Reagent and method for identifying and distinguishing D-amino acid oligopeptide-containing isomer

Abstract

The invention discloses a reagent and method applied to recognition and distinguishing of D-amino acid-containing oligopeptide isomers, and belongs to the field of biological detection.The reagent comprises oligopeptide with the concentration of 10 <-1 >-10 <-9 > mol / L, oligopeptide diastereomer containing D-amino acid, cucurbituril with the concentration of 10 <-1 >-10 <-9 > mol / L and metal salt with the concentration of 10 <-1 >-10 <-9 > mol / L. Preparing a 10 <-5 > mol / L ternary complex ionic solution from oligopeptide, an oligopeptide diastereomer containing D-amino acid, cucurbituril and metal salt according to a volume ratio of 1: 1: 1; according to the peptide-cucurbituril-metal ion ternary complex prepared by the preparation method disclosed by the invention, the D-amino acid oligopeptide-containing isomer can be simply, conveniently and quickly identified and distinguished without complex chromatographic pre-separation or chemical derivatization.

Description

Technical Field

[0001] A reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids and a preparation method thereof. Background Art

[0002] Almost all kinds of proteins in organisms are composed of L-amino acids, and D-amino acids were initially thought to be non-existent. It was not until the early 1980s that biologically active D-amino acid oligopeptides were discovered in opioid peptides secreted from the skin of South American tree frogs. Since then, D-amino acid residues of different properties and positions have been found in the venom or nerve tissues of various mollusks, crustaceans and vertebrates. More surprisingly, D-amino acids have been detected in free form in various human bodies, and D-amino acids have been detected from some peptides and proteins. In particular, different D-amino acid residues have been detected from various aged protein tissues (including teeth, eyes, skin and brain), and significant levels of D-amino acid residues have also been found in tissue proteins related to diseases such as cataracts and Alzheimer's disease. For example, isomerization of aspartic acid at specific positions in amyloid proteins may lead to abnormal aggregation or decomposition. It is generally believed that the origins of these D-amino acid-containing polypeptides may include isomerase-mediated post-translational modification, aging process, oxidative stress or disease-related non-enzymatic racemization, and abnormal metabolism of D-amino acids.

[0003] The chiral transformation of amino acid residues from L to D configuration will significantly change the structure of related peptides and proteins and the binding of adaptive receptors. For example, some endogenous neuropeptides D-amino acid oligopeptides may show better biological activity and significantly enhanced receptor binding affinity, while L-type may show lower performance or even completely lack activity, even if both forms of peptides are present in the same ganglion. Other studies have shown that the selectivity between different receptors can be regulated by L to D residue isomerization in endogenous neuropeptides, that is, different diastereoisomers preferentially activate different receptor targets. Some studies have shown that the isomerization modification of D-amino acid peptides makes the peptides more difficult to be degraded by endogenous peptidases. Since the isomerization rate of the d-form of the substrate peptide is slower than that of the l-isomer, the stability of the D-amino acid peptide is increased. Therefore, the D-amino acid peptides in some biological venoms allow them to stay longer in the victim to increase pain. In addition, some D-amino acid peptides show lower physiological toxicity levels, while other diastereoisomers with amino acid residues reversed to the D conformation may show the opposite trend. These phenomena indicate that increasing the recognition and differentiation of D-amino acid oligopeptide isomers affected by amino acid residue isomerization can help us reveal the mechanisms of related physiological or pathological processes, such as selective activation of receptors in cell signaling pathways, targets of disease conditions, etc., and help discover potential therapeutic interventions or develop specific drugs.

[0004] However, in mass spectrometry-based proteomics research, the identification and recognition of D-amino acid oligopeptides has always been and remains a challenging task, because the LD isomerization of amino acids does not cause any changes in the physicochemical properties of the molecules, such as isoelectric point, sequence or mass-to-charge ratio. At present, the analysis and characterization techniques for isomeric peptides such as D-amino acid oligopeptides include: Liquid Chromatography (LC), Capillary Electrophoresis (CE), Mass Spectrometry (MS) and Ion Mobility Spectrometry Mass Spectrometry (IMS MS). Among them, the combination of ion mobility spectrometry and mass spectrometry (IMSMS) as a fast and efficient powerful isomer analysis platform provides an additional structural analysis dimension-Collision Cross Section (CCS), which can then resolve isomeric species that cannot be distinguished by traditional mass spectrometry. Ion Mobility Spectrometry (IMS) is a technology that uses electric fields to drive the differences in the migration behaviors of gas-phase ions in neutral gases to achieve gas-phase ion separation and identification. The mobility differences caused by the collision cross sections of different ions are distributed in positions with different electric field strengths. The selective scanning or release of ions can be achieved by adjusting the electric field distribution. Therefore, for D-amino acid oligopeptides that have different collision cross-section values ​​and mobilities from L-amino acid-containing peptides, IMS can be used to identify isomers and accurately locate D-amino acid residues. However, due to the limited resolution of IMS, direct analysis based on ion mobility spectrometry technology still faces challenges for biological molecules such as oligopeptides and their D-amino acid oligopeptide isomers that have very small structural differences.

[0005] Cucurbituril (CB[n] for short, n=5-8, 10 and 14), also known as cucurbituril, is the fourth generation of macrocyclic molecules discovered after calixarene. It is a macrocyclic compound obtained by condensation reaction of glycoluril and methylene under acidic conditions. It is hydrophobic inside and hydrophilic outside, with a large cavity in the middle and small at both ends. It has a high degree of symmetry and is shaped like a gourd, with open cavities at both ends and carbonyl groups on the upper and lower edges. These structural features enable it to bond to a variety of inorganic and organic cations through hydrophobic interaction, hydrogen bonding, ion-dipole bonding, etc., and form metal complexes through coordination with metals. It has a strong bonding affinity, and the bonding stability constant can even reach more than 10 to the 10th power. Cucurbituril has the characteristics of high rigidity, strong thermal stability and highly specific host-guest bonding ability, so it can identify metal ions, organic molecules, neutral molecules, etc. through supramolecular action. However, cucurbituril has large steric hindrance and it is difficult to introduce new functional groups, which also limits the development of cucurbituril. The most important property of cucurbituril is that its ports and cavities have negative electrostatic potential. These carbonyl groups are excellent donors for densely charged metal cations; at the same time, the high polarity of the carbonyl bond is also beneficial to bonding; and for densely charged metal cations, it itself is an excellent acceptor. Therefore, cucurbituril has the ability to selectively include and coordinate a variety of organic cations and metal ions. Secondly, the ring wall of cucurbituril contains 4 times the number of N in its structural units. The cavity formed by them is hydrophobic and can include organic molecules, gas molecules and other small guest molecules. Thirdly, its carbonyl O and N can form hydrogen bonds and interact with the existing metal ions to form self-assembled molecular capsules or host-guest entities.

[0006] The present invention solves such a problem by preparing a peptide-cucurbituril-metal ion ternary complex. Summary of the invention

[0007] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids and a method for preparing the same. The prepared peptide-cucurbituril-metal ion ternary complex can identify and distinguish isomers of oligopeptides containing D-amino acids simply and quickly without the need for complex chromatographic pre-separation or chemical derivatization.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids, comprising: a concentration of 10 -1 -10 - 9 mol / L oligopeptide and its diastereomer containing D-amino acid, the concentration is 10 -1 -10 -9 mol / L cucurbituril and concentration of 10-1 -10 -9 10 mol / L metal salt; oligopeptide and oligopeptide diastereomer containing D-amino acid, cucurbituril and metal salt are prepared in a volume ratio of 1:1:1. -5 mol / L ternary complex ion solution.

[0010] In the aforementioned reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids, the cucurbituril is one or more of cucurbituril[6], cucurbituril[7], cucurbituril[8]; and the metal salt includes: Li salt, Na salt, K salt, Rb salt, Cs salt, Mg salt, Ca salt, Sr salt and Ba salt.

[0011] The aforementioned reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids further comprises: a solvent with a concentration of 0.1% to 99.9%.

[0012] Further preferably, the solvent of the aforementioned reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids includes one or a mixture of methanol, ethanol, acetonitrile, ether or acetone. The choice and concentration of the solvent are not limited, as long as the oligopeptide to be analyzed, the diastereomer of the oligopeptide containing D-amino acids and the cucurbituril can be dissolved, it is suitable for the present invention.

[0013] Further preferably, a reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids comprises: an oligopeptide and its diastereomers containing D-amino acids at a concentration of 10-4 -10-6, a cucurbituril at a concentration of 10-4 -10-6 mol / L, and a metal salt at a concentration of 10-4 -10-6 mol / L; the oligopeptide and its diastereomers containing D-amino acids, cucurbituril and the metal salt are prepared into a 10-5 mol / L ternary complex ion solution in a volume ratio of 1:1:1.

[0014] Further preferably, a reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids, wherein the oligopeptide is ME-1: YGGFM, and the diastereomer of the oligopeptide containing D-amino acids is ME-2: D YGGFM, ME-3: YGG D FM and ME-4: YGGF D M, cucurbituril is cucurbituril[8](CB[8]), metal salt is Li salt, and ternary complex ion is [ME+CB[8]+Li+H] 2+ .

[0015] A method for identifying and distinguishing isomers of oligopeptides containing D-amino acids, comprising the steps of:

[0016] S1, mixing the oligopeptide and its D-amino acid-containing oligopeptide diastereomer at the concentration described in the claim, cucurbituril and metal salt in a volume ratio of 1:1:1 to prepare a ternary non-covalent complex solution of peptide-cucurbituril-metal ion;

[0017] S2, using an ionization method capable of producing gas-phase ions, generating gas-phase ions from a ternary non-covalent complex solution of a peptide-cucurbituril-metal ion;

[0018] S3, using an ion mobility spectrometry experimental device to measure the ion mobility and collision cross-section area of ​​the positive ion of the ternary non-covalent complex containing peptide-cucurbituril-metal ion, and the structural information of the diastereomer can be obtained based on the measurement results of the ion mobility spectrometry.

[0019] The aforementioned method for identifying and distinguishing isomers of oligopeptides containing D-amino acids can generate an ionization method for gas phase ions, including: an electrospray ionization ion source, a laser-assisted desorption ionization ion source, and a desorption electrospray ionization ion source.

[0020] The aforementioned method for identifying and distinguishing isomers of oligopeptides containing D-amino acids, wherein the experimental device for ion mobility spectrometry is an ion mobility spectrometry-mass spectrometry instrument, and the experimental device for ion mobility spectrometry has an electrospray ion source containing a positive ion or negative ion mode.

[0021] Further preferred, a method for identifying and distinguishing isomers of oligopeptides containing D-amino acids, the instrument parameters of the ion mobility spectrometry-mass spectrometry instrument are electrospray ion source; positive ion mode; sample injection flow rate: 3 μL / min; electrospray voltage: 3600 V; nebulizing gas: 0.3 bar; drying gas flow rate: 3.0 L / min; drying gas temperature: 200°C.

[0022] The present invention is beneficial in that:

[0023] The present invention provides a reagent and method for identifying and distinguishing isomers of oligopeptides containing D-amino acids, by simply mixing an oligopeptide and its diastereomer containing D-amino acids, cucurbituril and a metal salt to form a mixed aqueous solution, then using an electrospray ionization ion source to generate a ternary non-covalent complex ion of peptide-cucurbituril-metal ion, and then using ion mobility spectrometry to obtain its mobility and collision cross-sectional area, thereby achieving identification of oligopeptide isomers and identification of the position of D-amino acid residues;

[0024] Furthermore, if the sample contains multiple different oligopeptide isomers at the same time, the method provided by the present invention can also obtain their relative contents.

[0025] The method for identifying and distinguishing isomers of oligopeptides containing D-amino acids provided by the present invention is simple and quick, and does not require complicated chromatographic pre-separation or chemical derivatization. The method can also be used for identifying and distinguishing isomers of other oligopeptides containing D-amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The model oligopeptide of the present invention (ME-1: YGGFM) and its corresponding three oligopeptide isomers containing D-amino acids (ME-2: D YGGFM;ME-3:YGG D FM; ME-4: YGGF D M) chemical structure diagram;

[0027] Figure 2 Schematic diagram of the chemical structure of cucurbituril of the present invention;

[0028] Figure 3 The mass spectra (a) and the corresponding ion mobility diagram (b) of the comparative experimental oligopeptide and its D-amino acid oligopeptide isomers of the present invention;

[0029] Figure 4 The mass spectra (a) and (b) of the oligopeptide of the present invention and its oligopeptide diastereomer containing D-amino acid, cucurbituril and metal salt mixed solution and the corresponding ion mobility diagrams (c) and (d);

[0030] Figure 5 ion mobility diagrams of neurotropic oligopeptides and diastereomeric complexes of oligopeptides containing D-amino acids according to some embodiments of the present invention (a: GDLFD; b: WKYMVDM; c: YDRFG). DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] A method for identifying and distinguishing isomers of oligopeptides containing D-amino acids, comprising the following steps:

[0033] S1, the concentration is 10 -1 -10 -9 mol / L oligopeptide and its diastereomer containing D-amino acid, the concentration is 10 -1 -10 -9 mol / L cucurbituril and concentration of 10 -1 -10 -9 10 mol / L of metal salt; the oligopeptide and its oligopeptide diastereomer containing D-amino acid, cucurbituril and metal salt are prepared in a volume ratio of 1:1:1 -5 mol / L ternary complex ion solution;

[0034] S2, using an ionization method that can produce gas-phase ions to produce gas-phase ions from a ternary non-covalent complex solution of a peptide-cucurbituril-metal ion; the ion source is an electrospray ionization ion source (ESI), a laser-assisted desorption ionization ion source (MALDI), a desorption electrospray ionization ion source (DEI) or other ternary non-covalent complex positive ions that can produce peptide-cucurbituril-metal ions.

[0035] S3, using an ion mobility spectrometry experimental device to measure the ion mobility and collision cross-section of the positive ions of the ternary non-covalent complex containing peptide-cucurbituril-metal ion, the structural information of the diastereomer can be obtained based on the measurement results of the ion mobility spectrometry. The experimental device of the ion mobility spectrometry is an ion mobility spectrometry or a composite experimental device containing an ion mobility spectrometry such as an ion mobility spectrometry-mass spectrometry instrument. For TIMS MS calibration, the sample solution is tested by manual injection. Instrument parameters: electrospray ion source; positive ion mode; sample injection flow rate: 3μL / min; electrospray voltage: 3600V; nebulizer gas: 0.3bar; drying gas flow rate: 3.0L / min; drying gas temperature: 200℃; and the test results are analyzed.

[0036] As a preferred embodiment, the oligopeptide and its diastereomer containing D-amino acids are a model oligopeptide (ME-1: YGGFM) and its corresponding three oligopeptide isomers containing D-amino acids (ME-2: DYGGFM; ME-3: YGGDFM; ME-4: YGGFDM), and their chemical structures are shown in the following figure. Figure 1 It should be noted that the oligopeptides that can be implemented in the present invention are not limited to the above-mentioned model oligopeptide ME-1 and its oligopeptide isomers containing D-amino acids, and can be extended to other oligopeptides and their oligopeptide diastereomers containing D-amino acids, such as Figure 5 The neurotropic oligopeptides and their D-amino acid containing oligopeptide diastereomers are shown.

[0037] The cucurbituril used is one of cucurbituril[6](CB[6]), cucurbituril[7](CB[7]), cucurbituril[8](CB[8]), and its chemical structure is shown in the schematic diagram. Figure 2 shown.

[0038] The metal salt used is any one containing metal ions Li + 、Na + , K + , Rb + , Cs +Mg +2 , Ca +2 , Sr +2 and Ba +2 Soluble salts such as one of LiCl, NaCl, KCl, RbCl, CsCl, MgCl2, CaCl2, SrCl2 and BaCl2, or LiNO3, NaNO3, KNO3, RbNO3, CsNO3, Mg(NO3)2, Ca(NO3)2, Sr(NO3)2 and Ba(NO3)2, etc.

[0039] The solvent used is a mixed solution containing water and one or more other liquids. Other solutions refer to other organic liquids other than water, including but not limited to methanol, ethanol, acetonitrile, ether, acetone and other organic liquid compounds. The proportion of each of the two or more liquid compounds is 0.1% to 99.9%. At the same time, it must be able to dissolve the oligopeptides to be analyzed, the diastereomers of the oligopeptides containing D-amino acids, and cucurbiturils. The selection and concentration of the above solvents are not limited, as long as they can dissolve the oligopeptides to be analyzed, the diastereomers of the oligopeptides containing D-amino acids, and cucurbiturils, they are applicable to the present invention.

[0040] In order to verify the technical effect of the present invention, the mass spectrometry and ion mobility spectrometry experiments of the model oligopeptide and its oligopeptide diastereomer containing D-amino acid were carried out using the ion mobility spectrometry-mass spectrometry instrument TIMS MS produced by Bruke Company. The results of the mass spectrometry and ion mobility spectrometry are shown in FIG. Figure 3 As shown in FIG. 1 , it can be seen that the four oligopeptide isomers could not be separated due to their same mass-to-charge ratio and similar collision cross-section. Subsequently, mass spectrometry and ion mobility spectrometry experiments of the peptide-cucurbituril-metal ion ternary non-covalent complex prepared in the above specific example were performed by IMS MS. The mass spectrometry and ion mobility spectrometry results are shown in FIG. Figure 4 As shown, it can be seen that the four oligopeptide isomers - cucurbituril [8] -Li + The complexes can be effectively separated from each other due to their different collision cross-sections. In addition, other neuropeptides and their diastereomers containing D-amino acids can be effectively separated by IMS MS after forming non-covalent complexes with cucurbituril and metal ions. The ion migration spectra are shown in Figure 5 shown.

[0041] In summary, the reagents and methods of the present invention are simple, quick, and can identify and distinguish isomers of oligopeptides containing D-amino acids without complicated chromatographic pre-separation or chemical derivatization.

[0042] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids, characterized in that: include: The concentration is 10 -1 -10 -9 mol / L oligopeptide and its diastereomer containing D-amino acid, the concentration is 10 -1 -10 -9 mol / L cucurbituril and a concentration of 10 -1 -10 -9 10 mol / L of metal salt; the oligopeptide and its oligopeptide diastereomer containing D-amino acid, cucurbituril and metal salt are prepared in a volume ratio of 1:1:1 -5 mol / L ternary complex ion solution.

2. A reagent for identifying and distinguishing isomers of oligopeptides containing D-amino acids according to claim 1, wherein the cucurbituril is one or more of cucurbituril [6], cucurbituril [7], and cucurbituril [8]; and the metal salt comprises: Li salt, Na salt, K salt, Rb salt, Cs salt, Mg salt, Ca salt, Sr salt and Ba salt.

3. A reagent for identifying and distinguishing isomers of D-amino acid oligopeptides according to claim 1, characterized in that: Also includes: Solvents available in concentrations from 0.1% to 99.9%.

4. A reagent for identifying and distinguishing isomers of D-amino acid oligopeptides according to claim 1, characterized in that: The solvent includes: one or a mixture of methanol, ethanol, acetonitrile, ether or acetone.

5. A reagent for identifying and distinguishing isomers of D-amino acid oligopeptides according to claim 1, characterized in that: include: The concentration is 10 -4 -10 -6 The oligopeptide and its diastereomer containing D-amino acid, the concentration is 10 -4 -10 - 6 mol / L cucurbituril and a concentration of 10 -4 -10 -6 10 mol / L of metal salt; the oligopeptide and its oligopeptide diastereomer containing D-amino acid, cucurbituril and metal salt are prepared in a volume ratio of 1:1:1 -5 mol / L ternary complex ion solution.

6. A reagent for identifying and distinguishing isomers of D-amino acid oligopeptides according to claim 1, characterized in that: The oligopeptide is ME-1:YGGFM, and the oligopeptide diastereomer containing D-amino acid is: ME-2: D YGGFM, ME-3: YGG D FM and ME-4: YGGF D M, the cucurbituril is cucurbituril[8](CB[8]), the metal salt is Li salt, and the ternary complex ion is [ME+CB[8]+Li+H] 2+ .

7. A method for identifying and distinguishing isomers of oligopeptides containing D-amino acids, characterized in that: Includes steps: S1, mixing the oligopeptide and its D-amino acid-containing oligopeptide diastereomer at the concentration described in the claim, cucurbituril and metal salt in a volume ratio of 1:1:1 to prepare a ternary non-covalent complex solution of peptide-cucurbituril-metal ion; S2, using an ionization method capable of producing gas-phase ions, generating gas-phase ions from a ternary non-covalent complex solution of a peptide-cucurbituril-metal ion; S3, using an ion mobility spectrometry experimental device to measure the ion mobility and collision cross-section area of ​​the positive ion of the ternary non-covalent complex containing peptide-cucurbituril-metal ion, and the structural information of the diastereomer can be obtained based on the measurement results of the ion mobility spectrometry.

8. A method for identifying and distinguishing isomers of oligopeptides containing D-amino acids according to claim 7, characterized in that: The ionization method capable of generating gas phase ions comprises: an electrospray ionization ion source, a laser-assisted desorption ionization ion source, and a desorption electrospray ionization ion source.

9. A method for identifying and distinguishing isomers of oligopeptides containing D-amino acids according to claim 7, characterized in that: The experimental device of the ion mobility spectrometry is an ion mobility spectrometry-mass spectrometry instrument, and the experimental device of the ion mobility spectrometry has an electrospray ion source containing a positive ion or negative ion mode.

10. A method for identifying and distinguishing isomers of oligopeptides containing D-amino acids according to claim 9, characterized in that: The instrument parameters of the ion mobility spectrometry-mass spectrometry instrument are: electrospray ion source; positive ion mode; sample injection flow rate: 3 μL / min; electrospray voltage: 3600V; Atomizing gas: 0.3 bar; Drying gas flow rate: 3.0 L / min; Drying gas temperature: 200°C.

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