CN110988244A

CN110988244A - Nexiridin peptide map analysis method and application thereof

Info

Publication number: CN110988244A
Application number: CN201911338579.1A
Authority: CN
Inventors: 李帅; 苏春所; 刘美; 卓芳芳
Original assignee: SHANGHAI JINGFENG PHARMACEUTICAL CO Ltd
Current assignee: SHANGHAI JINGFENG PHARMACEUTICAL CO Ltd
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2020-04-10
Anticipated expiration: 2039-12-23
Also published as: CN110988244B

Abstract

The invention provides a peptide map analysis method of nesiritide and application thereof. The method comprises the following steps: reducing and alkylating nesiritide, adding endopeptidase for enzymolysis, detecting by using LC-UV-MS, and attributing polypeptide fragments and/or amino acid residues formed after the nesiritide is subjected to enzymolysis according to collected peptide diagram data. According to the method for analyzing the peptide graph, provided by the invention, the nesiritide is subjected to reduction and alkylation, then is subjected to enzymolysis by using endopeptidase to form a plurality of polypeptide fragments and amino acid residues, and the polypeptide fragments and the amino acid residues are correctly corresponding to peaks in the peptide graph through LC-UV-MS detection, so that the purpose of accurately judging whether the polypeptide chain of the nesiritide has the phenomena of deletion, misconnection or repetition and the like is achieved.

Description

Nexiridin peptide map analysis method and application thereof

Technical Field

The invention belongs to the technical field of drug analysis, particularly relates to peptide map analysis of a recombinant polypeptide drug, and particularly relates to a peptide map analysis method of nesiritide and application thereof.

Background

The traditional treatment method of Heart Failure is 'Heart strengthening, diuresis and blood vessel expansion', and comprises medicaments such as diuretics, positive inotropic medicaments, β receptor blockers, nitrates and the like, although the traditional treatment method can relieve the clinical symptoms of patients with Heart Failure, the curative effect is limited, and myocardial fibrosis, ventricular remodeling and the like caused by the Heart Failure cannot be reversed, so a new clinical medicament capable of fundamentally treating the Heart Failure is necessarily searched.

Nesiritide (Nesiritide acetate) is a recombinant human brain natriuretic peptide (rhBNP) synthesized by using a recombinant DNA technology, can be combined with a natriuretic titanium receptor, has the functions of expanding blood vessels and promoting urination, can obviously improve the hemodynamic index of a heart failure patient, and reverses ventricular remodeling, thereby relieving clinical symptoms and reducing hospitalization rate and death rate.

The nesiritide has the same 32 amino acid sequence with the natural natriuretic peptide secreted by ventricular muscle, and the 10 th amino acid and the 26 th amino acid (both cysteine) are connected by a disulfide bond to form a ring containing 17 amino acids.

CN103275207A discloses a method for preparing nesiritide, which is simple in operation, low in cost, short in preparation period and/or high in total yield (> 25%), and is beneficial for large-scale production. The method for preparing nesiritide comprises the following steps: preparing 5 fragments, coupling said fragments in sequence to obtain linear nesiritide, followed by oxidation to obtain nesiritide, optionally including the step of purifying nesiritide. However, the nesiritide prepared by the method needs to be detected, and the arrangement structure of the peptide chain is confirmed to be the same as the theoretical structure so as to be applied to clinical treatment. At present, the identification method aiming at the recombinant protein or polypeptide drugs is mainly peptide mapping analysis.

Peptide Mapping is based on the molecular weight of protein and polypeptide and the characteristics of amino acid composition, a proteolytic enzyme (generally endopeptidase) with strong specificity is used to act on a special Peptide chain site to split the polypeptide into small fragments, and a characteristic fingerprint is formed by a certain separation and detection means. Peptide map analysis has important significance for polypeptide structure research and characteristic identification. Quality control of bioengineering products is the key to guarantee the product quality, wherein peptide mapping is an important analysis method for researching the primary structure of protein. Trypsin (Trypsin) is a common endopeptidase that cleaves the carboxyl side of lysine and arginine residues in polypeptide chains. For the inspection of peptide diagrams of recombinant polypeptide drugs, two methods, namely trypsin cracking-reverse liquid chromatography and cyanogen bromide cracking, are included in the pharmacopoeia of China.

Currently, for peptide mapping analysis of nesiritide, trypsin cleavage-reverse liquid chromatography is generally used for analyzing nesiritide, and after analysis is finished, fragments of nesiritide after decomposition need to be collected, but the process is time-consuming. When peaks of a liquid chromatogram are assigned, it is generally considered that a part of small molecule fragments (with large polarity) appear peaks after relatively large molecules (with small polarity), however, according to a retention mechanism of reverse phase chromatography, the fragments with large polarity should be retained on a reverse phase chromatography column weakly and appear peaks before, so that the analysis of the prior art has deviation in segment assignment.

Therefore, it is necessary to provide a new identification method with high accuracy and simple steps for detecting nesiritide, so as to realize accurate analysis of nesiritide peptide maps.

Disclosure of Invention

In view of the problems in the prior art, the invention provides a nesiritide peptide map analysis method, which can be used for simply, timely and accurately corresponding a fragment obtained by enzymolysis of nesiritide to a peak in a peptide map, wherein a small molecular fragment with high polarity belongs to a front peak in the peptide map, a large molecular fragment with low polarity belongs to a rear peak in the peptide map, and an obtained analysis result is more accurate. In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for analyzing a peptide map of nesiritide, comprising the steps of:

reducing and alkylating nesiritide, adding endopeptidase for enzymolysis, detecting by using LC-UV-MS, and attributing polypeptide fragments and/or amino acid residues formed after the nesiritide is subjected to enzymolysis according to collected peptide diagram data.

In the present invention, the disulfide bond of nesiritide is cleaved and reduced to a thiol group, and the thiol group is blocked with an alkylating agent in order to prevent the free thiol group from forming a disulfide bond again. Through reduction and alkylation treatment, nesiritide becomes a free extension peptide chain in solution, and is suitable for enzymolysis of endopeptidase. After enzymolysis, the LC-UV-MS is directly used for detecting the polypeptide fragment of the nesiritide after enzymolysis, the fraction collection link in the prior art is reduced, the analysis time is greatly shortened, the polypeptide fragment of the nesiritide corresponds to the peak in the peptide diagram through the peptide diagram and mass spectrum results, the accuracy of the analysis result is high, and if the fragment and the chromatographic peak can correspond to each other, the primary structure of the nesiritide is proved to be correct, and phenomena such as deletion, misconnection or repetition do not occur.

As a preferred technical scheme of the invention, the peptide map analysis method comprises the following steps:

(1) reducing nesiritide using a reducing agent, and then alkylating the reduced nesiritide by adding an alkylating agent;

(2) carrying out enzymolysis on the alkylated nesiritide by using endopeptidase to obtain a polypeptide fragment and/or an amino acid residue;

(3) and detecting the polypeptide fragments by using LC-UV-MS, and attributing the polypeptide fragments and/or amino acid residues formed after the nesiritide is subjected to enzymolysis according to the collected peptide diagram data.

Preferably, the mobile phase of the LC-UV-MS comprises mobile phase a and mobile phase B, wherein the mobile phase a is an aqueous formic acid solution, and the mobile phase B is an acetonitrile formic acid solution.

Preferably, the volume fraction of formic acid in the aqueous formic acid solution is 0.1 to 1%, and may be, for example, 0.1%, 0.15%, 0.2%, 0.3%, 0.5%, 0.8%, 1%, or the like.

Preferably, the volume fraction of formic acid in the formic acid acetonitrile solution is 0.1-1%, for example, 0.1%, 0.15%, 0.2%, 0.3%, 0.5%, 0.8%, 1%, etc.

Preferably, the LC-UV-MS detection column temperature is 55-60 deg.C, such as 55 deg.C, 56 deg.C, 57 deg.C, 58 deg.C, 59 deg.C or 60 deg.C.

Preferably, the chromatographic column used in the LC-UV-MS is an alkylsilane bonded silica reverse phase chromatographic column, preferably an octadecylsilane bonded silica reverse phase chromatographic column.

As a preferred technical scheme of the invention, the LC-UV-MS adopts gradient elution.

Preferably, the parameters of the gradient elution are: a first stage in which the mobile phase A volume fraction is 95-99% (e.g., 95%, 95.5%, 96%, 97%, 98%, or 99%, etc.), the mobile phase B volume fraction is 1-5% (e.g., 1%, 1.5%, 2%, 3%, 4%, or 5%, etc.), for 2-2.5min (e.g., 2min, 2.1min, 2.2min, 2.3min, 2.4min, or 2.5min, etc.), a second stage in which the mobile phase A volume fraction is 95-99% (e.g., 95%, 95.5%, 96%, 97%, 98%, or 99%, etc.), the mobile phase B volume fraction is 1-5% (e.g., 1%, 1.5%, 2%, 3%, 4%, or 5%, etc.), for 6-7min (e.g., 6%, 6.2min, 6.4min, 6.5min, 6.8min, or 7min, etc.), and a third stage in which the mobile phase A volume fraction is 50-70% (e.g., 50-70%) 55%, 56%, 60%, 62%, 65%, or 70%, etc.), a mobile phase B volume fraction of 30-50% (e.g., may be 30%, 35%, 40%, 44%, 45%, or 50%, etc.), for 2-2.5min (e.g., may be 2min, 2.1min, 2.2min, 2.3min, 2.4min, or 2.5min, etc.), a fourth stage, a mobile phase a volume fraction of 1-5% (e.g., may be 1%, 1.5%, 2%, 3%, 4%, or 5%, etc.), a mobile phase B volume fraction of 95-99% (e.g., may be 95%, 95.5%, 96%, 97%, 98%, or 99%, etc.), for 4-5min (e.g., may be 4min, 4.2min, 4.4min, 4.6min, 4.8min, or 5min, etc.), a fifth stage, a mobile phase a volume fraction of 1-5% (e.g., may be 1%, 1.5%, 2%, 3%, 4.5%, or 5%), etc.), the volume fraction of mobile phase B is 95-99% (e.g., 95%, 95.5%, 96%, 97%, 98%, 99%, etc.) for 0.2-0.3min (e.g., 0.2min, 0.22min, 0.24min, 0.26min, 0.28min, 0.3min, etc.), and in the sixth stage, the volume fraction of mobile phase a is 95-99% (e.g., 95%, 95.5%, 96%, 97%, 98%, 99%, etc.), and the volume fraction of mobile phase B is 1-5% (e.g., 1%, 1.5%, 2%, 3%, 4%, 5%, etc.) for elution to 15-20min (e.g., 15min, 16min, 17min, 18min, 19min, 20min, etc.).

The ultraviolet detection wavelength of the LC-UV-MS is 210-220nm, and can be 210nm, 212nm, 214nm, 215nm, 216nm, 218nm, 220nm and the like.

Preferably, the liquid phase detection part in the LC-UV-MS uses UPLC for detection.

In the invention, the Liquid phase detection part in the LC-UV-MS can use Ultra Performance Liquid Chromatography (UPLC) to be combined with the mass spectrum, so that the obtained result is more accurate.

Preferably, the mass spectrum detection mode in the LC-UV-MS is a Q-TOF positive ion mode.

Preferably, the mass spectrum detection voltage in the LC-UV-MS is 2800-3000V, and can be 2800V, 2810V, 2850V, 2900V, 2910V, 2920V, 2950V, 2980V, 3000V and the like.

Preferably, the detection temperature of the mass spectrum in the LC-UV-MS is 300-350 ℃, for example, 300 ℃, 305 ℃, 310 ℃, 320 ℃, 330 ℃, 340 ℃ or 350 ℃ and the like can be realized.

In the present invention, the reduced and alkylated nesiritide is cleaved in disulfide bond, and the polypeptide chain is Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-His, and after cleavage with trypsin (which cleaves the carboxy side of lysine and arginine residues in the polypeptide chain), it is theoretically decomposed into 8 amino acid residues or polypeptide fragments, specifically: SPK, MVQGSGCFGR, K, MDR, ISSSSGLGGCK, VLR, R, and H. Wherein, theoretically, the mass-to-charge ratio (m/z) of each segment is respectively as follows: SPK m/z 331.20, MVQGSGCFGR m/z 1098.46, K m/z 147.11, MDR m/z 421.19, ISSSSGLGCK m/z 938.46, VLR m/z 387.27, R m/z 175.12, and H m/z 156.08.

Preferably, the peptide map analysis method further comprises a step of detecting using HILIC.

For amino acid residues or small peptides with high polarity that are not easily separated, such as R, H and SPK, it can be further confirmed by means of Hydrophilic Interaction Chromatography (HILIC).

As a preferred embodiment of the present invention, the reduction and alkylation processes in the peptide mapping analysis method are performed in a buffer.

Preferably, the buffer is an ammonium bicarbonate buffer.

Preferably, the molar concentration of ammonium bicarbonate in the ammonium bicarbonate buffer is 0.1-0.4mol/L, such as 0.1mol/L, 0.15mol/L, 0.18mol/L, 0.2mol/L, 0.25mol/L, 0.3mol/L, 0.35mol/L, or 0.4mol/L, and the like, preferably 0.1 mol/L.

In a preferred embodiment of the present invention, the reducing agent is dithiothreitol.

Preferably, the molar concentration of dithiothreitol is 0.1-0.4mol/L, and may be, for example, 0.1mol/L, 0.15mol/L, 0.18mol/L, 0.2mol/L, 0.25mol/L, 0.3mol/L, 0.35mol/L, or 0.4mol/L, etc., preferably 0.1 mol/L.

Preferably, the alkylating agent is iodoacetamide.

Preferably, the iodoacetamide has a molar concentration of 0.25 to 0.5mol/L, and may be, for example, 0.25mol/L, 0.3mol/L, 0.32mol/L, 0.35mol/L, 0.4mol/L, 0.42mol/L, 0.45mol/L, or 0.5mol/L, and the like, and is preferably 0.25 mol/L.

Preferably, the peptide map analysis method further comprises a step of detecting by using MS after the alkylation is completed.

In the invention, after nesiritide is reduced and alkylated, mass spectrometry can be used for analyzing whether a disulfide bond in nesiritide is completely opened, five characteristic peaks appear in the fully reduced and alkylated nesiritide through mass spectrometry, the mass-to-charge ratios are 291, 597, 716, 895 and 1194 respectively, and the mass spectrogram of the nesiritide is obviously different from that of the nesiritide which is not reduced and alkylated.

In a preferred embodiment of the present invention, the endopeptidase is trypsin and/or chymotrypsin, preferably chymotrypsin.

In a second aspect, the present invention also provides a use of the peptide mapping analysis method according to the first aspect for detecting and/or identifying nesiritide.

The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between any of the above-recited numerical ranges not recited, and for the sake of brevity and clarity, the present invention is not intended to be exhaustive of the specific numerical values encompassed within the range.

Compared with the prior art, the invention has at least the following beneficial effects:

the invention establishes a peptide map analysis method based on LC-MS (liquid chromatography-mass spectrometry), which can accurately correspond an enzymolysis fragment of nesiritide to a peak in a peptide map, and has short detection time.

Drawings

Figure 1 is a mass spectrum of nesiritide after reduction and alkylation in example 1.

Figure 2 is a mass spectrum of nesiritide without reduction and alkylation.

Figure 3 is a liquid chromatographic assay of nesiritide obtained after detection using LC-UV-MS in example 1.

Figure 4 is a mass spectrum of nesiritide after reduction and alkylation in example 2.

Fig. 5 is a liquid chromatography detection chart of nesiritide obtained in comparative example 1.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.

In the following examples, samples of nesiritide used may be prepared as follows:

firstly, carrying out fermentation culture on recombinant escherichia coli capable of producing nesiritide, collecting cultured thalli, and carrying out bacterium breaking and filtering; performing affinity chromatography on the filtrate, collecting chromatography eluate, performing enzyme digestion with EK enzyme (enterokinase), and performing cation chromatography and hydrophobic chromatography on the enzyme digestion product; and (3) carrying out gel filtration on the chromatographic eluent by G25, and then further carrying out cation chromatography to finally obtain the nesiritide stock solution.

Example 1

The present embodiment provides a method for analyzing a peptide map of nesiritide, which specifically includes the following steps:

(1) reduction and alkylation of nesiritide samples

The sample is diluted to 1.5mg/mL by water and added into a 1.5mL centrifuge tube, 100 μ L of 0.1mol/L ammonium bicarbonate buffer solution is added, 11.56 μ L of 0.1mol/L DTT (dithiothreitol) is added, the mixture is mixed evenly and incubated for 1h at 56 ℃, and after the incubation, the mixture is placed in a water bath at 20 ℃ and cooled for 1 min.

12.6. mu.L of 0.25mol/L iodoacetamide solution was added to the reduced sample, mixed well and incubated at 20 ℃ for 1h in the dark.

After the reaction is finished, mass spectrometry is used for confirming that the reduction and the alkylation are complete (as shown in figure 1), four mass spectrum peaks can be observed in the figure, and compared with the mass spectrum of the nesiritide which is not reduced and alkylated in figure 2, the reduction and the alkylation are complete, and the disulfide bond of the nesiritide is completely opened.

(2) Enzymolysis with trypsin

To the alkylated solution was added 6.75. mu.L of 1mg/mL Trypsin (purchased from Sigma), shaken well and incubated at 37 ℃ for 6 h. The reaction was stopped by adding 14. mu.L of 1% formic acid (final concentration 0.1%).

(3) Detection Using LC-UV-MS

Centrifuging at 12000rpm for 1min to collect polypeptide fragments after enzymolysis, and adding into a liquid phase-mass spectrometer (G2-XSQTOF) for detection.

Detection conditions for LC-UV-MS:

the chromatographic column is C18 chromatographic column (ACQUITY BEH 1.7 × 150mm), and the method comprises gradient elution with 0.1% formic acid water solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B at flow rate of 0.3 mL/min; the column temperature was 60 ℃.

The mobile phase gradient elution conditions (volume ratio) of the liquid chromatography were as follows:

time (min)	Phase A (%)	Phase B (%)
			0	95	5
2	95	5
			8	70	30
10	5	95
			14	5	95
14.2	95	5
			15	95	5

Detection a liquid chromatography detection map of nesiritide is obtained, as shown in figure 1. In fig. 1, the abscissa represents the peak-appearance time, and the ordinate represents the peak-appearance intensity. As can be seen from FIG. 1 combined with the results of mass spectrometry, in FIG. 1, the peak with a peak-off time of 1.626min represents the polypeptide fragment VLR, its m/z is 387.2789, the peak with a peak-off time of 5.217min represents the polypeptide fragment ISSSSGLGCK, its m/z is 995.4988, the peak with a peak-off time of 6.361min represents the polypeptide fragment MVQGSGCFGR, and its m/z is 1098.4985.

Polypeptide fragments or amino acid residues not retained in the LC include SPK (m/z-331.21), K (m/z-147.06), MDR (m/z-421.19), R (m/z-175.12) and H (m/z-156.07), which fragments are more polar and therefore analyzed using the hilic (hydrophic Interaction Liquid chromatography) format.

Finally, all amino acids on nesiritide can correspond to peaks on a peptide diagram, which indicates that the detected nesiritide polypeptide chain is complete, and also indicates that the analysis method provided by the embodiment can detect all polypeptide fragments or amino acid residues of nesiritide.

Meanwhile, as can be seen from the results of the examination, in addition to the eight amino acid residues or polypeptide fragments theoretically produced, other polypeptide fragments were detected, including QGSGCFGR (m/z-868.39), GSGCFGR (m/z-740.33), MVQGSGCFG (m/z-885.38), mdsisss (m/z-882.41) and mdsisss (m/z-795.38). It is noted that cleavage of the polypeptide chain of nesiritide with Trypsin is a non-leaky cleavage, but a cleavage may occur to obtain another polypeptide fragment.

Example 2

(1) reduction and alkylation of nesiritide samples

The sample is diluted to 1.5mg/mL by water and added into a 1.5mL centrifuge tube, 100 μ L of 0.4mol/L ammonium bicarbonate buffer solution is added, 11.56 μ L of 0.4mol/L DTT (dithiothreitol) is added, the mixture is mixed evenly and incubated for 1h at 56 ℃, and after the incubation, the mixture is placed in a water bath at 25 ℃ and cooled for 1 min.

12.6. mu.L of 0.5mol/L iodoacetamide solution was added to the reduced sample, mixed well and incubated at 25 ℃ for 1h in the dark.

After the reaction is finished, MS detection is adopted to confirm that the reduction and alkylation are complete (as shown in fig. 4), four mass spectrum peaks can be observed in the figure, and compared with the mass spectrum without reduction and alkylation in fig. 2, the reduction and alkylation are complete, and the disulfide bond of nesiritide is completely opened.

(2) Chymotrypsin (Chymotrypsin) for enzymolysis

To the post-alkylation solution was added 6.75. mu.L of 1mg/mL Chymotrypsin, shaken well and incubated at 37 ℃ for 6 h. The reaction was stopped by adding 14. mu.L of 1% formic acid (final concentration 0.1%).

(3) Detection Using LC-UV-MS

Detection conditions for LC-UV-MS:

time (min)	Phase A (%)	Phase B (%)
			0	99	1
2	99	1
			8	50	50
10	1	99
			14	1	99
14.2	99	1
			15	99	1

Example 3

(1) reduction and alkylation of nesiritide samples

The sample is diluted to 1.5mg/mL by water and added into a 1.5mL centrifuge tube, 100 mu L of 0.2mol/L ammonium bicarbonate buffer solution is added, 11.56 mu L of 0.2mol/L DTT is added, the mixture is mixed evenly and incubated for 1h at 56 ℃, and after the incubation, the mixture is placed in a water bath at 15 ℃ and cooled for 1 min.

12.6. mu.L of 0.5mol/L iodoacetamide solution was added to the reduced sample, mixed well and incubated at 15 ℃ for 1h in the dark.

(2) Chymotrypsin (Chymotrypsin, available from Sigma) for enzymatic hydrolysis

To the post-alkylation solution was added 6.75. mu.L of 2mg/mL Chymotrypsin, shaken well and incubated at 37 ℃ for 6 h. The reaction was stopped by adding 14. mu.L of 1% formic acid (final concentration 0.1%).

(3) Detection Using LC-UV-MS

Detection conditions for LC-UV-MS:

Comparative example 1

The comparative example provides a peptide mapping analysis method of nesiritide, which specifically comprises the following steps:

detection conditions for LC-UV: the chromatographic column is a C18 chromatographic column (Agilent 1260Zobax SB-Aq C18, 3.5 mu m column, 4.6 multiplied by 100mm), formic acid aqueous solution with the volume ratio of 0.1 percent is taken as a mobile phase A, acetonitrile and water with the volume ratio of 9:1 are added, formic acid with the volume fraction of 0.1 percent is taken as a mobile phase B, gradient elution is adopted, and the flow rate is 0.75 mL/min; the column temperature was 25 ℃.

Fig. 5 shows a liquid chromatography detection chart of nesiritide, in which the abscissa represents the peak time, and the peak times of 6 peptides are: 6.115min, 6.267min, 6.830min, 7.132min, 7.441min and 8.663 min. Fractions from the liquid phase elution were collected and subjected to MS analysis, which required a total of 12 h. And obtaining a peptide graph of nesiritide by an LC-UV method, collecting fractions corresponding to the peptide fragments of all the fractions, and attributing the peptide fragments of all the fractions by using MS.

By combining the analysis of the embodiment 1 and the analysis of the comparative example 1, the analysis method provided by the invention can accurately correspond the fragment of nesiritide after enzymolysis to the peak in the peptide map, the small molecular fragment with high polarity belongs to the front peak in the peptide map, the large molecular fragment with low polarity belongs to the rear peak in the peptide map, and the obtained analysis result is more accurate. The method adopts the efficient and sensitive UPLC-MS coupling technology in the liquid chromatography-mass spectrometry, greatly shortens the analysis time, reduces unnecessary fraction collection links, and simultaneously ensures the analysis accuracy.

The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A method for analyzing a peptide map of nesiritide, which is characterized by comprising the following steps:

2. The peptide map analysis method according to claim 1, comprising the steps of:

3. The peptide graph analysis method according to claim 1 or 2, wherein the mobile phase of the LC-UV-MS comprises a mobile phase a and a mobile phase B, wherein the mobile phase a is an aqueous solution of formic acid, and the mobile phase B is an acetonitrile solution of formic acid;

preferably, the volume fraction of formic acid in the aqueous formic acid solution is 0.1 to 1 percent;

preferably, the volume fraction of formic acid in the formic acid acetonitrile solution is 0.1-1%;

preferably, the detection column temperature of the LC-UV-MS is 55-60 ℃;

4. The peptide mapping analysis method of any of claims 1-3, wherein the LC-UV-MS employs gradient elution:

preferably, the parameters of the gradient elution are: the first stage, the volume fraction of mobile phase A is 95-99%, the volume fraction of mobile phase B is 1-5%, the duration is 2-2.5min, the second stage, the volume fraction of mobile phase A is 95-99%, the volume fraction of mobile phase B is 1-5%, the duration is 6-7min, the third stage, the volume fraction of mobile phase A is 50-70%, the volume fraction of mobile phase B is 30-50%, the duration is 2-2.5min, the fourth stage, the volume fraction of mobile phase A is 1-5%, the volume fraction of mobile phase B is 95-99%, the duration is 4-5min, the fifth stage, the volume fraction of mobile phase A is 1-5%, the volume fraction of mobile phase B is 95-99%, the duration is 0.2-0.3min, the sixth stage, the volume fraction of mobile phase A is 95-99%, and the volume fraction of mobile phase B is 1-5% and the elution is 15-20 min.

5. The peptide graph analysis method of any one of claims 1-4, wherein the UV detection wavelength of LC-UV-MS is 210-220 nm;

preferably, the liquid phase detection part in the LC-UV-MS uses UPLC for detection;

preferably, the mass spectrum detection mode in the LC-UV-MS is a Q-TOF positive ion mode;

preferably, the mass spectrum detection voltage in the LC-UV-MS is 2800-3000V;

preferably, the detection temperature of the mass spectrum in the LC-UV-MS is 300-350 ℃;

6. The peptide map analysis method according to any one of claims 1 to 5, wherein the reduction and alkylation processes are carried out in a buffer;

preferably, the buffer is an ammonium bicarbonate buffer;

preferably, the molar concentration of ammonium bicarbonate in the ammonium bicarbonate buffer is 0.1-0.4mol/L, preferably 0.1 mol/L.

7. The peptide mapping analysis method of any of claims 1-6, wherein the reducing agent is dithiothreitol;

preferably, the molar concentration of dithiothreitol is 0.1-0.4mol/L, preferably 0.1 mol/L.

8. The peptide graph analysis method of any one of claims 1 to 7, wherein the alkylating agent is iodoacetamide;

preferably, the molar concentration of the iodoacetamide is 0.25-0.5mol/L, preferably 0.25 mol/L;

9. The method for peptide mapping analysis according to any of claims 1-8, wherein the endopeptidase is trypsin and/or chymotrypsin, preferably chymotrypsin.

10. Use of a peptide mapping assay according to any of claims 1 to 9 for the detection and/or identification of nesiritide.