CN111948315A

CN111948315A - Sequence analysis method and identification method of insulin or insulin analogue

Info

Publication number: CN111948315A
Application number: CN202010812328.9A
Authority: CN
Inventors: 王建伟; 孙跃权; 周奕含
Original assignee: Changchun Institute of Applied Chemistry of CAS
Current assignee: Changchun Institute of Applied Chemistry of CAS
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2020-11-17

Abstract

The invention relates to the field of drug analysis, in particular to a sequence analysis method and an identification method of insulin or insulin analogues. The invention adopts a proper disulfide bond reduction method to reduce the double-chain structure of the insulin into two independent peptide chains, obtains the most abundant mass spectrum fragment information by liquid chromatography separation and high-resolution tandem mass spectrometry analysis and adopting proper secondary mass spectrometry collision energy, matches with a theoretical sequence to obtain 100 percent of sequence coverage rate and provides powerful technical support for the quality control and the fake-fighting work of the insulin.

Description

Sequence analysis method and identification method of insulin or insulin analogue

Technical Field

The invention relates to the field of drug analysis, in particular to a sequence analysis method and an identification method of insulin or insulin analogues.

Background

In the last decade, biotechnological drugs have shown significant advantages in the treatment of several diseases, among which a large number of star drugs have emerged. Unlike traditional small molecule chemical drugs, biotechnological drugs generally have the characteristics of large molecular weight, complex structure, poor stability, large batch-to-batch variation and the like, so that quality control is more complicated than that of chemical drugs, and a series of analysis techniques covering different analysis fields are required to effectively characterize the physicochemical properties of the drugs. The clear requirements of the technical guide principles of research and development and evaluation of biotechnological drugs (trial), and the amino acid sequences of candidate drugs for protein and polypeptide drugs should be the same as those of reference drugs in principle; in pharmaceutical research and evaluation, it is required to perform alignment test studies for amino acid sequence determination, and the alignment test studies can be directly performed with a known reference drug sequence.

Conventional protein sequence analysis methods include N-terminal analysis methods such as dinitrofluorobenzene method, dansyl chloride method, Edman degradation method and aminopeptidase method; c-terminal analysis methods such as hydrazinolysis, reduction and carboxypeptidase methods. These methods are complicated in operation procedure and large in workload. With the rapid development of mass spectrometry, high-resolution mass spectrometry with the advantages of high mass precision, wide dynamic range and the like makes the sequence analysis of biotechnological drugs more convenient. The principle of mass spectrometry for protein sequence analysis is based on the fragmentation of peptide chains through mass spectrometer collisions to generate regular fragments (fig. 1), which are then assembled into the amino acid sequence of the peptide fragments based on the high resolution mass spectral information of these fragments.

Insulin and the like are one of the most effective diabetes treatment drugs, and the usage amount of insulin preparations in diabetes treatment drugs is the first. Insulin and its analogs are classified according to their duration of action into rapid-acting insulin analogs (including recombinant human insulin, insulin lispro, insulin aspart and insulin glulisine) and long-acting insulin analogs (including insulin glargine, insulin detemir and insulin degluin). The insulin analogs are prepared by partially modifying the amino acid sequence and structure of human insulin by using genetic engineering technology, and are similar to human insulin in sequence. Therefore, in the conventional method for identifying insulin by analyzing a peptide map obtained by the product of the polypeptide enzymolysis of insulin by the restriction endonuclease by using high performance liquid chromatography, for the identification of insulin analogues with almost the same sequence, the retention time of enzyme digestion products is very close, so that certain difficulty exists in the actual identification operation. In addition, the enzyme digestion reaction has the risk of introducing artificial modification, which leads to interference of analysis results.

At present, the method for identifying insulin by using mass spectrometry is to firstly carry out enzymolysis on complete insulin into polypeptide by using restriction endoprotease, then obtain a peptide map by using liquid chromatography or liquid chromatography-mass spectrometry combined analysis, and compare the peptide map with a reference substance peptide map.

The current method for identifying insulin comprises the following steps: 1. carrying out enzymolysis on insulin by Glu-C endoprotease to obtain polypeptide, analyzing characteristic enzymolysis products by using liquid chromatography, and recording a chromatogram map, wherein the peptide map of the test solution is consistent with that of the reference solution. 2. The Glu-C enzyme and the Lys-C enzyme are adopted for overnight reaction, various insulins are hydrolyzed specifically, and a peptide diagram containing insulin characteristic fragments is established by utilizing a liquid chromatography-mass spectrometry combined technology and is used for determining various insulins.

1. The prior art requires the use of expensive sequencing grade restriction endonucleases.

2. In the prior art, the enzymolysis reaction time is long, and the identification period is long compared with that of a reference substance.

3. In the prior art, the enzymolysis reaction has the risk of introducing artificial modification, so that an interference peak appears to influence result judgment.

Disclosure of Invention

Accordingly, the present invention provides methods for sequence analysis and identification of insulin or insulin analogs. The invention adopts a proper disulfide bond reduction method to reduce the double-chain structure of the insulin into two independent peptide chains, obtains the most abundant mass spectrum fragment information by liquid chromatography separation and high-resolution tandem mass spectrometry analysis and adopting proper secondary mass spectrometry collision energy, matches with a theoretical sequence to obtain 100 percent of sequence coverage rate and provides powerful technical support for the quality control and the fake-fighting work of the insulin.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a sequence analysis method of insulin or an analogue thereof, which comprises the steps of mixing the insulin or the analogue thereof to be detected with acid, reducing the mixture into a single chain by a disulfide bond reduction reagent and a protein denaturant, and carrying out sequence analysis by ultra-high performance liquid chromatography-high resolution tandem mass spectrometry;

the disulfide bond reducing agents include, but are not limited to: one or the combination of the two of tri (2-carboxyethyl) phosphine or dithiothreitol;

the protein denaturant is not limited to: one or the combination of two of guanidine hydrochloride or urea.

In some embodiments of the invention, the acid is hydrochloric acid, the concentration of hydrochloric acid is 10mmol/L, and the insulin or analog thereof to be tested is diluted to 1mg/mL with hydrochloric acid.

In some embodiments of the invention, the guanidine hydrochloride concentration is 6mol/L and the urea concentration is 8 mol/L.

In some embodiments of the invention, the tris (2-carboxyethyl) phosphine is present at a concentration of 50mmol/L and the dithiothreitol is present at a concentration of 50 mmol/L.

In some embodiments of the invention, the temperature of the reduction is 40 to 45 ℃ and the time is 40 min.

In some embodiments of the invention, the fragmentation pattern used in the hplc-hplc mass spectrometry includes, but is not limited to, HCD and/or CID.

In some embodiments of the invention, the analysis conditions of the ultra performance liquid chromatography-high resolution tandem mass spectrometry are as follows:

liquid chromatography conditions: a C18 chromatography column; the column temperature is 45 ℃; the mobile phase A is acetonitrile solution containing 0.1 percent of formic acid, and the mobile phase B is aqueous solution containing 0.1 percent of formic acid; linear gradient elution: 0-3 min, 3% A; 3-4 min, 3% -10% A; 4-9 min, 10% -30% A; 9-15 min, 30% A; 15-17 min, 30% -50% A; 17.1-22 min, 95% A; 22.1-25 min, 3% A; flow rate: 0.3 mL/min; sample introduction amount: 2 mu L of the solution;

mass spectrum conditions: an electrospray ion source; a positive ion detection mode; the scanning range m/z is 50-2000; the primary mass spectral resolution is 60000FWHM (m/z400), and the secondary mass spectral resolution is 15000FWHM (m/z 400); secondary mass fragmentation mode: collision-induced Dissociation (CID), high energy Collision-induced Dissociation (HCD); the Collision Energy (NCE) is: 10 to 45.

In some embodiments of the invention, the insulin or analog thereof includes, but is not limited to: recombinant human insulin raw material, biosynthetic human insulin injection, protamine biosynthetic human insulin injection (premixed 30R), protamine biosynthetic human insulin injection (premixed 50R), insulin aspart raw material, insulin aspart injection, insulin aspart 30 injection, insulin aspart 50 injection, insulin lispro raw material, insulin lispro injection, insulin glulisine raw material, insulin glulisine injection, insulin glargine raw material, insulin glargine injection, insulin detemir raw material, insulin detemir injection, insulin degum raw material, insulin degum injection.

In some embodiments of the invention, insulin aspart is reduced to insulin aspart A and insulin aspart B chains; the mass spectrum fragmentation mode of the insulin aspart A chain is HCD, and the collision energy is 20; the fragmentation mode of the mass spectrum of the B chain of insulin aspart is HCD, and the collision energy is 25.

In some embodiments of the invention, deglutaric insulin is reduced to a deglutaric insulin a chain and a deglutaric insulin B chain; the mass spectrum fragmentation mode of the insulin deglutamide A chain is HCD, and the collision energy is 20; the mass spectrometric fragmentation pattern of degluin B chain is HCD with a collision energy of 25.

The identification method of insulin or its analogue comprises collecting fragment ions of the substance to be tested according to the sequence analysis method, analyzing protein sequence, and matching with the theoretical sequence of insulin or its analogue; if the matching is complete, the object to be tested is the insulin or the analogue thereof; otherwise, no insulin or analog thereof is determined.

The invention provides a rapid insulin identification method based on sequence analysis, which is used for identifying insulin and determining whether an insulin sequence is accurate. The invention adopts a proper disulfide bond reduction method to reduce the double-chain structure of the insulin into two independent peptide chains, obtains the most abundant mass spectrum fragment information by liquid chromatography separation and high-resolution tandem mass spectrometry analysis and adopting proper secondary mass spectrometry collision energy, matches with a theoretical sequence to obtain 100 percent of sequence coverage rate and provides powerful technical support for the quality control and the fake-fighting work of the insulin.

The beneficial effects of the invention include but are not limited to:

the invention uses chemical reagent, and has low cost.

The invention has the advantages of reduction reaction for 40 minutes, no need of comparison with a reference substance and high identification speed.

The invention has rapid reduction reaction, does not relate to sequence change and has more accurate result.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the fragmentation pattern and fragment ion structure of peptide chains in mass spectrometry;

FIG. 2 shows a chromatogram of deglutaric insulin reduction product;

FIG. 3 shows the effect of time and temperature on deglutaric insulin reduction reaction;

FIG. 4 shows a total ion flow diagram after insulin aspart reduction;

FIG. 5 shows a first order high resolution mass spectrum of Peak 1(B chain) after reduction of insulin aspart;

FIG. 6 shows a first order high resolution mass spectrum of Peak 2(A chain) after reduction of insulin aspart;

FIG. 7 shows the effect of collision energy on insulin aspart A chain double or triple charge peak fragmentation in CID (A) and HCD (B) modes;

FIG. 8 shows the HCD secondary high resolution mass spectrum and fragmentation pattern of the insulin aspart A chain (m/z 1192.51);

FIG. 9 shows the effect of collision energy on fragmentation of three, four or five charge peaks of the B chain of insulin aspart in CID (A) and HCD (B) modes;

FIG. 10 shows the HCD secondary high resolution mass spectrum and fragmentation pattern of insulin aspart B chain (m/z 862.92);

FIG. 11 shows a total ion flow graph after deglutaric insulin reduction;

FIG. 12 shows a first order high resolution mass spectrum of Peak 1 after reduction of deglutaric insulin;

FIG. 13 shows a first order high resolution mass spectrum of Peak 2 after reduction of deglutaric insulin;

FIG. 14 shows the effect of collision energy on dual or triple charge peak fragmentation of insulin degluded A chains in CID (A) and HCD (B) modes;

FIG. 15 shows the HCD secondary high resolution mass spectrum and fragmentation pattern of insulin deglutamide A chain (m/z 1192.51);

FIG. 16 shows the effect of collision energy on tri-, tetra-or pentacharge peak fragmentation of insulin degluded B-chain in CID (A) and HCD (B) modes;

FIG. 17 shows the HCD second order high resolution mass spectrum and fragmentation pattern of insulin deglutamide B chain (m/z 862.92).

Detailed Description

The invention discloses a sequence analysis method and an identification method of insulin or insulin analogues, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Insulin analogues: the amino acid sequence and structure of human insulin are modified locally by means of gene engineering technology, and the product has no clinical effect difference in quality, safety and effectiveness. Insulin analogs are currently available: fast-acting insulin analogs (including insulin lispro, insulin aspart and insulin glulisine) and long-acting insulin analogs (including insulin glargine, insulin detemir and insulin degluin).

The invention aims to provide a method for quickly identifying insulin, which is used for identifying the insulin and determining whether the insulin sequence is accurate or not. The invention adopts a proper disulfide bond reduction method to reduce the double-chain structure of the insulin into two independent peptide chains, obtains the most abundant mass spectrum fragment information by liquid chromatography separation and high-resolution tandem mass spectrometry analysis and adopting proper secondary mass spectrometry collision energy, matches with a theoretical sequence to obtain 100 percent of sequence coverage rate and provides powerful technical support for the quality control and the fake-fighting work of the insulin.

The invention provides a rapid insulin identification method based on sequence analysis, which comprises the following steps:

a) diluting various insulin analogs to 1mg/mL by using 10mmol/L hydrochloric acid solution;

b) adding 100 mu L of the solution into a centrifugal tube, adding guanidine hydrochloride and tri (2-carboxyethyl) phosphine, and heating and incubating for 40min to obtain a reduction product;

in the present invention, the preferred concentration of guanidine hydrochloride is 6mol/L, the preferred concentration of tris (2-carboxyethyl) phosphine is 50mmol/L, the preferred heating temperature is 40 ℃, and the preferred incubation time is 40 min. The existence of disulfide bonds in proteins can affect the fragmentation of peptide chains in secondary mass spectra of the proteins, and abundant fragment ions cannot be obtained for sequence analysis, so that the disulfide bonds need to be reduced and opened before mass spectrum detection. Currently used disulfide bond reducing agents include Dithiothreitol (DTT) and tris (2-carboxyethyl) phosphine (TCEP). During the reduction reaction, if excessive reduction (such as high temperature or long time reaction) occurs, undesirable substances are generated, so that the selection of proper reaction temperature and time is a precondition for ensuring the accuracy of sequence analysis. In addition to the disulfide bond between the two chains, the disulfide bond in one chain is also present in the A chain in the structure of insulin, so that the reduction method of combining TCEP with protein denaturant guanidine hydrochloride is adopted in the experiment, and the reduction temperature and the reduction time are further optimized. Fig. 2 shows a chromatogram of the reduction product of degu insulin by ultra performance liquid chromatography-high resolution tandem mass spectrometry, and specific chromatogram peak assignments are shown in table 1. Peak 1 is the A chain with intra-chain disulfide bonds, peak 2 is the A chain after reduction, peaks 4, 5, 6 are the reduction products with two disulfide bonds between the A chain and the B chain, peak 7 is the B chain after reduction, and peaks 8, 9 are the reduction products with two one-sulfur bonds between the A chain and the B chain. As shown in figure 3, 50mmol/L TCEP reacts with 6mol/L guanidine hydrochloride at 45 ℃, the content of deglutated insulin is rapidly reduced, the content of A chain and B chain after reduction is rapidly increased, and the deglutated insulin can be fully reduced into two peptide chains within 40 min. Since the disulfide bond structures of insulin are all the same, the above disulfide bond reduction conditions are applicable to all reduction reactions of insulin.

Table 1 first order mass spectral information of degu insulin reduction products

Note: denotes a disulfide bond

c) The reduction product is analyzed by ultra performance liquid chromatography-high resolution tandem mass spectrometry, and the specific analysis conditions are as follows:

liquid chromatography conditions: c₁₈A chromatographic column; the column temperature is 45 ℃; the mobile phase A is acetonitrile solution containing 0.1 percent of formic acid, and the mobile phase B is aqueous solution containing 0.1 percent of formic acid; linear gradient elution: 0-3 min, 3% A; 3-4 min, 3% -10% A; 4-9 min, 10% -30% A; 9-15 min, 30% A; 15-17 min, 30% -50% A; 17.1-22 min, 95% A; 22.1-25 min, 3% A; flow rate: 0.3 mL/min; sample introduction amount: 2 μ L.

Mass spectrum conditions: an electrospray ion source; a positive ion detection mode; the scanning range m/z is 50-2000; the primary mass spectral resolution is 60000FWHM (m/z400), and the secondary mass spectral resolution is 15000FWHM (m/z 400); secondary mass fragmentation mode: collision-induced Dissociation (CID), high energy Collision-induced Dissociation (HCD); collision Energy (Normalized Collision Energy, NCE) optimization Range: 10 to 45.

d) The insulin is reduced and then injected into ultra high performance liquid chromatography-high resolution tandem mass spectrometry for sequence analysis. In order to obtain more insulin fragment ions, the present invention compares two commonly used tandem mass spectrometry fragmentation modes: collision Induced Dissociation (CID) and high energy collision induced cleavage (HCD). CID is a fragmentation mode commonly used for peptide fragment sequence analysis in biological mass spectrometry, and b-type fragment ions and y-type fragment ions are mainly generated after activation collision of peptide fragments so as to analyze sequences. CID mainly fragments at the backbone of the peptide chain and has an "1/3 effect" in the ion trap, where fragment ions less than 28% of the parent ion mass to charge ratio are not detectable. HCD is a highly efficient mode of peptide chain fragmentation and is widely used in orbitrap mass spectrometers. HCD is more susceptible to secondary fragmentation than CID and therefore more fragment ions are likely to be generated, enabling analysis of side chain modifications of peptide chains. In addition, the ion detection device utilizes the orbit trap to detect ions, overcomes the '1/3 effect' of CID in the ion trap, and has wider scanning range, thereby being capable of detecting the ions in a low mass-to-charge ratio region. Insulin analogs, insulin detemir and insulin degluder, contain side chain modifications, so the present invention employs HCD fragmentation and optimizes collision energy so that it can detect all amide bond breaks to 100% sequence coverage.

e) According to the invention, TCEP is combined with guanidine hydrochloride to reduce insulin double chains, and HCD mass spectrum fragmentation mode is combined to obtain abundant fragment ions for sequence analysis of non-protein modified parts of protein main chains and side chains. The method is accurate and convenient, powerful technical support is provided for quality control and fake making work of the insulin, and the established method can be used for sequence analysis of the insulin.

The raw materials and reagents used in the method for sequence analysis and the method for identifying insulin or an analog thereof according to the present invention are commercially available.

The insulin source is commercially available, the hydrochloric acid, TCEP and guanidine hydrochloride sources are commercially available, and the experimental instrument is a UPLC-Orbitrap valves Pro LC MS (liquid chromatography-Mass spectrometer) and is provided with an ESI ion source.

The invention is further illustrated by the following examples:

example 1 sequence analysis of insulin aspart

Diluting the insulin aspart injection with 10mmol/L hydrochloric acid solution to 1mg/mL, adding 100 μ L into a centrifugal tube, adding guanidine hydrochloride and 50mmol/L TCEP with final concentrations of 6mol/L respectively, incubating at 45 deg.C for 40min, reducing disulfide bond of insulin aspart to open, and separating two peptide chains. The reduction product is analyzed by ultra performance liquid chromatography-high resolution tandem mass spectrometry, and the sample volume is 2 mu L. The instrumental analysis conditions were as described above. The preferable post-mass spectrum fragmentation mode of the insulin aspart A chain is HCD, and the preferable post-collision energy is 20; the preferred post-mass fragmentation mode of B chain insulin aspart is HCD, and the preferred post-collision energy is 25.

The detection result of the liquid chromatography-mass spectrometry is shown in fig. 4, and fig. 4 is a total ion flow graph of the insulin aspart after reduction provided in example 1, wherein the peak 1 peak time is 9.26min, and the peak 2 peak time is 10.20 min.

The primary mass spectrum of peak 1 has mainly three, four and five charge peaks (as shown in fig. 5), and the primary mass spectrum of peak 2 has mainly two and three charge peaks (as shown in fig. 6). The monoisotopic molecular weights are obtained by deconvolution calculation of the first-order mass spectrum peak data of peak 1 and peak 2, and by combining the theoretical molecular weights of the A chain and the B chain of insulin aspart (see table 2 for details), the following results are obtained: peak 1 is insulin aspart B chain, and Peak 2 is insulin aspart A chain.

TABLE 2 Primary high resolution Mass Spectrometry information of insulin aspart reduction products

The insulin aspart A chain is composed of 21 amino acids and contains 20 amido bonds, and a primary mass spectrum of the insulin aspart A chain mainly has double charge peaks and triple charge peaks. For the mass spectrum peak of the primary mass spectrum, CID and HCD collision energy are optimized to detect as many amide bond breaks as possible, as shown in fig. 7, in the CID mode, the ion fragments of the a chain are abundant, and the detected amide bond break number increases with the increase of NCE, and can reach 18 at most. In HCD mode, fragment ions of the a chain are more abundant. Where the collision energy of the HCD was 20, fragmentation of 20 amide bonds by a double charge peak (m/z 1192.51) was detected with 100% sequence coverage (FIG. 8). The sequence is consistent with the theoretical sequence of the insulin aspart A chain, and the peptide chain is proved to be the insulin aspart A chain. The mass spectral peak assignments for the specific HCD cleavage fragments are shown in table 3.

TABLE 3 HCD Secondary Mass Spectroscopy fragmentation Peak List of insulin aspart A chain

The B chain of insulin aspart consists of 30 amino acids, contains 29 amido bonds, and has a primary mass spectrum mainly comprising three charge peaks, four charge peaks and five charge peaks. CID and HCD collision energies were optimized for the mass spectral peaks of the primary mass spectrum to detect as many amide bond breaks as possible. As shown in FIG. 9, the maximum number of amide bond breaks detected in the CID mode can reach 22. In the HCD mode, fragment ions of the B chain are more abundant, and at the NCE of 30, the HCD mode can detect the fragmentation of 29 amide bonds generated by a four-charge peak (m/z 862.92), the sequence coverage rate is 100 percent (figure 10), and the sequence is consistent with the theoretical sequence of the B chain of insulin aspart, so that the peptide chain is proved to be the B chain of the insulin aspart. The mass spectral peak assignments for the specific HCD cleavage fragments are shown in table 4.

TABLE 4 HCD Secondary Mass Spectroscopy fragmentation Peak List of insulin aspart B chain

After the insulin aspart is reduced by TCEP combined with guanidine hydrochloride, the detection is carried out by using ultra-high performance liquid chromatography-high resolution tandem mass spectrometry to obtain abundant fragment ions such as b-type and y-type ions for analyzing the protein sequence of the insulin aspart, the obtained result is completely matched with the theoretical sequence of the insulin aspart, and whether the sequence composition is correct or not is accurately identified.

Example 2 sequence analysis of deglutaric insulin

Diluting deglutated insulin injection with 10mmol/L hydrochloric acid solution to 1mg/mL, adding 100 μ L into a centrifuge tube, adding guanidine hydrochloride and TCEP with final concentration of 6mol/L and 50mmol/L respectively, incubating at 45 deg.C for 40min, reducing disulfide bond of deglutated insulin to open, and separating two peptide chains. The reduction product is analyzed by ultra performance liquid chromatography-high resolution tandem mass spectrometry, and the sample volume is 2 mu L. The instrumental analysis conditions were as described above. The preferred post-mass spectrum fragmentation mode of the degummed insulin A chain is HCD, and the preferred post-collision energy is 20; degluin B chain is preferably fragmented by HCD in a post-mass spectrometric manner, preferably with a post-collision energy of 25.

Fig. 11 shows the detection result of the liquid chromatography-mass spectrometry, and fig. 5 is a total ion flow graph of the deglutaric insulin reduced provided in example 2, in which the peak 1 out time is 10.21min, and the peak 2 out time is 16.65 min.

The primary mass spectrum of peak 1 has mainly double charge peaks and triple charge peaks (as shown in fig. 12), and the primary mass spectrum of peak 2 has mainly triple charge peaks, quadruple charge peaks and quintuplet charge peaks (as shown in fig. 13). The monoisotopic molecular weights were calculated by deconvolution of the first-order mass spectrum peak data of peak 1 and peak 2, and by combining the theoretical molecular weights of deglu insulin a chain and B chain (see table 5 for details), it can be seen that: peak 1 is degluin A chain and peak 2 is degluin B chain.

TABLE 5 first order high resolution Mass Spectrometry information of insulin degu reduction products

The deglutated insulin A chain consists of 21 amino acids and contains 20 amido bonds, and a primary mass spectrum of the deglutated insulin A chain mainly has double charge peaks and triple charge peaks. HCD collision energy was optimized for the mass spectral peaks of the primary mass spectrum to detect as many amide bond breaks as possible. As shown in FIG. 14, in the CID mode, the ion fragments of the A chain are abundant, and the number of detected amide bond breaks increases with the increase of NCE, and can reach 18. In HCD mode, fragment ions of the a chain are more abundant. Where the collision energy of the HCD was 20, fragmentation of 20 amide bonds by a double charge peak (m/z 1192.51) was detected with 100% sequence coverage (FIG. 15). The measured sequence is consistent with the theoretical sequence of the insulin A chain of deglutition, and the peptide chain is proved to be the insulin A chain of deglutition. The mass spectral peak assignments for the specific HCD cleavage fragments are shown in table 6.

TABLE 6 HCD secondary Mass Spectroscopy fragmentation Peak List of deglutaric insulin A chain

The insulin deglutamide B chain consists of 30 amino acids, contains 29 amido bonds, and mainly has a three-charge peak, a four-charge peak and a five-charge peak in a primary mass spectrum. CID and HCD collision energies were optimized for the mass spectral peaks of the primary mass spectrum to detect as many amide bond breaks as possible. As shown in fig. 16, in the CID mode, the number of amide bond breaks detected can reach 19 at the maximum, and only fragmentation can occur in the main chain. In the HCD mode, fragment ions are more abundant, and not only can the amide bond breakage of the main chain be detected, but also the amide bond breakage of non-protein sequence modification on the amino group of the side chain at the C terminal can be detected. At NCE of 25, all 30 amide bond breaks resulting from the five charge peak (m/z 746.18) were detected, and sequence coverage reached 100% (FIG. 17). Where the collision energy of the HCD was 25, fragmentation of 29 amide bonds by a four charge peak (m/z 862.92) was detected with 100% sequence coverage (fig. 17). The measured sequence is consistent with the theoretical sequence of the insulin B chain of deglutition, and the peptide chain is proved to be the insulin B chain of deglutition. The mass spectral peak assignments for the specific HCD cleavage fragments are shown in table 7.

Table 7 HCD secondary mass spectrum fragmentation peak list of degluin B chain

After TCEP is combined with guanidine hydrochloride for reduction, the deglutated insulin is detected by using ultra-high performance liquid chromatography-high resolution tandem mass spectrometry to obtain abundant b, y type and other fragment ions for analyzing a protein main chain sequence and a side chain non-protein modified sequence of the deglutated insulin, the obtained result is completely matched with a theoretical sequence of the deglutated insulin, and whether the sequence composition is accurate or not is accurately identified.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The sequence analysis method of the insulin or the analogues thereof is characterized in that the insulin or the analogues thereof to be detected is mixed with acid, reduced into a single chain by a disulfide bond reduction reagent and a protein denaturant, and subjected to sequence analysis by ultra-high performance liquid chromatography-high resolution tandem mass spectrometry;

2. The sequence analysis method according to claim 1, wherein the acid is hydrochloric acid having a concentration of 10mmol/L, and the insulin or the analog thereof to be tested is diluted to 1mg/mL with hydrochloric acid.

3. The sequence analysis method according to claim 1 or 2, wherein the concentration of guanidine hydrochloride is 6mol/L and the concentration of urea is 8 mol/L.

4. The sequence analysis method of any one of claims 1 to 3, wherein the concentration of tris (2-carboxyethyl) phosphine is 50mmol/L and the concentration of dithiothreitol is 50 mmol/L.

5. The sequence analysis method according to any one of claims 1 to 4, wherein the temperature of the reduction is 40 to 45 ℃ and the time is 40 min.

6. The sequence analysis method of any one of claims 1 to 5, wherein the fragmentation pattern of mass spectrometry used in the HPLC-HSS comprises but is not limited to HCD and/or CID.

7. The sequence analysis method of any one of claims 1 to 6, wherein the conditions for the HPLC-HSS analysis are as follows:

8. The sequence analysis method of any one of claims 1 to 7, wherein the insulin or an analogue thereof includes but is not limited to: recombinant human insulin raw material, biosynthetic human insulin injection, protamine biosynthetic human insulin injection (premixed 30R), protamine biosynthetic human insulin injection (premixed 50R), insulin aspart raw material, insulin aspart injection, insulin aspart 30 injection, insulin aspart 50 injection, insulin lispro raw material, insulin lispro injection, insulin glulisine raw material, insulin glulisine injection, insulin glargine raw material, insulin glargine injection, insulin detemir raw material, insulin detemir injection, insulin degum raw material, insulin degum injection.

9. The sequence analysis method according to claim 8, wherein insulin aspart is reduced to an insulin aspart A chain and an insulin aspart B chain; the mass spectrum fragmentation mode of the insulin aspart A chain is HCD, and the collision energy is 20; the mass spectrum fragmentation mode of the B chain of insulin aspart is HCD, and the collision energy is 25;

deglutated insulin is reduced to a deglutated insulin a chain and a deglutated insulin B chain; the mass spectrum fragmentation mode of the insulin deglutamide A chain is HCD, and the collision energy is 20; the mass spectrometric fragmentation pattern of degluin B chain is HCD with a collision energy of 25.

10. The method for identifying insulin or an analogue thereof, characterized in that fragment ions are obtained from an analyte according to the sequence analysis method of any one of claims 1 to 9, and are matched with the theoretical sequence of the insulin or the analogue thereof through protein sequence analysis; if the matching is complete, the object to be detected is the insulin or the analogue thereof; otherwise, the test substance is not insulin or its analog.