition, observe the full spectrum to ensure sufficient water peak suppression, meaning the peptide sample signal should be relatively sharp and have a good signal-to-noise ratio. For peptides that have correctly folded into higher-order structures, the NH signal in the main chain region (e.g., 6.5-10 ppm) should be well dispersed and should be comparable to the expected amount of NH in the molecule. Meanwhile, peak linewidths provide information on chemical and conformational exchanges in the peptide molecule; generally, peak broadening indicates that the sample may have undergone oligomerization or even aggregation in solution. Furthermore, the pH of the sample solution also affects the amount of NH signal in the main chain and the peak linewidth; therefore, neutral to slightly acidic solution conditions (e.g., pH 5.0-6.0) should be selected whenever possible.

[0070] According to a specific embodiment of the present invention, a 2D spectrum of a peptide sample is acquired, and the sweep width of the 2D spectrum should cover the 1D spectrum, i.e. 1 The signal range observed in H-spectroscopy. 2D spectra are acquired to assign amino acid signals, thereby analyzing and locating disulfide bonds. Common experiments include... 1 H- 1 H COSY, TOCSY, NOESY and natural abundance 1 H- 13 CHSQC. The mixing time for acquiring TOCSY spectra is typically 80-120 msec, and for NOESY spectra, it is typically 200-500 msec. For peptides, if weak or sparse correlation peaks are observed in the NOESY spectrum, it may be due to rapid turbulence in the solution; in this case, ROESY spectra can be acquired instead. 1 H- 13 C HSQC spectra are mainly used to analyze information related to non-exchangeable protons. Therefore, they need to be measured in D2O solution to minimize the interference of water peak signals on the assignment of amino acid HA regions. Before testing, the sample can be fully lyophilized and recovered, and then reconstituted with D2O solvent.

[0071] According to a specific embodiment of the present invention, using the collected data... 1 Spectral analysis was performed using H-spectrum and 2D spectra. Homonuclear correlation spectra were also utilized.1 H- 1 H COSY and TOCSY can identify amino acid signals within residues. Combined with NOESY spectra, important information about the sequence linking of each amino acid can be determined. Then, based on the assigned... 1 H chemical shift, completing heteronuclear-related HSQC spectra 13 Assigning C chemical shifts is useful because the latter chemical shifts have a wider distribution range and can be used to identify overlapping or uncertain assignments. 1 The H signal is used for reconfirmation. Specifically, C β The chemical shift value is highly sensitive to the state of cysteine, i.e., whether it is in a reduced state (free -SH) or an oxidized state (disulfide bond -SS-). According to 1 H- 13 C HSQC spectrum for cysteine ​​C β Attribution information allows us to determine its current state. If C... β If the chemical shift value is less than 32.0 ppm, cysteine ​​exists in its reduced form; if C β If the chemical shift value is greater than 35.0 ppm, cysteine ​​exists in its oxidized form. If it falls within the range of 32.0-35.0 ppm, the oxidation / reduction state of cysteine ​​needs further determination. For example, if the polypeptide molecule contains only one cysteine, then that cysteine ​​is in its reduced state; if the polypeptide molecule contains multiple cysteines, only one C... β If the chemical shift value is between 32.0 and 35.0 ppm, the cysteine ​​can be determined to be in an oxidized state; if the polypeptide molecule contains multiple cysteine ​​residues and multiple C atoms... β If the chemical shift is in the intermediate range, the corresponding redox state still needs to be judged comprehensively based on the actual situation.

[0072] According to a specific embodiment of the present invention, spatially close proximity is determined by observing the NOE signal in the NOESY spectrum, based on dipolar coupling information. 1 H, 1 H. Specifically, the NOE signal between the β-methylene protons in the side chain portion of the two cysteine ​​residues is observed to determine whether a covalent disulfide bond has formed between them. The C of the two cysteine ​​residues... β The distance between atoms ranges from The mean is Therefore, the interproton distance of the β-methylene group in the pairing disulfide bond between residues will always be less than [the distance between the protons of the β-methylene group]. Ensure that a relatively obvious NOE-related signal can be observed in the spectrum.

[0073] For cysteine-rich peptide molecules, there may be severe chemical shift stacking, making it impossible to target H+. βAssigning values ​​based on displacement can lead to ambiguity in the identification of NOE correlation peaks. In such cases, multidimensional heteronuclear correlation experiments, such as 3D... 13 C-edited HSQC-NOESY reduces the complexity of spectral overlap, thus solving the problem. However, this method requires prior analysis of the sample molecules. 13 Carbon atoms can be used for stable isotopic labeling, which can be achieved through culture medium feeding. 13 Recombinant peptides can be prepared using C-glucose as the sole carbon source for spectral analysis. Alternatively, peptide solid-phase synthesis (SPPS) can be used to selectively introduce cysteine ​​residues. 13 Carbon atoms can be used for stable isotopic labeling, making 2D sampling simpler. 13 The C-edited NOESY experiment (i.e., 3DHSQC-NOESY spectral projection) is used to solve the problem of disulfide bond localization.

[0074] In addition, for certain toxic peptides rich in cysteine, cysteine ​​H may be present. β cysteine ​​H that is not covalently linked β NOE (non-oxygenated outlet) occurs because local structural congestion causes spatial proximity, leading to incorrect conclusions when locating disulfide bonds based on this "false" NOE signal. In such cases, scalar coupling can be used to determine the disulfide bond connection information. Considering... 33 S is not an active NMR nucleus, a problem that can be solved by obtaining selenocysteine ​​(through solid-phase synthesis or recombinant expression feeding). Replacing the disulfide bond (-SS-) with a diselenocysteine ​​bond (-Se-Se-) generally does not affect the peptide structure and function. Because... 77 Se has a very wide chemical shift distribution (~3000 ppm), so signal overlap is rare. By acquiring heteronuclear correlation spectra such as 2D... 1 H, 77 Se-HMQC, 1 H, 77 Se-HMBC spectrum, observe the correlation peaks of moderate intensity. 3 J Hβ-Seγ (~2Hz) can determine the disulfide bond linkage information in the original polypeptide. If performing [analysis] on cysteine... 77 Stable isotope labeling of se allows for the collection of homonuclear samples. 77 Se- 77 Se COSY spectrum, by extraction 1 J Seγ-Seγ (~35Hz) information can directly determine the disulfide bond bridging status within the original polypeptide molecule.

[0075] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0076] Example 1: Analysis of disulfide bond linkage information of the peptide drug linaclotide based on nuclear magnetic resonance technology

[0077] Linaclotide, marketed as Linzess (purchased from Ironwood Pharmaceuticals, USA), is an FDA-approved drug in August 2012 for clinical use as a locally acting guanylate cyclase-C (GC-C) agonist. Linaclotide activates the gastrointestinal surface receptor GG-C, leading to increased levels of cyclic guanosine monophosphate (cGMP). cGMP, as a second messenger, plays an important role in the regulation and secretion of intestinal fluid and can be used to treat constipation-predominant irritable bowel syndrome and chronic idiopathic constipation. This polypeptide drug consists of 14 amino acids, containing 6 cysteine ​​residues forming 3 pairs of intramolecular disulfide bonds. The structural formula is shown below. Figure 1 The experimental flowchart for analyzing disulfide bond information in peptide samples using nuclear magnetic resonance (NMR) technology is shown below. Figure 2 The specific steps are as follows:

[0078] 1. Pretreatment of peptide drugs

[0079] The sample was dissolved in an aqueous solution, and the pH was adjusted to 5.0 dropwise with 1M sodium hydroxide solution. D2O was then added for field locking (final concentration 10%), and the final concentration of the peptide drug was 14.5mM.

[0080] 2. Conduct nuclear magnetic resonance experiments

[0081] The entire NMR experiment was conducted at low temperature (280K) to reduce the tumbling motion of peptide molecules in solution and thus better obtain the NOE signal. A 600MHz spectrometer with a liquid helium high-sensitivity probe was used for the NMR experiment. The pretreated peptide drug was added to the NMR tube, and locking, shimming, and tuning preparation steps were performed. The p1 90° pulse width was calculated to maximize net magnetization transfer, and a pulse sequence for suppressing the water peak was selected. Specifically, the water gate method (3919 pulse sequence) was used in the water peak suppression experiment, and continuous optimization and adjustment were performed. 1 The frequency at the center of the H-spectrum window overlaps with the frequency of the water peak signal, thereby minimizing interference from the water peak signal.

[0082] 3. Collect 1D of polypeptide drugs 1 H spectrum

[0083] See results Figure 3 It can be seen that the sample drug signal has a good signal-to-noise ratio and is clear, and the peak line width is not abnormal. The NH signal in the main chain is well dispersed in the 6.5-10.5 ppm region and the signal quantity is close to that expected, indicating that the polypeptide molecule has folded into the correct higher-order structure.

[0084] 4. Acquire 2D spectra of peptide drugs

[0085] Setting the scan width can cover 1 All signals were detected in the H spectrum. The mixing time for the TOCSY spectrum was set to 90 msec, and the mixing time for the NOESY spectrum was set to 500 msec. The drug was then thoroughly freeze-dried for recovery, and the resulting lyophilized powder was reconstituted with D2O before being analyzed. 1 H spectrum and 1 H- 13 C HSQC spectrum, with indirect dimension width set to 75 ppm, can fully cover side chains. 13 C chemical shift range.

[0086] Figure 4-7 These are the peptide drugs. 1 H- 1 H COSY, TOCSY, NOESY 1 H- 13 C HSQC spectral signal attribution diagram, using 1 H- 1 H COSY and TOCSY spectra can assign signals to each amino acid spin system, and combined with NOESY spectra, signals related to sequence connections between amino acids can be identified, such as NH... i+1 -αH i NH i+1 -NH i NH i+1 -βH i Relevant signals. Utilizing 1 H- 13 C HSQC spectra can be used for direct connections 1 H 13 C signal is used for attribution;

[0087] 5. Analyze relevant spectra to obtain disulfide bond information.

[0088] Determining the number of disulfide bonds: by 13 By assigning the C signal, we can determine the C values ​​of C1, C5, C6, C10, and C13. β The chemical shifts, ranging from 34.9 to 40.3 ppm, indicate that these cysteines exist in disulfide bond form (oxidized state). The C2 group... βThe chemical shift (32.7 ppm) falls within the intermediate region of the oxidation / reduction state. Considering that five of the six cysteine ​​residues in the molecule are already in the oxidized state, the remaining C2 can be determined to be in the oxidized state rather than a free thiol group. Therefore, it can be concluded that this polypeptide drug contains three pairs of intramolecular disulfide bonds.

[0089] Location of disulfide bonds: Observing the NOE signal between the β-methylene protons of two cysteine ​​residues can determine whether the two cysteine ​​residues are covalently linked by a disulfide bond. This can be achieved through the NOESY spectrum of the polypeptide drug. Figure 6 Obvious correlation peaks were observed between C1-HB# / C6-HB and C2-HB / C10-HB#, and moderate correlation peaks were also observed between C1-HA / C6-HB, C1-HB# / C6-HA, C2-HA / C10-HB#, and C2-HB / C10-HA, suggesting that disulfide bonds are formed between C1 and C6, and between C2 and C10. For C5-C13, due to the overlap of their respective HB signals (C5-HB3 ~ 2.66 ppm, C13-HB# ~ 2.63 ppm), it was not easy to directly observe the NOE signal between the β-methylene protons of the two residues. However, the moderate intensity correlation peaks between C5-HA / C13-HB# and C5-HB / C13-HA indicate the presence of a disulfide bond between C5 and C13. Therefore, it can be determined that the polypeptide contains three pairs of intramolecular disulfide bonds, formed between C1 and C6, C2 and C10, and C5 and C13, respectively.

[0090] The above analytical results are consistent with the reported disulfide bond pairing mode in the peptide drug linaclotide. Using existing techniques for de novo analysis and solution structure calculation, the analysis of the peptide drug linaclotide typically takes several months, while the analytical method provided by this invention only requires 1-2 weeks, and is non-destructive to the sample structure, achieving efficient acquisition of disulfide bond information in the peptide.

[0091] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0092] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for analyzing disulfide bonds in a polypeptide based on nuclear magnetic resonance technology, characterized by, The method comprises: (1) pretreating a polypeptide sample to obtain a nuclear magnetic sample; (2) collecting 1D 1 NMR spectra and 2D spectra of the NMR sample, and performing spectral analysis; wherein, The 2D spectra include 1 H- 1 H COSY, TOCSY, NOESY and 1 H- 13 C HSQC spectra; The spectrum analysis comprises: utilizing the 1 H- 1 H COSY, TOCSY, NOESY spectrum to attribute the amino acid signals of the polypeptide sample; utilizing the 1 H- 13 C HSQC spectrum to attribute the 13 C chemical shifts, judging the existence and quantity of disulfide bond in the polypeptide sample according to the C β chemical shifts; determining the pairing mode of the disulfide bond according to the NOE signals in the NOESY spectrum; The C β Chemical shift determines the presence and number of disulfide bonds in polypeptide samples, including: if C β Chemical shift value is less than 32.0 ppm, cysteine exists in reduced form, indicating that no disulfide bond is formed; if C β Chemical shift value is greater than 35.0 ppm, cysteine exists in oxidized form, indicating that a disulfide bond is formed; if in the intermediate range of 32.0-35.0 ppm, cysteine exists in an intermediate state of oxidation / reduction; determining the pairing mode of the disulfide bond according to the NOE signal between the β-methylene protons of the two cysteine residues comprises determining the pairing mode of the disulfide bond according to the NOE signal between the β-methylene protons of the two cysteine residues.

2. The method of claim 1, wherein, The nuclear magnetic sample comprises a field locking reagent; The pH value of the nuclear magnetic sample is 5.0-6.0; The nuclear magnetic sample comprises a phosphate buffer solution; The field locking reagent comprises deuterated reagent heavy water; The nuclear magnetic sample comprises an internal standard; The internal standard comprises 4,4-dimethyl-4-silapentane-1-sulfonic acid or 3-trimethylsilylpropionic acid.

3. The method of claim 2, wherein, The purity of the polypeptide sample is more than 95%; The final concentration of the polypeptide sample is greater than 1mM; The concentration of the field locking reagent is not less than 5%.

4. The method of claim 1, wherein, Step (2) comprises: acquiring 1D 1 H spectra and 2D spectra of the NMR sample by NMR test. The nuclear magnetic resonance test comprises: adding the nuclear magnetic sample into a nuclear magnetic tube, performing a preparation step, calling a pulse sequence for suppressing a water peak, The preparation step comprises a field locking, shimming and tuning step; The pulse sequence comprises at least one selected from a pre-saturation pulse sequence, a water gate suppression pulse sequence and a gradient excitation etching pulse sequence; The nuclear magnetic resonance test comprises: testing the 90° pulse width after performing the preparation step; The nuclear magnetic resonance test is performed at 280K-298K.

5. The method of claim 1, wherein, The scanning spectral width in the acquisition of the 2D spectrum in step (2) is set to be able to cover the 1D 1 The frequency of all signals of the H spectrum The scanning spectrum width is 14-18ppm; The mixing time for collecting the TOCSY spectrum is set to 80-120msec; The mixing time for collecting the NOESY spectrum is set to 200-500msec.

6. The method of claim 1, wherein, The polypeptide sample is freeze-dried and reconstituted with D2O before the acquisition of the HSQC spectrum. 1 H- 13 The polypeptide sample is freeze-dried and reconstituted with D2O before the acquisition of the HSQC spectrum. The 1 H- 13 The indirect dimension spectral width was set to 5-80 ppm for HSQC spectral data acquisition.

7. The method of claim 1, wherein, Utilizing the 1 H- 1 H COSY, TOCSY, NOESY spectra for the assignment of amino acid signals of the polypeptide sample include: 1 H- 1 H COSY, TOCSY spectra for the assignment of intra-amino acid spin systems, combined with the NOESY spectrum for the identification of the sequence of connections between the amino acids; The 1 H- 13 C chemical shifts in the 13 C chemical shifts were assigned by 1 H chemical shifts. The 1 H- 13 CHSQC spectrum 13 C chemical shifts were assigned by 8. The method of claim 1, wherein, Step (2) comprises acquiring at least one of a polypeptide sample ROESY, 2D 13 C-edited NOESY, 3D 13 C-edited HSQC-NOESY spectrum.

9. The method of claim 1, wherein, The method comprises: substituting the disulfide bond in the structure of the polypeptide sample with a diselenide bond, collecting a 2D spectrum after preparing the sample, and determining the connection information of the disulfide bond in the original structure; The 2D spectra include at least one selected from the group consisting of 2D 1 H, 77 Se-HMQC, 1 H, 77 Se-HMBC, 77 Se- 77 Se COSY spectra. said determining the connection information of disulfide bonds in the original structure comprises: analyzing the 2D 1 H, 77 Se-HMQC and / or 1 H, 77 correlation peaks in Se-HMBC spectrum 3 J Hβ-Seγ information determines the connection information of disulfide bonds in the original structure; and / or extracting the 77 Se- 77 Se COSY spectrum 1 J Seγ -Seγ Information determines the connection information of disulfide bond.

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