Method for rapidly distinguishing and determining leucine and isoleucine in polypeptide sequence based on tandem multistage high-resolution mass spectrometry

Abstract

The invention relates to the technical field of polypeptide detection, in particular to a method for rapidly distinguishing and determining leucine and isoleucine in a polypeptide sequence based on tandem multistage high-resolution mass spectrometry. The method comprises the following steps: (1) performing HCD fragmentation on parent ions or daughter ions of polypeptide to generate imine ions related to leucine and / or isoleucine; (2) carrying out CID fragmentation on the imine ions, and detecting relative peak intensities of m / z 44 + / -0.2 and m / z 69 + / -0.2 generated by fragmentation in a mass axis range of m / z 40-90; and (3) judging the types of leucine and isoleucine in the polypeptide according to the relationship between the relative peak intensities of m / z 44 + / -0.2 and m / z 69 + / -0.2. The method provided by the invention is higher in fragmentation efficiency and confidence degree, and provides a basis for rapid and accurate identification of leucine and isoleucine.

Description

Technical Field

[0001] This invention relates to the field of peptide detection technology, and in particular to a method for rapidly distinguishing and determining leucine and isoleucine in a peptide sequence based on tandem multi-stage high-resolution mass spectrometry. Background Technology

[0002] Leucine and isoleucine are isomers, exhibiting identical mass spectrometric peaks, making them difficult to distinguish. When a leucine site is present in the amino acid sequence of a polypeptide, replacing it with isoleucine often alters drug safety and efficacy, and vice versa. Therefore, distinguishing between leucine and isoleucine sites is crucial, whether for sequencing newly discovered polypeptides in natural products or differentiating drug isomeric impurities.

[0003] Currently, there are two main analytical techniques for distinguishing leucine and isoleucine within peptide sequences. One is amino acid sequencing, which relies on Edman degradation. This involves chemically degrading amino acids sequentially from the peptide sequence and then identifying the types of amino acids that are degraded. However, this method has several limitations. First, amino acid sequencing requires large sample volumes and is time-consuming, making high-throughput analysis difficult. Second, Edman degradation cannot sequence cyclized peptides or peptides with N-terminal modifications. Cycling and N-terminal modifications are common drug modification techniques in peptide drugs, so Edman degradation cannot meet the requirements for peptide drug sequencing for various structural types, and therefore cannot distinguish between leucine and isoleucine sites within the sequence.

[0004] The second method utilizes characteristic ions generated by multi-stage tandem mass spectrometry (MS / MS) to distinguish leucine and isoleucine sites in peptide sequences. This analytical technique first requires determining the number of sites in the target peptide sequence that require differentiation between leucine and isoleucine. If only one site requires differentiation, HCD (High-energy collision dissociation) is used to fragment the peptide precursor ion containing both leucine and isoleucine sites, generating a secondary fragment ion—an imine ion—with an m / z of 86.1 associated with either leucine or isoleucine. This imine ion is then further subjected to HCD-MS. 3Fragmentation. If tertiary fragment ions with an m / z of 69±0.2 are generated, then the imine ion with an m / z of 86.1 is specifically generated by isoleucine, therefore the polypeptide precursor ion sequence contains only isoleucine. If tertiary fragment ions with an m / z of 69±0.2 cannot be generated, then the imine ion with an m / z of 86.1 is specifically generated by leucine, therefore the polypeptide precursor ion sequence contains only leucine, and this can be used for differentiation. If there are multiple sites in the polypeptide sequence that require differentiation between leucine and isoleucine, then ETD (Electron Transfer Dissociation), HECD (Hot Electron Captured Dissociation), or EThCD (Electron-Transfer / Higher-Energy Collision Dissociation) are used to fragment the polypeptide precursor ion, generating z-ions with leucine or isoleucine sites as terminals. These z-ions are then further subjected to HCD-MS. 3 Fragmentation produces w ions. The terminal amino acid of the z ion is determined by observing the mass loss shift between the z ion and its generated w ions. When the mass loss shift between the z ion and its generated w ions is 43 Da, the terminal amino acid of the z ion is leucine; when the mass loss shift between the z ion and its generated w ions is 29 Da, the terminal amino acid of the z ion is isoleucine.

[0005] The analytical techniques for distinguishing leucine and isoleucine sites in peptide sequences using characteristic ions generated by multistage tandem mass spectrometry (MS / MS) have significant limitations. Firstly, for sites requiring differentiation between only one leucine and isoleucine, HCD MS is used instead. 3 Fragmenting imine ions with m / z 86.1 was performed to observe whether tertiary fragment ions with m / z 69 were generated, in order to determine whether the sequence contained leucine or isoleucine. Literature review and experimental analysis revealed that both amino acids can produce mass spectrometry peaks at m / z 69 ± 0.2, thus failing to effectively distinguish between leucine and isoleucine in the polypeptide sequence. Furthermore, for polypeptides with multiple sites requiring differentiation between leucine and isoleucine, ETD-HCD MS was used. 3The ETD (Extra-Through-Depletion) fragmentation method involves fragmenting specific sites to generate z-ions that can identify leucine / isoleucine. The mass loss shift of w-ions produced by further fragmentation is then observed to determine leucine and isoleucine. However, this fragmentation method has significant limitations. ETD fragmentation efficiency is highly dependent on charge valence; higher charge valence results in more thorough fragmentation. Many peptide drugs, especially antimicrobial peptides, are short peptides, often exhibiting a monovalent charge valence in mass spectrometry. ETD fragmentation of these monovalent precursor ion peaks generally does not produce any effective fragmentation, nor does it generate specific types of z-ions. Therefore, for short peptide drugs containing multiple leucine and isoleucines, the above method is completely ineffective in identifying and distinguishing each leucine and isoleucine isomer site within the peptide sequence.

[0006] Based on this, the present invention is proposed. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for rapidly distinguishing and determining leucine and isoleucine in a polypeptide sequence based on tandem multi-stage high-resolution mass spectrometry.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for rapidly distinguishing and determining leucine and isoleucine in a polypeptide sequence based on tandem multi-stage high-resolution mass spectrometry, comprising the following steps: (1) The precursor or daughter ions of the polypeptide are fragmented by HCD to generate imine ions related to leucine and / or isoleucine; (2) The imine ions were subjected to CID fragmentation, and the relative peak intensities of m / z 44±0.2 and m / z 69±0.2 generated by fragmentation were detected in the mass axis range of m / z 40~90; (3) Based on the relationship between the relative peak intensities of m / z 44±0.2 and m / z 69±0.2, determine the types of leucine and isoleucine in the polypeptide.

[0009] Preferably, the number of sites containing leucine and isoleucine in the precursor ion of the polypeptide in step (1) is ≤2.

[0010] Preferably, the number of leucine and isoleucine sites in the daughter ion of step (1) is ≤2; When the parent ion of a polypeptide contains more than 2 but less than or equal to 4 sites of leucine and isoleucine, the parent ion of the polypeptide is first fragmented by CID or HCD to generate daughter ions, so that the number of leucine and isoleucine sites in the daughter ions is ≤2.

[0011] Preferably, when the precursor ion of the polypeptide contains more than 4 sites containing leucine and isoleucine, the precursor ion of the polypeptide is first enzymatically digested or fragmented until each peptide segment contains less than or equal to 4 sites containing leucine and isoleucine, and then the detection is performed according to the method described above.

[0012] Preferably, the normalized collision energy during HCD fragmentation in step (1) is 34~36.

[0013] Preferably, the normalized collision energy during CID fragmentation in step (2) is 30~32.

[0014] Preferably, when the parent or daughter ion of the polypeptide contains one leucine or isoleucine site: The relative peak intensity at m / z 44±0.2 is much greater than that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide is leucine. The relative peak intensity at m / z 69±0.2 is much greater than that at m / z 44±0.2, indicating that the parent or daughter ion of the polypeptide is isoleucine.

[0015] Preferred, When the parent or daughter ion of a polypeptide contains two leucine and / or isoleucine sites: The relative peak intensity at m / z 44±0.2 is much greater than that at m / z 69±0.2, indicating that the parent ion or daughter ion of the polypeptide contains two leucines. The relative peak intensity at m / z 69±0.2 is much greater than that at m / z 44±0.2, and the relative peak intensity at m / z 44±0.2 is less than 18% of that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide contains two isoleucines. The relative peak intensity at m / z 69±0.2 is greater than that at m / z 44±0.2, and the relative peak intensity at m / z 44±0.2 is more than 25% of that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide contains one isoleucine and one leucine.

[0016] Preferably, the polypeptide is a polypeptide drug; the polypeptide drug is leucine enkephalin and its isomers, teriparatide and its isomers, oxytocin and its isomers, antimicrobial peptide surfactant and its homologues.

[0017] This invention also provides the application of the method in the preparation of products that rapidly and accurately determine leucine and isoleucine in polypeptide sequences.

[0018] The present invention has the following advantages: This invention utilizes multi-stage mass spectrometry based on HCD / CID (High-energy collision dissociation or collision-induced dissociation) to fragment polypeptides or peptides containing leucine or isoleucine. By leveraging the relative intensities of fragment ions generated by imine ions (m / z 44±0.2 and m / z 69±0.2), the leucine and isoleucine sites and amino acid types within the polypeptide amino acid sequence can be rapidly and accurately identified. This method is simple, easy to implement, and not limited by the number of leucine and isoleucine sites within the polypeptide sequence or the charge valence state of the analyzed polypeptide in mass spectrometry, making it applicable to polypeptide molecules of any structure. It can provide a basis for the rapid differentiation and detection of leucine and isoleucine isomeric amino acids in the identification of leucine / isoleucine isomer peptide impurities during the development of polypeptide and protein mass spectrometry sequencing methods, sequence characterization of natural or synthetic polypeptide products in drug development, and the identification of leucine / isoleucine isomeric peptide impurities in drug quality control. Attached Figure Description

[0019] Figure 1 The fragmentation of a polypeptide imine ion containing one leucine or isoleucine in the m / z 40–90 range produces daughter ions (A: YGGFX primary mass spectrum; B: YGGFX secondary mass spectrum fragmentation producing an imine ion at m / z 86.1; C: YGGFX imine ion fragmentation in CID mode; D: Relative peak intensity of YGGFX daughter ions; E: HVXDS primary mass spectrum; F: HVXDS secondary mass spectrum fragmentation producing an imine ion at m / z 86.1; G: HVXDS imine ion fragmentation in CID mode; H: Relative peak intensity of HVXDS daughter ions). Figure 2 The mass spectra of the model peptide containing two leucines and / or isoleucines are shown below (A is the molecular weight of the peptide obtained by primary mass spectrometry; B is the molecular weight of the reduced oxytocin and its sequence isomers by primary mass spectrometry; C is the fragmentation of secondary mass spectrometry by CID fragmentation to produce daughter ions; D is the imine ion with m / z 86.1 produced by HCD fragmentation of the parent ion of the reduced oxytocin and its sequence isomers). Figure 3 This section describes the fragmentation of imine ions of secondary fragment ions and imine ions of the parent polypeptide containing two leucines and / or isoleucines (oxytocin and its sequence isomers) in the m / z 40–90 range to produce daughter ions (A, D, and G are all examples). Figure 2 The Cb3 daughter ion, after fragmentation by HCD, produces an imine ion with m / z 86.1, which, after fragmentation by CID, produces imine ions with m / z 44±0.2 and m / z 69±0.2; B, E, and H are... Figure 2The y3 daughter ion in C is fragmented by HCD to produce an imine ion with m / z 86.1, and then fragmented in CID mode to produce m / z 44±0.2 and m / z 69±0.2; Figures C, F and I are all... Figure 2 The polypeptide precursor ion after disulfide bond reduction in D is directly fragmented by HCD to produce an imine ion with m / z 86.1. Subsequent fragmentation in CID mode produces imine ions with m / z 44±0.2 and m / z 69±0.2. Figure 4 This is a liquid phase separation diagram of the four peptide fragments produced after double digestion of teriparatide; Figure 5 The results show the detection of HR7, LF7, and VR5 peptides after enzyme digestion (A, D, and G represent the molecular weight of the peptides in primary mass spectrometry; B, E, and H represent the imine ions of the peptides; C, F, and I represent the m / z values ​​of 44±0.2 and 69±0.2 respectively, resulting from fragmentation in CID mode). Figure 6 The mass spectra of the XK9 peptide (where X represents an undetermined leucine or isoleucine) obtained by further digestion of SK13 (obtained by double digestion of teriparatide and its two isomers with trypsin and lysine protease) with Glu-C protease are shown in the image (A represents the molecular weight of the polypeptide obtained by primary mass spectrometry; B represents the fragmentation of the peptide by CID fragmentation in secondary mass spectrometry). Figure 7 To characterize the leucine and isoleucine sites in the XK9 peptide segment of teriparatide and its two isomers (where X represents an undetermined leucine or isoleucine), b2, y4, and y7 daughter ions were selected for subsequent analysis (A, D, and G represent the m / z values ​​of 44±0.2 and 69±0.2 respectively, resulting from the fragmentation of the imine ion generated by the b2 ion of the XK9 peptide segment of teriparatide and its two isomers under CID mode; B, E, and H represent the m / z values ​​of 44±0.2 and 69±0.2 respectively, resulting from the fragmentation of the imine ion generated by the y7 ion of the XK9 peptide segment of teriparatide and its two isomers under CID mode; C, F, and I represent the m / z values ​​of 44±0.2 and 69±0.2 respectively, resulting from the fragmentation of the imine ion generated by the y4 ion of the XK9 peptide segment of teriparatide and its two isomers under CID mode). Figure 8 The chemical structure of surfactant; Figure 9These are mass spectra of the antimicrobial peptide surfactant and its homologues. FA represents the hydrophobic fatty acid side chain (A is the molecular weight of the peptide obtained by primary mass spectrometry of surfactant; B is the molecular weight of the peptide produced by CID fragmentation of surfactant by secondary mass spectrometry; C is the molecular weight of the peptide obtained by primary mass spectrometry of homologue 1; D is the molecular weight of the peptide produced by CID fragmentation of homologue 1 by secondary mass spectrometry; E is the molecular weight of the peptide obtained by primary mass spectrometry of homologue 2; F is the molecular weight of the peptide produced by CID fragmentation of homologue 2 by secondary mass spectrometry). Figure 10 Mass spectra of antimicrobial peptide surfactant and its homologues generated by HCD fragmentation to produce internal fragmented ions and daughter ions (A: HCD fragmentation of surfactant secondary mass spectrometry to produce internal fragmented ions and daughter ions; B: HCD fragmentation of homologue 1 secondary mass spectrometry to produce internal fragmented ions and daughter ions; C: HCD fragmentation of homologue 2 secondary mass spectrometry to produce internal fragmented ions and daughter ions). Figure 11 To characterize the leucine and isoleucine sites in the antimicrobial peptide surfactant and its homologues, the internal fragmentation ion y6b3 and the daughter ion y2 were selected for subsequent analysis (A, C, and E represent the m / z 44±0.2 and m / z 69±0.2 cases of the imine ions generated by the y6b3 ions of surfactant and its two homologues in CID mode fragmentation; B, D, and F represent the m / z 44±0.2 and m / z 69±0.2 cases of the imine ions generated by the y2 ions of surfactant and its two homologues in CID mode fragmentation). Detailed Implementation

[0020] In this invention, when the precursor ion of the polypeptide contains disulfide bonds, the disulfide bonds can be reduced to make the mass spectrum clearer. Alternatively, reduction can be omitted. After reducing the disulfide bonds, the polypeptide with reduced disulfide bonds can be fragmented to obtain daughter ions. These daughter ions are then subjected to HCD fragmentation to generate imine ions. The imine ions are then fragmented in CID mode, and the relative peak intensities at m / z 44±0.2 and m / z 69±0.2 generated by fragmentation are detected within the mass axis range of m / z 40~90. Based on the relationship between the relative peak intensities at m / z 44±0.2 and m / z 69±0.2, the types of leucine and isoleucine in the polypeptide can be determined.

[0021] In this invention, when the number of leucine and isoleucine sites in the precursor ion of the polypeptide is greater than 4, the precursor ion of the polypeptide needs to be enzymatically digested or fragmented first. The enzyme used for enzymatic digestion is one or more of trypsin, lysine protease, and Glu-C protease. During enzyme digestion, the mass ratio of enzyme to polypeptide is 1:20~30; The enzyme digestion temperature is 35~39℃; The enzyme digestion time is 2-3 hours; The fragmentation is CID / HCD fragmentation.

[0022] When the peptide contains ≤2 sites of leucine and isoleucine, HCD fragmentation is performed directly to generate imine ions. The imine ions are then subjected to CID fragmentation. Within the m / z mass axis range, the relative peak intensities of m / z 44±0.2 and m / z 69±0.2 generated by fragmentation are detected. Based on the relationship between the relative peak intensities of m / z 44±0.2 and m / z 69±0.2, the types of leucine and isoleucine in the peptide are determined.

[0023] The criteria for determining whether a peptide contains one leucine or isoleucine site are as follows: The relative peak intensity at m / z 44±0.2 is much greater than that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide is leucine. The relative peak intensity at m / z 69±0.2 is much greater than that at m / z 44±0.2, indicating that the parent or daughter ion of the polypeptide is isoleucine.

[0024] Criteria for determining whether a peptide contains two leucine and / or isoleucine sites: The relative peak intensity at m / z 44±0.2 is much greater than that at m / z 69±0.2, indicating that the parent ion or daughter ion of the polypeptide contains two leucines. The relative peak intensity at m / z 69±0.2 is much greater than that at m / z 44±0.2, and the relative peak intensity at m / z 44±0.2 is less than 18% of that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide contains two isoleucines. The relative peak intensity at m / z 69±0.2 is greater than that at m / z 44±0.2, and the relative peak intensity at m / z 44±0.2 is more than 25% of that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide contains one isoleucine and one leucine.

[0025] When the peptide contains more than 2 but less than or equal to 4 sites of leucine and isoleucine, it is first fragmented by CID or HCD to generate daughter ions, ensuring that the number of leucine and isoleucine sites in the daughter ions is ≤2. Then, the daughter ions are fragmented by HCD to generate imine ions. The imine ions are then fragmented by CID. Within the m / z mass axis range, the relative peak intensities of m / z 44±0.2 and m / z 69±0.2 generated by fragmentation are detected. Based on the relationship between the relative peak intensities of m / z 44±0.2 and m / z 69±0.2, the types of leucine and isoleucine in the peptide are distinguished according to the above judgment criteria, thereby obtaining the types of leucine and isoleucine in the polypeptide.

[0026] In this invention, when the parent ion of the polypeptide contains more than 2 and less than or equal to 4 sites of leucine and isoleucine, the parent ion of the polypeptide is first fragmented by CID or HCD to generate daughter ions, such that the number of leucine and isoleucine sites in the daughter ions is ≤2. Then, the daughter ions are fragmented by HCD to generate imine ions. The imine ions are then fragmented by CID. Within the m / z mass axis range, the relative peak intensities of m / z 44±0.2 and m / z 69±0.2 generated by fragmentation are detected, and the relationship between the relative peak intensities of m / z 44±0.2 and m / z 69±0.2 is used.

[0027] The criteria for determining whether a polypeptide's parent ion contains one leucine or isoleucine site are as follows: The relative peak intensity at m / z 44±0.2 is much greater than that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide is leucine. The relative peak intensity at m / z 69±0.2 is much greater than that at m / z 44±0.2, indicating that the parent or daughter ion of the polypeptide is isoleucine.

[0028] The criteria for determining whether a polypeptide's parent ion contains two leucine and / or isoleucine sites are as follows: The relative peak intensity at m / z 44±0.2 is much greater than that at m / z 69±0.2, indicating that the parent ion or daughter ion of the polypeptide contains two leucines. The relative peak intensity at m / z 69±0.2 is much greater than that at m / z 44±0.2, and the relative peak intensity at m / z 44±0.2 is less than 18% of that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide contains two isoleucines. The relative peak intensity at m / z 69±0.2 is greater than that at m / z 44±0.2, and the relative peak intensity at m / z 44±0.2 is more than 25% of that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide contains one isoleucine and one leucine.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] In this embodiment of the invention, the Orbitrap Eclipse 3-in-1 high-resolution mass spectrometer manufactured by Thermo Fisher Scientific is used, employing only the HCD / CID (High-energy collision dissociation or collision-induced dissociation) multi-stage mass spectrometry fragmentation mode.

[0031] Example 1

[0032] Detection of the presence of one leucine or isoleucine in a polypeptide.

[0033] The detection was performed using leucine enkephalin and its isoform YGGFX (SEQ ID NO.1) and laboratory-customized peptide and its isoform HVXDS (SEQ ID NO.2) as model peptides, where X represents leucine or isoleucine.

[0034] The molecular weight of the model peptide was first determined by full-scan high-resolution mass spectrometry in positive ion mode. Then, secondary mass spectrometry was performed on the model peptide. The precursor ion was fragmented in HCD mode (normalized collision energy NCE energy of 35) to generate an imine ion with m / z 86.1. Subsequently, the imine ion was fragmented in CID mode (normalized collision energy NCE energy of 31). The resulting mass spectrometry results are shown below. Figure 1 As shown.

[0035] The parameters for mass spectrometry detection are as follows: During full-scan high-resolution mass spectrometry detection, the Orbitrap mass spectrometer operating parameters are: resolution 60,000, mass range m / z 200~2000, RF lens voltage 50%, standard AGC target, and maximum ion injection time 50 milliseconds.

[0036] ddMS detection by secondary mass spectrometry 2The detector type is Orbitrap, the isolation window is 6 m / z, the resolution is 30,000, the mass range is 40~2000 m / z, and the maximum ion injection time is 100 ms.

[0037] Level 3 ddMS 3 The detector type is selected as ion trap, MS, and MS. 2 The precursor ions were set with isolation windows of 6 m / z and 3 m / z, respectively, with a mass range of m / z 40~100 and a maximum injection time of 100 ms.

[0038] Figure 1 The results showed that after the model peptide generated an imine ion at m / z 86.1, further fragmentation of this ion within the m / z 40-90 mass axis revealed that model peptides containing only one leucine ion produced a daughter ion at m / z 44±0.2 after imine ion fragmentation, with almost no or trace amounts of a daughter ion at m / z 69±0.2; model peptides containing only one isoleucine ion produced a daughter ion at m / z 69±0.2 after imine ion fragmentation, with a certain intensity of the m / z 44±0.2 daughter ion (its relative intensity is about 15% of that of m / z 69±0.2). Based on this, for peptides containing one leucine or isoleucine ion, the identification criterion for the leucine or isoleucine site is: by comparing the relative peak intensities of m / z 44±0.2 and m / z 69±0.2, the leucine or isoleucine in the peptide sequence can be determined. When m / z 44±0.2... m / z 69±0.2 indicates a leucine residue in the polypeptide sequence; when m / z 69±0.2... m / z 44±0.2, the polypeptide sequence contains isoleucine.

[0039] “ "" indicates much greater than.

[0040] Example 2

[0041] Detection of the presence of two leucine and / or isoleucine residues in a polypeptide.

[0042] Taking oxytocin and its sequence isomers as an example, the molecular weight of the model peptide is obtained by primary mass spectrometry. Since oxytocin and its sequence isomers contain one disulfide bond, disulfide bond reduction is required to obtain better secondary mass spectrum quality. Alternatively, the disulfide bond can be removed and mass spectrometry analysis can be performed directly.

[0043] To reduce the disulfide bonds in oxytocin and its two isomers, a corresponding peptide solution (50 μg / g) was prepared and reacted with tris(2-carboxyethyl)phosphine (TCEP) at a molar ratio of 1:10 (peptide:TCEP). The reaction solution was placed in a metal bath and shaken at 600 rpm for 4 hours at room temperature. The reduced solution was immediately analyzed by mass spectrometry.

[0044] The reduced oxytocin and its sequence isomers were fragmented in CID mode (normalized collision energy NCE energy was 32) to produce b3 / y3 daughter ions (producing daughter ions containing one leucine or isoleucine site for subsequent fragmentation to determine the leucine or isoleucine position).

[0045] The generated daughter ions were fragmented by HCD (normalized collision energy NCE energy of 35) to produce imine ions with m / z 86.1, and then fragmented by CID mode (NCE energy of 31) to produce peak intensities of m / z 44±0.2 and m / z 69±0.2.

[0046] Mass spectrometry instrument parameters: The parameters for the first-stage high-resolution mass spectrometry full scan are consistent with those for the first-stage mass spectrometry in Example 1; the parameters for the second-stage ddMS... 2 Acquisition: The activation type was set to CID, and the rest were the same as the second-stage mass spectrometry detection parameters in Example 1; the third-stage ddMS... 3 and Level 4 ddMS 4 The collected parameters correspond to the second-level ddMS in the method of Example 1. 2 and Level 3 ddMS 3 .

[0047] The mass spectrometry detection results are shown in Figures 2 and 3.

[0048] Figure 3 Criteria for determining whether sites A, 3B, 3D, 3E, 3G, and 3H contain one leucine / isoleucine residue.

[0049] Figure 3 C, 3F, and 3I correspond to the criteria of sites containing two leucine and / or isoleucine (for oxytocin without disulfide bonds).

[0050] The method for determining the two leucine and / or isoleucine sites in oxytocin and its sequence isomers is based on: Figure 2As shown in C, oxytocin and its sequence isomers, after being fragmented and reduced by CID, can generate daughter ions b3 and y3, each containing one different site. Since the sequences corresponding to the b3 and y3 daughter ions contain only one leucine or isoleucine site, the b3 and y3 corresponding to each of the reduced oxytocin and its sequence isomers were detected according to the method in Example 1. The results are as follows. Figure 3 As shown in A, 3B, 3D, 3E, 3G, and 3H. For oxytocin, further HCD-CID fragmentation of the b3 and y3 ions revealed the multi-stage fragmentation mass spectrum corresponding to b3 (…). Figure 3 In A), m / z 69±0.2 is much larger than m / z 44±0.2, while its y3 corresponds to a multi-stage fragmented mass spectrum ( Figure 3 B) The m / z value of 44±0.2 is much greater than that of 69±0.2, therefore it can be clearly determined that the polypeptide sequence corresponding to b3 in oxytocin contains isoleucine, while the polypeptide sequence corresponding to y3 contains leucine. The same method can be used to quickly determine the two leucine and isoleucine sites within the molecular sequence of the two sequence isomers. Figure 3 D and Figure 3 E indicates that the polypeptide sequences corresponding to b3 and y3 in sequence isomer 1 both contain isoleucine; from Figure 3 G and Figure 3 H indicates that the polypeptide sequences corresponding to b3 and y3 in sequence isomer 2 both contain leucine. Based on this conclusion, oxytocin and its sequence isomers were directly fragmented and reduced using HCD to generate imine ions, such as... Figure 2 As shown in D, the generated imine ions undergo further CID fragmentation. This provides a direct basis for determining whether the composition contains two leucine and / or isoleucine sites.

[0051] Oxytocin and its sequence isomers were subjected to primary mass spectrometry (PMS) to obtain the molecular weight of the model peptide. Then, the model peptide was subjected to secondary mass spectrometry (PMS). The precursor ion of the peptide was fragmented in HCD mode (normalized collision energy NCE energy of 35) to generate an imine ion with m / z 86.1. Subsequently, the imine ion was fragmented in CID mode (normalized collision energy NCE energy of 31). The molecular weights of the model peptides were then determined. Figure 3 From the figures C, 3F, and 3I, it can be seen that for the two leucine and isoleucine sites, when m / z 44±0.2 An m / z value of 69±0.2 indicates that the precursor or daughter ion of the polypeptide sequence contains two leucine residues; when m / z is 69±0.2... If m / z 44±0.2 is less than 18% of m / z 69±0.2, it indicates that the precursor or daughter ion of the polypeptide sequence contains two isoleucines. If m / z 69±0.2 > m / z 44±0.2, and m / z 44±0.2 is more than 25% of m / z 69±0.2, it indicates that the precursor or daughter ion of the polypeptide sequence contains one isoleucine and one leucine.

[0052] “ " " indicates much greater than, and ">" indicates greater than.

[0053] Example 3

[0054] Taking the peptide drug teriparatide as an example for detection

[0055] Taking teriparatide, a polypeptide drug containing six leucine and isoleucine sites, and its two sequence isomers as examples, the sequence of teriparatide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ ID NO.3), the sequence of isomer 1 is SVSEIQLMHNIGKHLNSMERVEWLRKKLQDVHNF (SEQ ID NO.4), and the sequence of isomer 2 is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ ID NO.5). The isomer positions of the two isomers are the third and first positions of the first three leucine or isoleucine sites, respectively, to simulate the isomer impurities produced by leucine or isoleucine isomerization commonly seen in the synthesis of polypeptide drugs.

[0056] Because its amino acid sequence contains more than 4 leucine and isoleucine sites, it is necessary to first perform enzymatic digestion to obtain smaller peptides, and then analyze each peptide individually. First, teriparatide was prepared by dissolving it in 50 mM Tris-HCl solution to a concentration of approximately 100 μg / g. Teriparatide was then double-digested with trypsin and lysine protease Lys-C at an enzyme:peptide ratio of 1:25 at 37°C for 3 hours. The resulting peptide fragments and liquid chromatography separation diagram are shown below. Figure 4 As shown.

[0057] Figure 4The results showed that the four cleaved peptides obtained by double enzyme digestion were SK13 (amino acid sequence SVSEIQLMHNLGK (SEQ ID NO.6) or SVSEIQLMHNIGK (SEQ ID NO.7) or SVSELQLMHNLGK (SEQ ID NO.8)), HR7 (amino acid sequence HLNSMER (SEQ ID NO.9)), VR5 (amino acid sequence VEWLR (SEQ ID NO.10)), and LF7 (amino acid sequence LQDVHNF (SEQ ID NO.11)).

[0058] Since HR7, VR5, and LF7 contain only one leucine or isoleucine site in their sequences, each peptide was detected according to the method in Example 1. The results are as follows: Figure 5 As shown. Figure 5 The results showed that the imine ions generated by the three peptides HR7, VR5, and LF7 fragmented at m / z 44±0.2 in the m / z 40–90 range. The m / z value is 69±0.2, therefore all three peptide sequences contain leucine.

[0059] The SK13 fraction was further enriched and digested with Glu-C enzyme to obtain XK9, XK9-1, and XK9-2 peptides, where X represents undetermined leucine or isoleucine (amino acid sequences IQLMHNLGK (SEQ ID NO.12), IQLMHNIGK (SEQ ID NO.13), and LQLMHNLGK (SEQ ID NO.14), respectively). Each peptide was added to a 50 mM ammonium bicarbonate aqueous solution and digested at 37°C for 2 h at an enzyme-to-peptide mass ratio of 1:25. The obtained XK9 peptide contained three undetected sites, which can be considered as a combination of cases containing only one leucine or isoleucine site and cases containing two leucine or isoleucine sites. First, a second-order mass spectrum is obtained through CID second-order fragmentation (normalized collision energy NCE energy is 32). The three sites are then fragmented to produce a b2 daughter ion containing only one left-hand position, a y7 daughter ion containing both the middle and right-hand positions, and a y4 daughter ion containing the right-hand position. Figure 6 As shown in B.

[0060] These daughter ions were further fragmented by HCD (normalized collision energy NCE energy of 35) to generate imine ions with m / z 86.1. Finally, the imine ions were fragmented by CID (normalized collision energy NCE energy of 31) to generate characteristic daughter ions. The types and positions of amino acids were determined according to the identification methods of Examples 1 and 2. The results are as follows. Figure 7 As shown.

[0061] Figure 7Images A, 7B, and 7C show that for the XK9 peptide (generated by teriparatide digestion), according to the criteria in Example 1, the m / z of the sequence corresponding to b2 after fragmentation is 69 ± 0.2. m / z 44±0.2 (7A), identified as isoleucine; according to the judgment criteria in Example 2, the m / z of the sequence corresponding to y7 after fragmentation is 44±0.2. The m / z value is 69±0.2 (7B), and both corresponding sites are leucine residues; according to the criteria in Example 1, the m / z value of the sequence corresponding to y4 after fragmentation is 44±0.2. m / z 69±0.2 (7C). Based on the judgment, the rightmost position is leucine, so the middle position is also leucine.

[0062] For the XK9-1 peptide (generated by digestion of sequence isoform 1), based on the same criteria, the m / z of the sequence corresponding to b2 after fragmentation is 69±0.2. m / z 44±0.2 (7D) corresponds to isoleucine; the fragmented sequence of y7 produces m / z 69±0.2 > m / z 44±0.2 (7E), with the relative peak intensity of m / z 44±0.2 being more than 25% of that of m / z 69±0.2. The two corresponding sites are leucine and isoleucine; the fragmented sequence of y4 produces m / z 69±0.2. m / z 44±0.2 (7F). Based on the judgment, the rightmost position is isoleucine, therefore the middle position is leucine.

[0063] For the XK9-2 peptide (generated by enzyme digestion of sequence isoform 2), based on the same criteria, the m / z of the sequence corresponding to b2 after fragmentation is 44±0.2. m / z 69±0.2 (7G), representing leucine; the m / z of the y7 sequence after fragmentation is 44±0.2. m / z 69±0.2 (7H), corresponding to two leucine residues; the m / z of the sequence corresponding to y4 after fragmentation is 44±0.2. m / z 69±0.2 (7I), based on the judgment, the corresponding rightmost position is leucine, therefore the middle position is leucine.

[0064] The mass spectrometry parameters were set according to those in Example 2.

[0065] In summary, the six leucine and six isoleucine sites in teriparatide and its sequence isomers were characterized completely and accurately.

[0066] Example 4

[0067] Taking antimicrobial peptides-surfactants as an example for detection

[0068] Take surfactants, an antimicrobial peptide containing 3-4 leucine and isoleucine sites, and its two homologues as examples. Surfactants are a family of cyclic lipopeptides synthesized by Bacillus subtilis as a secondary metabolite. They possess antibacterial, antifungal, and antiviral activities, while also exhibiting surfactant properties. The chemical structure of surfactants is as follows: Figure 8 As shown, its structure consists of an amphiphilic heptapeptide chain and a hydrophobic 13-16 carbon β-hydroxy fatty acid, which together form a cyclic lactone structure through amide and ester bonds. For the hydrophobic 13-16 carbon β-hydroxy fatty acid, the terminal methyl group can also exist in three isomeric forms. In the heptapeptide amino acid sequence of surfactant, positions 1 and 5 are fixed with L-glutamic acid and L-aspartic acid, respectively; positions 3 and 6 are fixed with D-leucine; and positions 2, 4, and 7 can be any one of L-leucine, L-isoleucine, or L-valine.

[0069] Taking the laboratory-customized isoC14-EXLVDLX (SEQ ID NO.15) and its homologues 1 isoC14-EVLVDLX (SEQ ID NO.16) and 2 isoC13-EXLVDLX (SEQ ID NO.17) as examples, where X represents undetermined leucine or isoleucine. Due to the unique structure of the surfactant lactone ring, its mass spectrometry fragmentation exhibits certain regularities. Without any pretreatment reactions, direct secondary mass spectrometry fragmentation of the three surfactant molecules using HCD / CID yields a series of b-ions confirming the heptapeptide amino acid sequence and internal fragmentation ions generated by multiple fragmentation, such as... Figure 9 As shown, both the primary mass spectrum and the subsequent CID secondary mass spectrum confirm the heptapeptide amino acid sequences of the three surfactants mentioned above. However, secondary mass spectrometry alone cannot confirm whether the amino acids at positions 2 and / or 7 are leucine or isoleucine. Therefore, multi-stage mass spectrometry analysis is required for the three surfactant molecules to fully characterize the heptapeptide amino acid sequences.

[0070] Since the number of leucine and isoleucine sites in its amino acid sequence is ≤4 and the fragmentation of its tandem mass spectrometry exhibits a certain regularity, no pretreatment steps such as enzyme digestion are required, and multi-stage tandem mass spectrometry analysis can be performed directly.

[0071] First, secondary mass spectrometry with relatively high intensity of internally fractured fragment ions was obtained through HCD secondary fragmentation (normalized collision energy NCE energy of 35). This fragmentation yielded a Y6b3 internally fractured ion containing an unidentified leucine / isoleucine site at position 2 and a Y2 daughter ion containing an unidentified leucine / isoleucine site at position 7. Figure 10As shown in A, 10B, and 10C.

[0072] These daughter ions were further fragmented by HCD (normalized collision energy NCE energy of 35) to generate imine ions with m / z 86.1. Finally, the imine ions were fragmented by CID (normalized collision energy NCE energy of 31) to generate characteristic daughter ions. The types and positions of the 2nd and 7th amino acids were determined according to the identification method in Example 2 and the characteristics of the surfactant molecule structure. The results are as follows: Figure 11 As shown.

[0073] Figure 11 As shown in A and 11B, for the surfactant isoC14-EXLVDLX, according to the judgment criteria in Example 2, the m / z values ​​of the fragmented sequences of the internal fractured ions of y6b3 and the corresponding y2 daughter ions after fragmentation are all 44±0.2. The m / z value is 69±0.2, and both corresponding sites in the sequence are leucine. Since the 3rd and 6th positions of the heptapeptide amino acid sequence of surfactant are fixed D-leucine, it is determined that the amino acids at positions 2 and 7 are L-leucine. In conclusion, the complete amino acid sequence of surfactant isoC14-EXLVDLX is confirmed to be isoC14-ELLVDLL.

[0074] For homologue 1 isoC14-EVLVDLX, according to the judgment criteria in Example 1, the m / z of the fragmented ion sequence within y6b3 after fragmentation is 44±0.2. The m / z value was 69 ± 0.2 (11C), confirming that the third amino acid in homologue 1 was indeed a fixed D-leucine. Based on the criteria in Example 2, the m / z value of the fragmented sequence corresponding to the y2 daughter ion was 44 ± 0.2. The m / z value is 69±0.2 (11D), and both positions in the corresponding sequence are leucine. Since the 6th amino acid position of the heptapeptide sequence of surfactant is a fixed D-leucine, the 7th amino acid is determined to be L-leucine. In summary, the complete amino acid sequence of homologue 1 isoC14-EVLVDLX is confirmed to be isoC14-EVLVDLL.

[0075] Figure 11E and 11F show that, for homologue 2 isoC13-EXLVDLX, according to the judgment criteria in Example 2, the m / z 69±0.2 generated after the fragmentation of the sequences corresponding to the internal fracture ion of y6b3 and the y2 daughter ion is greater than m / z 44±0.2, and the relative peak intensity of m / z 44±0.2 is greater than 25% of that of m / z 69±0.2. The corresponding sequences contain one leucine and one isoleucine at each of the two sites. Since the 3rd and 6th positions of the heptapeptide amino acid sequence of surfactant are fixed D-leucines, it is determined that the amino acids at positions 2 and 7 are both L-isoleucines. In conclusion, the complete amino acid sequence of homologue 2 isoC13-EXLVDLX is confirmed to be isoC13-EILVDLI.

[0076] Mass spectrometry parameters except for the second-stage ddMS 2 During acquisition, the activation type was set to HCD, the normalized collision energy was set to 35, and the rest were set according to the mass spectrometry parameters in Example 2.

[0077] In summary, the 3-4 leucine and isoleucine sites in the antimicrobial peptide surfactant and its homologues were characterized completely and accurately.

[0078] The method of this invention can clearly characterize the position and type of leucine and isoleucine in a polypeptide sequence.

[0079] As demonstrated in the above examples, the fragmentation of the HCD / CID used is not dependent on the charge number, resulting in higher fragmentation efficiency and easier acquisition of characterization sites containing leucine or isoleucine. Furthermore, by using the relative intensities of m / z 44±0.2 and m / z 69±0.2, the multi-stage mass spectrometry technique based on HCD / CID was successfully applied to the characterization of peptides containing multiple leucine and isoleucine sites. Compared to previous methods, this approach offers higher confidence levels, and the combination of enzyme digestion and multi-stage mass spectrometry fragmentation can rapidly and accurately identify all leucine and isoleucine sites in any peptide. This provides a basis for the rapid and accurate identification of leucine and isoleucine.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapidly distinguishing and determining leucine and isoleucine in a polypeptide sequence based on tandem multi-stage high-resolution mass spectrometry, characterized in that, Includes the following steps: (1) The precursor or daughter ions of the polypeptide are fragmented by HCD to generate imine ions related to leucine and / or isoleucine; (2) The imine ions were subjected to CID fragmentation, and the relative peak intensities of m / z 44±0.2 and m / z 69±0.2 generated by fragmentation were detected in the mass axis range of m / z 40~90; (3) Based on the relationship between the relative peak intensities of m / z 44±0.2 and m / z 69±0.2, determine the types of leucine and isoleucine in the polypeptide.

2. The method according to claim 1, characterized in that, The polypeptide in step (1) contains ≤2 sites of leucine and isoleucine in its parent ion.

3. The method according to claim 1, characterized in that, In step (1), the number of leucine and isoleucine sites in the daughter ion is ≤2; When the parent ion of a polypeptide contains more than 2 but less than or equal to 4 sites of leucine and isoleucine, the parent ion of the polypeptide is first fragmented by CID or HCD to generate daughter ions, so that the number of leucine and isoleucine sites in the daughter ions is ≤2.

4. The method according to claim 1, characterized in that, When the precursor ion of a polypeptide contains more than 4 sites containing leucine and isoleucine, the precursor ion of the polypeptide is first enzymatically digested or fragmented until each peptide segment contains less than or equal to 4 sites containing leucine and isoleucine, and then detected according to the method described in any one of claims 1 to 3.

5. The method according to claim 1, characterized in that, The normalized collision energy during the HCD fragmentation in step (1) is 34~36.

6. The method according to claim 1, characterized in that, The normalized collision energy at the time of CID fragmentation in step (2) is 30~32.

7. The method according to claim 1, characterized in that, When the parent or daughter ion of a polypeptide contains one leucine or isoleucine site: The relative peak intensity at m / z 44±0.2 is much greater than that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide is leucine. The relative peak intensity at m / z 69±0.2 is much greater than that at m / z 44±0.2, indicating that the parent or daughter ion of the polypeptide is isoleucine.

8. The method according to claim 1, characterized in that, When the parent or daughter ion of a polypeptide contains two leucine and / or isoleucine sites: The relative peak intensity at m / z 44±0.2 is much greater than that at m / z 69±0.2, indicating that the parent ion or daughter ion of the polypeptide contains two leucines. The relative peak intensity at m / z 69±0.2 is much greater than that at m / z 44±0.2, and the relative peak intensity at m / z 44±0.2 is less than 18% of that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide contains two isoleucines. The relative peak intensity at m / z 69±0.2 is greater than that at m / z 44±0.2, and the relative peak intensity at m / z 44±0.2 is more than 25% of that at m / z 69±0.2, indicating that the parent or daughter ion of the polypeptide contains one isoleucine and one leucine.

9. The method according to claim 1, characterized in that, The polypeptide is a polypeptide drug; the polypeptide drug is leucine enkephalin and its isomers, teriparatide and its isomers, oxytocin and its isomers, antimicrobial peptide surfactant and its homologues.

10. The use of the method according to any one of claims 1 to 8 in the preparation of products for rapidly and accurately determining leucine and isoleucine in a polypeptide sequence.

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