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CN-121978195-A - Method for rapidly distinguishing and determining leucine and isoleucine in polypeptide sequence based on tandem multi-stage high-resolution mass spectrometry

CN121978195ACN 121978195 ACN121978195 ACN 121978195ACN-121978195-A

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 steps of (1) carrying out HCD fragmentation on parent ions or child ions of polypeptides to generate imine ions related to leucine and/or isoleucine, (2) carrying out CID fragmentation on the imine ions, detecting relative peak intensities of m/z 44+/-0.2 and m/z 69+/-0.2 generated by fragmentation within a mass axis range of m/z 40-90, and (3) judging the types of leucine and isoleucine in the polypeptides according to the relative peak intensity relation of m/z 44+/-0.2 and m/z 69+/-0.2. The method has higher fragmentation efficiency and confidence level, and provides a basis for rapid and accurate identification of leucine and isoleucine.

Inventors

  • WU PEIZE
  • LI MING

Assignees

  • 中国计量科学研究院

Dates

Publication Date
20260505
Application Date
20260323

Claims (10)

  1. 1. A method for rapidly distinguishing and determining leucine and isoleucine in a polypeptide sequence based on tandem multi-stage high-resolution mass spectrometry, which is characterized by comprising the following steps: (1) Subjecting parent or daughter ions of the polypeptide to HCD fragmentation to produce imine ions associated with leucine and/or isoleucine; (2) CID fragmentation is carried out on the imine ions, and relative peak intensities of m/z 44+/-0.2 and m/z 69+/-0.2 generated by fragmentation are detected within a mass axis range of m/z 40-90; (3) The types of leucine and isoleucine in the polypeptide are judged according to the relative peak intensity relation of m/z 44+/-0.2 and m/z 69+/-0.2.
  2. 2. The method of claim 1, wherein the parent ion of the polypeptide of step (1) comprises leucine and isoleucine at a position of 2 or less.
  3. 3. The method of claim 1, wherein the positions of leucine and isoleucine in the ion of step (1) are 2 or less; When the sites of leucine and isoleucine in the parent ion of the polypeptide are more than 2 and less than or equal to 4, CID or HCD fragmentation is carried out on the parent ion of the polypeptide to generate child ions, so that the sites of leucine and isoleucine in the child ions are less than or equal to 2.
  4. 4. The method according to claim 1, wherein when the site containing leucine and isoleucine in the parent ion of the polypeptide is greater than 4, the parent ion of the polypeptide is first subjected to cleavage or fragmentation, and the cleavage or fragmentation is performed until the site containing leucine and isoleucine in each peptide fragment is equal to or less than 4, and the detection is performed according to the method of any one of claims 1 to 3.
  5. 5. The method of claim 1, wherein the normalized collision energy at the time of HCD fragmentation of step (1) is 34-36.
  6. 6. The method of claim 1, wherein the normalized collision energy at CID fragmentation in step (2) is 30-32.
  7. 7. The method of claim 1, wherein when the parent or child ion of the polypeptide contains 1 leucine or isoleucine site: The relative peak intensity of m/z 44+/-0.2 is far greater than that of m/z 69+/-0.2, which indicates that leucine is in parent ion or child ion of the polypeptide; The relative peak Jiang Yuan for m/z 69+ -0.2 was greater than the relative peak intensity for m/z 44+ -0.2, indicating that isoleucine was in the parent or daughter ion of the polypeptide.
  8. 8. The method of claim 1, wherein the step of determining the position of the substrate comprises, When the parent or child ion of the polypeptide contains 2 leucine and/or isoleucine sites: the relative peak intensity of m/z 44+/-0.2 is far greater than that of m/z 69+/-0.2, which indicates that 2 leucine are contained in the parent ion or the child ion of the polypeptide; The relative peak Jiang Yuan of m/z 69+ -0.2 is greater than the relative peak intensity of m/z 44+ -0.2, and the relative peak intensity of m/z 44+ -0.2 is less than 18% of the relative peak intensity of m/z 69+ -0.2, indicating that 2 isoleucine are contained in the parent or child ion of the polypeptide; The relative peak intensity of m/z 69+ -0.2 is greater than that of m/z 44+ -0.2, and the relative peak intensity of m/z 44+ -0.2 is more than 25% of that of m/z 69+ -0.2, which indicates that the parent ion or the child ion of the polypeptide contains 1 isoleucine and 1 leucine.
  9. 9. The method of claim 1, wherein the polypeptide is a polypeptide drug, and wherein the polypeptide drug is leucine enkephalin and its isomers, teriparatide and its isomers, oxytocin and its isomers, antimicrobial peptide surfactants and its homologs.
  10. 10. Use of the method of any one of claims 1-8 for the preparation of a product for rapid and accurate determination of leucine and isoleucine in a polypeptide sequence.

Description

Method for rapidly distinguishing and determining leucine and isoleucine in polypeptide sequence based on tandem multi-stage high-resolution mass spectrometry Technical Field 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. Background Leucine and isoleucine are isomers of each other, and their mass spectral peaks in mass spectra are identical, resulting in both being harder to resolve. When leucine sites are present in the polypeptide amino acid sequence, substitution of leucine for isoleucine at the same site often results in altered safety and efficacy of the drug and vice versa. Thus, the distinction between leucine and isoleucine sites is important, whether it is sequencing of the newly discovered polypeptide in the natural product or the distinction between drug isomer impurities. At present, two analysis techniques mainly exist for distinguishing leucine from isoleucine in a polypeptide sequence, one is to perform amino acid sequencing by an amino acid sequencer, and the principle is to sequentially degrade amino acids from the polypeptide sequence by chemical reaction in a Edman degradation mode, and sequentially detect the types of the degraded amino acids to distinguish. However, the method has more limitations, firstly, the sample amount required for amino acid sequencing is larger, the required time is longer, and high-throughput analysis is difficult to realize, secondly, edman degradation cannot sequence cyclized polypeptides and polypeptides modified at the N end of an amino acid sequence, and cyclizing and N-end modification in polypeptide drugs are more common drug modification means, so that when the polypeptide drugs with various structure types are faced, edman degradation cannot meet the requirement of sequencing the polypeptide drugs, and naturally, leucine and isoleucine sites in the sequence cannot be distinguished. The second is to use characteristic ions generated by multistage tandem mass spectrometry to distinguish leucine and isoleucine sites in a polypeptide sequence, in the analysis technology, firstly, the number of sites needing to distinguish leucine from isoleucine in the polypeptide sequence to be detected needs to be judged, if only 1 site needing to distinguish leucine from isoleucine exists in the polypeptide sequence to be detected, HCD (High-energy collision dissociation) is utilized to crack polypeptide parent ions containing leucine and isoleucine sites, secondary fragment ion-imine ions with m/z of 86.1 related to leucine or isoleucine are generated, and then HCD-MS 3 is further carried out on the imine ions. If three-stage fragment ions with m/z of 69+/-0.2 are generated, imine ions with m/z of 86.1 are generated specifically for isoleucine, so that polypeptide parent ion sequences only contain isoleucine, if three-stage fragment ions with m/z of 69+/-0.2 cannot be generated, imine ions with m/z of 86.1 are generated specifically for leucine, so that polypeptide parent ion sequences only contain leucine, differentiation is carried out according to the fact, if a plurality of sites needing to differentiate leucine from isoleucine exist in polypeptide sequences to be detected, it is required to determine whether the amino acid of the z ion is isoleucine by utilizing the number of mass loss displacement between the z ion and w ion generated by the z ion, such as ETD (electron transfer dissociation Electron transfer dissociation), HECD (hot electron capture dissociation hot electron capture dissociation) or EThcD (electron transfer high energy collision dissociation electron-transfer/high-energy collision dissociation), z ion taking leucine or isoleucine site as a terminal group is generated, and then HCD-MS 3 is further carried out to generate w ion. The amino acid at the end of the z ion is leucine when the mass loss displacement between the z ion and the w ion generated by the z ion is 43 Da, and isoleucine when the mass loss displacement between the z ion and the w ion generated by the z ion is 29 Da. There are great limitations to analytical techniques for distinguishing between leucine and isoleucine sites in polypeptide sequences using characteristic ions generated by multistage tandem mass spectrometry. First, for the presence of 1 site discrimination for leucine and isoleucine, whether or not three-stage fragment ions having an m/z of 69 could be generated was observed by fragmenting an imine ion having an m/z of 86.1 by HCD MS 3, and whether leucine or isoleucine was present in the sequence was determined. The literature studies and experimental analysis show that both amino acids can generate mass spectrum peaks at m/z 69+/-0.2, so that leucine or isoleucine in a polypeptide sequence cannot be effectively distinguished. Secondly, for a polypeptide having a pl