Method for representing structure and effect of protein based on extreme ultraviolet laser dissociation and mass spectrometry

Abstract

The invention relates to an analysis method for characterizing structures and interaction of non-denatured proteins and compounds thereof based on pulse extreme ultraviolet laser dissociation and mass spectrometry detection. Protein interacts with large and small molecules such as metal ions, drug small molecules, protein, nucleic acid or polysaccharide to form a protein compound. Natural protein and protein compound samples in a biocompatible neutral solution enter a gas phase through a non-denaturing electrospray ionization technology, then radiation excitation is performed on the natural protein and protein compound samples through pulse extreme ultraviolet laser, dissociation is induced to generate protein structure characteristic fragment ions, and finally detection and spectrogram recording are performed on the fragment ions through mass spectrum. According to the invention, the technology for characterizing the structure and interaction of the non-denatured protein and the compound of the non-denatured protein by the extreme ultraviolet band laser is realized for the first time internationally, and the method has the remarkable advantages of high sensitivity, high flux, single amino acid site resolution and the like in the aspect of researching fine structures and conformation dynamic changes of protein-protein and protein-micromolecule interaction interfaces.

Description

Technical Field

[0001] The present invention belongs to the field of biomass spectrometry, and particularly relates to a method for efficiently ionizing and dissociating the structures and interactions of non-denatured proteins and their complexes by using extreme ultraviolet lasers. Background Art

[0002] Proteins are important components in organisms and play a key role in regulating cell activities and participating in cell processes related to growth, development, aging, and diseases. Therefore, the fine characterization of protein structures plays an important role in biochemical research and drug development.

[0003] Native mass spectrometry is a technique for determining the molecular weights, compositions, and stoichiometries of ligands of non-denatured proteins, that is, intact proteins in their native folded states and their non-covalent complexes or other biomolecules. Biomolecules are dissolved in a volatile non-denaturing solvent (usually ammonium acetate) and ionized in the ion source to determine their molecular properties. Combining native mass spectrometry with tandem mass spectrometry can obtain fine structural information of proteins and their complexes. In this process, a high-ion-strength solvent transfers the native-structured non-deformed intact proteins and their complexes to the gas phase through electrospray ionization. The precursor ions are mass-selected and fragmented through different ion activations to obtain more structural details.

[0004] Common ion activation techniques include collision-induced dissociation (CID), electron-mediated dissociation (EMD), and photodissociation. CID is the most traditional ion activation technique, which achieves dissociation through thousands of collisions between ions and neutral gas molecules. However, due to the long dissociation time scale and high thermal effect of this ion activation method, it often leads to the preferential cleavage of non-covalent bonds, making it difficult to obtain information on binding sites or specific structural changes of proteins. EMD involves the interaction of protonated proteins with low-energy electrons or negatively charged electron donors, and the transfer of electrons to peptide ions results in the dissociation of peptide bonds. This dissociation method can preserve non-covalent interactions, but it is currently only applicable to relatively small soluble polypeptides and proteins. Photodissociation is an emerging ion activation method that irradiates biomolecular ions with photons generated by lasers, causing the ions to fragment. According to the working wavelength of the laser, it can be divided into ultraviolet photodissociation (UVPD) and infrared multiphoton dissociation (IRMPD). UV single photons have high energy and short time scales, and can generate various types of fragment ions (a / x, b / y, and c / z and their variants), making it an ideal ion activation method. The currently most mature 193-nm UVPD has been proven to be able to provide sequence information, locate ligand binding sites, and detect protein structural changes (Brodbelt et al, J. Am. Chem. Soc. 2015, 137, 28, 9128 - 9135; Wang et al, J. Am. Chem. Soc. 2023, 145, 2, 1285 - 1291). However, since the commercially available argon fluoride excimer laser that can provide 193-nm UV laser currently has a pulse width of 10 ns and cannot adjust the wavelength and pulse width, further reducing the wavelength of the excitation light source, shortening the time scale, and increasing the energy of single photons to meet the requirements of different-wavelength high-brightness extreme ultraviolet light sources for the characterization of biomolecular systems is an effective method to achieve efficient dissociation of non-denatured proteins and their complexes and obtain sufficient sequence, modification, and structural feature fragments. Summary of the Invention

[0005] The present invention aims to overcome the problems of low dissociation efficiency of traditional ion activation techniques for macromolecular proteins and their complexes, easy destruction of the interactions between complexes, difficulty in structural analysis, and hard retention of interaction information, and to develop a method for structural analysis of non-denatured proteins and their complexes based on extreme ultraviolet lasers. This method combines non-denaturing mass spectrometry technology with extreme ultraviolet laser dissociation technology. The protein and its complex are transferred from the liquid phase to the gas phase through non-denaturing electrospray, maintaining the high-level structure of the protein while also maintaining the interaction between the protein and its ligand. A tunable extreme ultraviolet free electron laser with a wavelength of 50 nm to 150 nm is introduced into the mass spectrometry dissociation trap to ionize and dissociate the protein and its complex, generating fragment ions with fingerprint information that may or may not contain ligands, thereby achieving precise characterization of the structure of non-denatured proteins and their complexes.

[0006] To achieve the above object, the present invention provides, but is not limited to, the following new method for analyzing the structure of non-denatured proteins and their complexes based on extreme ultraviolet lasers:

[0007] (1) For the freeze-dried protein sample without salt, dissolve it in an ammonium acetate buffer solution with a concentration of 50 mmol / L to 2 mol / L, and the protein concentration is 5 μmol / L to 500 μmol / L. For the protein sample obtained by separation and purification, replace the buffer solution with an ammonium acetate buffer solution with a concentration of 50 mmol / L to 2 mol / L, and dilute the protein concentration to 5 μmol / L to 500 μmol / L with the ammonium acetate buffer solution of the same concentration;

[0008] (2) Inject the sample solution into the mass spectrometer through an ion source via a static microspray capillary with an inner diameter of 400 nm to 1 μm to form gas-phase ions of proteins and their complexes;

[0009] (3) Connect the pulsed extreme ultraviolet laser to the mass spectrometry dissociation trap through a high-vacuum pipeline. The protein ions formed in step (2) are aggregated in the mass spectrometry dissociation trap, and are ionized and dissociated by selecting a pulsed extreme ultraviolet laser with a specific wavelength. For the protein complex bound to a ligand, fragment ion information containing the ligand and fragment ion information containing the protein spatial structure can be obtained. For the protein ions not bound to the ligand, fragment ion information containing the protein spatial structure can be obtained;

[0010] (4) Perform deconvolution analysis on the mass spectrometry data obtained in step (3) to confirm the composition of the generated fragment ions and ligand information, and obtain the structural characterization information of the protein and its complexes.

[0011] Proteins interact with one or more small and large molecules such as metal ions, drug small molecules, proteins, nucleic acids, or polysaccharides (ligands) to form protein complexes.

[0012] The present invention realizes for the first time in the world the technology of using extreme ultraviolet band lasers to characterize the structure and interactions of non-denatured proteins and their complexes. It can simultaneously analyze the interaction interfaces and dynamic structural changes of non-denatured proteins and their complexes, and has significant advantages such as high sensitivity, high throughput, and single amino acid site resolution in studying the fine structures and conformational dynamic changes of protein-protein and protein-small molecule interaction interfaces.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention has realized the world's first technology for characterizing the structures of non-denatured proteins and their complexes using extreme ultraviolet (EUV) lasers. By using the short-pulse high-energy photon beam generated by EUV lasers to analyze the structures of proteins and their complexes, compared with traditional ion activation methods, the excitation and activation rate of protein ions in the present invention is increased by 7 to 10 orders of magnitude. The EUV lasers used in the present invention have a short time scale and low thermal effects, which can reduce the generation of secondary dissociation and internal fragment ions. The EUV free electron laser can achieve a wide range of continuous wavelength scanning and ionize and dissociate non-denatured proteins and their complexes in the range of 50 nm to 150 nm. The present invention can generate various types of fragment ions that are complementary to traditional ion activation methods, with rich molecular fingerprint information, capable of accurately locating ligand interaction information and identifying three-dimensional structure information. The operation of the present invention is simple, and no protein pretreatment methods are required except for replacing the buffer system. The present invention does not need to introduce any labeling reagents, avoiding the perturbation of protein conformation and the non-covalent interaction between proteins and ligands, and completely retaining the structural information of proteins and their complexes under physiological conditions. The sample consumption of the present invention is low, and only a micro-mole level of sample is required to complete the detection. Description of the Drawings

[0015] Figure 1 Shows the dissociation differences of the myoglobin-heme cofactor complex in Example 1 of the present invention and Comparative Examples 1, 2, and 3.

[0016] Figure 2 Shows the ionization and thermal effect differences of the myoglobin-heme cofactor complex in Example 2 and Examples 3 and 4 of the present invention.

[0017] Figure 3 Shows the dissociation differences of the myoglobin-heme cofactor complex in Example 2 and Examples 3 and 4 of the present invention.

[0018] Figure 4 Shows the binding region of myoglobin and heme cofactor found by the EUV laser dissociation method.

[0019] Figure 5 Shows the quantity differences of different fragment types in Example 2 and Examples 3 and 4 of the present invention.

[0020] Figure 6 Shows the dissociation of proteins by EUV free electron lasers with different wavelengths at the same pulse energy. Detailed Description of the Invention

[0021] The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art and researchers to further understand the present invention, but do not constitute any limitation to the present invention. Any modification made in any form within the scope of the claims of the present invention is still within the scope of protection of the claims of the present invention.

[0022] Example 1

[0023] Dissociation fragments of myoglobin-heme prosthetic group complex by extreme ultraviolet laser dissociation method 1. Sample preparation

[0024] Dissolve the myoglobin-heme prosthetic group complex in an ammonium acetate buffer solution with a concentration of 200 mmol / L, the protein concentration is 10 μmol / L, and the hydrogen ion concentration index is 6.8.

[0025] 2. Mass spectrometry (with quadrupole and linear ion trap) detection

[0026] The protein sample enters the ion source through a static microspray capillary made of quartz glass tube with an inner diameter of 600 nm to form protein ions (non-denaturing electrospray). In the positive ion mode, the chemical coordination number between the protein and its ligand and the strength of the interaction force are obtained through the mass-to-charge ratio information. Further, the quadrupole is used to select and enrich the myoglobin-heme prosthetic group complex with a mass-to-charge ratio of 2196.8 and 8 positive charges. The enriched protein ions are injected into the linear ion trap, and the pulsed extreme ultraviolet laser is introduced into the linear ion trap along the axial direction of the linear ion trap, and it is ionized and dissociated with the extreme ultraviolet laser along the axial direction of the linear ion trap, and data acquisition is carried out.

[0027] Working conditions of the extreme ultraviolet laser: wavelength: 70 nm; frequency: 10 Hz; single pulse energy: 23 μJ; pulse width: 1 ps.

[0028] Mass spectrometry conditions: electrospray ion source: positive ion mode; spray voltage: 800 V; source temperature: 275 °C; resolution: 240,000; RF Lens: 60%; AGC: 5e6; maximum injection time: 500 ms.

[0029] 3. Data analysis

[0030] The secondary mass spectrometry spectra containing fragment ions were deconvoluted using Xtract to obtain their molecular weights and mass-to-charge ratios. The collected data was opened with the ProSight Lite software developed by Northwestern University. The detected molecular weight was compared with the theoretical molecular weight with a tolerance of 20 ppm to confirm the types of fragment ions generated, that is, whether they are various types of characteristic fragment ions such as a-, x-, b-, y-, c-, z-, a+1, a-1, c-1, z-1, z+1, etc. and their neutral loss peaks. The actual detected fragment masses were compared with the theoretical fragment masses to obtain whether the fragments retained the information of the heme cofactor ligand molecules, thereby obtaining the amino acids where the heme cofactor interacted with myoglobin. For example, for a fragment ion with a mass-to-charge ratio of 1928.18 and 6 positive charges, its molecular weight was 615.17 larger than that of the corresponding sequence molecule, so it was identified as an amino acid chain that interacted with the heme cofactor ligand molecule.

[0031] Comparative Example 1

[0032] Dissociation fragments of myoglobin-heme cofactor complex by high-energy collision-induced dissociation method

[0033] The process and conditions were the same as those in Example 2. The difference between this comparative example and Example 1 was that in the mass spectrometry conditions of step 2, the fragmentation mode of the secondary scan was a single high-energy collision dissociation (without laser dissociation), and the fragmentation energy was 20%.

[0034] Comparative Example 2

[0035] Dissociation fragments of myoglobin-heme cofactor complex by electron transfer dissociation coupled with high-energy collision-induced dissociation method

[0036] The process and conditions were the same as those in Example 1. The differences between this comparative example and Example 1 and Comparative Example 2 were that in the mass spectrometry conditions of step 2, the fragmentation mode of the secondary scan was electron transfer dissociation coupled with high-energy collision dissociation (without laser dissociation). The time of electron transfer dissociation was 20 milliseconds, and the fragmentation energy of high-energy collision dissociation was 15%.

[0037] Comparative Example 3

[0038] Dissociation fragments of myoglobin-heme cofactor complex by 193-nm ultraviolet photodissociation method

[0039] The process and conditions were the same as those in Example 1. The differences between this comparative example and Example 1, Comparative Example 2 and Comparative Example 3 were that in the mass spectrometry conditions of step 2, the fragmentation mode of the secondary scan was 193-nm ultraviolet single-photon dissociation. The frequency of the ultraviolet light was 10 Hz, and the single-pulse energy was 1 mJ.

[0040] Figure 1Shows the fragment difference diagrams generated by the second-stage dissociation of Example 1 of the present invention and Comparative Examples 1, 2, and 3, where Figure 1 a The selected precursor ions are subjected to high-energy collision-induced dissociation fragmentation, Figure 1 b The selected precursor ions are subjected to electron transfer dissociation coupled with high-energy collision-induced dissociation fragmentation, Figure 1 c The selected precursor ions are subjected to 193-nm ultraviolet photodissociation fragmentation, Figure 1 d The selected precursor ions are subjected to 70-nm extreme ultraviolet laser dissociation fragmentation. In ultraviolet photodissociation and extreme ultraviolet photodissociation, the laser can use a single pulse or multiple pulses. In the present invention, a single pulse is used. In the figure, the mass-to-charge ratio is 616.18, and the peak with a single positive charge is the characteristic peak of the ligand heme. The higher this characteristic peak, the stronger the damage to the protein-ligand complex. Therefore, compared with high-energy collision-induced dissociation, electron transfer dissociation coupled with high-energy collision-induced dissociation, and 193-nm ultraviolet photodissociation, 70-nm extreme ultraviolet laser dissociation reduces the damage to the ligand-protein binding interface and significantly increases the number of fragments, thereby obtaining richer structural information.

[0041] Example 2

[0042] Detection of the interaction between myoglobin and heme cofactor by 75-nm, 0.1-μJ extreme ultraviolet free electron laser

[0043] 1. Sample preparation

[0044] The myoglobin-heme complex is dissolved in an ammonium acetate buffer solution with a concentration of 200 mmol / L, the protein concentration is 10 μmol / L, and the hydrogen ion concentration index is 6.8.

[0045] 2. Mass spectrometry detection

[0046] The protein sample enters the ion source through a static microspray capillary made of quartz glass tube with an inner diameter of 600 nm to form protein ions. The mass-to-charge ratio of the protein is determined in the positive ion mode. Further, a quadrupole is used to select and enrich the myoglobin-heme cofactor complex with a mass-to-charge ratio of 2196.8 and 8 positive charges. The enriched protein ions are injected into a linear ion trap, ionized and dissociated with an extreme ultraviolet laser, and data is collected.

[0047] Working conditions of the extreme ultraviolet laser: wavelength: 75 nm; frequency: 10 Hz; single pulse energy: 0.1 μJ; pulse width: 1 ps.

[0048] Mass spectrometry conditions: electrospray ion source: positive ion mode; spray voltage: 800 V; source temperature: 275 °C; resolution: 240,000; RF Lens: 60%; AGC: 5e6; maximum injection time: 500 ms.

[0049] 3. Data analysis

[0050] Use Xtract to deconvolute the secondary mass spectrometry map containing fragment ions to obtain its molecular weight and mass-to-charge ratio information. Open the collected data with the ProSight Lite software developed by Northwestern University, compare the detected molecular weight with the theoretical molecular weight with a tolerance of 20 ppm, and confirm the types of fragment ions generated, that is, whether they are various types of characteristic fragment ions such as a-, x-, b-, y-, c-, z-, a+1, a-1, c-1, z-1, z+1 and their neutral loss peaks. Compare the actually detected fragment mass with the theoretical fragment mass to obtain whether the fragment retains the information of the heme cofactor ligand molecule, so as to obtain the amino acids where the heme cofactor ligand interacts with myoglobin. For example, the fragment ion with a mass-to-charge ratio of 1928.18 and 6 positive charges has a molecular weight 615.17 larger than the corresponding sequence molecule, so it is identified as the amino acid chain that interacts with the heme cofactor ligand molecule.

[0051] Example 3

[0052] Detection of the interaction between myoglobin and heme cofactor by 75-nm 0.4-μJ extreme ultraviolet free electron laser

[0053] The process and conditions are the same as those in Example 2. The difference between this example and Example 2 lies in the working conditions of the extreme ultraviolet laser in step 2, and the single-pulse energy is 0.4 μJ.

[0054] Example 4

[0055] Detection of the interaction between myoglobin and heme cofactor by 75-nm 0.7-μJ extreme ultraviolet free electron laser

[0056] The process and conditions are the same as those in Example 2. The difference between this example and Example 2 lies in the working conditions of the extreme ultraviolet laser in step 2, and the single-pulse energy is 0.7 μJ.

[0057] Figure 2 Show the ionization and thermal effect differences of myoglobin in Example 2, Example 3 and Example 4 of the present invention, where Figure 2 a corresponds to the ionization and thermal effect of the myoglobin-heme complex by a 75-nm 0.1-μJ extreme ultraviolet free electron laser, Figure 2 b corresponds to the ionization and thermal effect of the myoglobin-heme complex by a 75-nm 0.4-μJ extreme ultraviolet free electron laser, Figure 2c corresponds to the ionization and thermal effect of myoglobin-heme complex by extreme ultraviolet free electron laser at 75 nm and 0.7 μJ. The peak with a mass-to-charge ratio of 2196.8 and 8 charges is the parent ion. The peaks with mass-to-charge ratios of 1952.7 and 9 charges, 1757.4 and 10 charges, and 1593.7 and 11 charges correspond to the first ionization peak, the second ionization peak, the third ionization peak, and the fourth ionization peak respectively. The ionization peaks with the loss of one or more electrons generated under the excitation of a single pulse of extreme ultraviolet free electron laser are unique phenomena of the extreme ultraviolet laser ion activation method different from the conventional activation method for analyzing protein structures, and the intensity of the photoionization peaks changes with the laser intensity. The unique protein structure information can be obtained from the extreme ultraviolet photoionization spectrum obtained through research. The peak with a mass-to-charge ratio of 616.2 and 1 charge is the heme prosthetic group ion. The low-abundance heme prosthetic group ion indicates that the thermal effect generated during the dissociation by extreme ultraviolet free electron laser is extremely low, and the structural information of proteins and their complexes under physiological conditions is basically retained.

[0058] Figure 3 Shows the dissociation differences of myoglobin in Example 2 and Examples 3 and 4 of the present invention. With the increase of energy, the myoglobin-heme complex has a higher dissociation efficiency, but the overall dissociation trend is very consistent. In the binding region between myoglobin and the heme prosthetic group, non-covalent interactions make the amino acids near the binding region relatively difficult to dissociate, and the binding efficiency is low. The parts of the myoglobin-heme complex that are prone to dissociation are usually not considered the interaction regions between myoglobin and the heme prosthetic group. Figure 4 Shows the binding region between myoglobin and the heme prosthetic group found by using extreme ultraviolet laser dissociation technology, where the binding region is shown in white and the unbound region is shown in black. Comparing with the reported crystal structure (PDB Code: 1YMB) of the myoglobin-heme complex, the binding region found by this method highly coincides with the binding region found by X-ray crystallography, reflecting the superiority of this method in protein complex structure detection. Figure 5 Shows the difference in the number of fragment types between Example 2 and Comparative Examples 4 and 5 of the present invention, and uses arrows to point out the apoprotein fragments, non-apoprotein fragments, and unmatched fragment results respectively. It shows that the non-apoprotein fragments are relatively few under 0.7 μJ, probably because the energy is too high to cause the heme prosthetic group to fall off, indicating that the degree of protein structure analysis by extreme ultraviolet free electron laser does not increase linearly with the laser pulse intensity.

[0059] Example 5

[0060] Selectivity of extreme ultraviolet free electron laser with different wavelengths for protein structure detection

[0061] 1. Sample preparation

[0062] Myoglobin and heme prosthetic group were dissolved in an ammonium acetate buffer solution with a concentration of 200 mmol / L, the protein concentration was 10 μmol / L, and the hydrogen ion concentration index was 6.8.

[0063] 2. Mass spectrometry detection

[0064] The protein sample enters the ion source through a static microspray capillary made of fused silica tube with an inner diameter of 600 nm to form protein ions. The mass-to-charge ratio of the protein is determined in the positive ion mode. Further, a quadrupole is used for the selection and enrichment of the myoglobin-heme prosthetic group complex with a mass-to-charge ratio of 2196.8 and 8 positive charges. The enriched protein ions are injected into a linear ion trap, ionized and dissociated with extreme ultraviolet laser, and data acquisition is carried out.

[0065] Working conditions of the extreme ultraviolet laser: wavelength: continuously variable from 75 nm to 140 nm; frequency: 10 Hz; pulse width: 1 ps.

[0066] Mass spectrometry conditions: electrospray ionization source: positive ion mode; spray voltage: 800 V; source temperature: 275 °C; resolution: 240,000; RF Lens: 60%; AGC: 5e6; maximum injection time: 500 ms.

[0067] 3. Data analysis

[0068] Xtract was used to deconvolute the tandem mass spectrum containing fragment ions to obtain its molecular weight and mass-to-charge ratio information. The collected data was opened with ProSight Lite software developed by Northwestern University, and the detected molecular weight was compared with the theoretical molecular weight with a tolerance of 20 ppm to confirm the types of fragment ions generated, that is, whether they are various types of characteristic fragment ions such as a-, x-, b-, y-, c-, z-, a+1, a-1, c-1, z-1, z+1 and their neutral loss peaks. The actually detected fragment mass was compared with the theoretical fragment mass to obtain whether the fragment retained the heme prosthetic group ligand molecule information, so as to obtain the amino acids where the heme prosthetic group ligand interacted with myoglobin. For example, the molecular weight of the fragment ion with a mass-to-charge ratio of 1928.18 and 6 positive charges was 615.17 larger than that of the corresponding sequence molecule, so it was identified as the amino acid chain interacting with the heme prosthetic group ligand molecule.

[0069] Figure 5The dissociation of myoglobin-heme complex at different sites by extreme ultraviolet free electron lasers with different wavelengths at the same pulse energy is shown. The higher the degree of black (deeper color), the higher the dissociation degree of the extreme ultraviolet laser on this site, and the higher the degree of white (lighter color), the lower the dissociation degree of the extreme ultraviolet laser on this site. The results show that the change of the extreme ultraviolet laser wavelength is not linearly related to the dissociation degree. Among them, the dissociation at 100 nm is relatively strong, and the dissociation of the protein structure by the extreme ultraviolet free electron laser is selective for different wavelengths.

Claims

1. A method for characterizing the structure and interaction of proteins and / or complexes based on extreme ultraviolet laser dissociation and mass spectrometry, characterized in that: It is an analytical method for characterizing the structure and interaction of non-denatured proteins and / or complexes based on extreme ultraviolet laser ionization dissociation and mass spectrometry detection. Native proteins and / or protein complexes enter the gas phase through non-denaturing electrospray. The electrospray ions of proteins and / or protein complexes with 1 to 5 different charge states after electrospray ionization are irradiated and excited by a pulsed extreme ultraviolet laser in the linear ion trap of the mass spectrometer to induce the generation of characteristic fragment ions of the structure. All fragment ions are detected by mass spectrometry and the spectra are recorded. The protein and / or complex structure or ligand binding information in the complex is obtained through data processing.

2. The method according to claim 1, wherein: The non-denatured protein after separation and purification uses an ultrafiltration tube to replace the buffer system with an ammonium acetate buffer solution with a concentration of 50 mmol / L to 2 mol / L (biocompatible neutral solution), the protein concentration is 5 μmol / L to 500 μmol / L, and the hydrogen ion concentration index is 2 to 10.

3. The method according to claim 1, wherein: The voltage for electrospray ionization is 500 to 2300 volts.

4. The method according to claim 1, characterized in that: The pulsed laser wavelength range is 50 nm to 150 nm (preferably 75 to 130 nm), the pulse width is 0.1 picosecond to 200 nanoseconds (preferably 0.1 to 10 picoseconds), the pulse repetition frequency is 1 Hz to 50 Hz (preferably 10 to 30 Hz), and the pulse energy is 0.1 μJ to 100 μJ (preferably 5 to 35 μJ).

5. The method according to claim 1 or 4, characterized in that: High-energy ultraviolet photons excite the protein backbone to a high-energy electron excited state on the femtosecond to picosecond scale, generating radical ions that lose one or more electrons, and initiating the overall fragmentation of the protein backbone mediated by radicals, retaining the non-covalent interactions and higher-order structure information of the protein in the fragment ions; the dissociation process can use a single or multiple laser pulses.

6. The method according to claim 1, characterized in that: Proteins and their complexes can stabilize the structure and maintain interactions in non-denaturing electrospray; the stoichiometry and affinity strength information of gas-phase protein and complex ions are obtained through mass spectrometry detection; the pulsed extreme ultraviolet laser is used for secondary dissociation of protein ions and protein complex ions respectively, and the dissociation fragments with or without ligands are detected by mass spectrometry and recorded in the secondary mass spectrometry spectrum.

7. The method according to any one of claims 1-6, characterized in that: Analysis of protein non-covalent interactions: It can be the interaction between a protein and one or more than two molecules including but not limited to metal ions, small drug molecules, proteins, nucleic acids, polysaccharides, etc. within and / or between molecules.

8. The method according to any one of claims 1-6, characterized in that: Protein structure and ligand binding information: Through extreme ultraviolet laser dissociation, various types of characteristic fragment ions such as a-, x-, b-, y-, c-, z-, a+1, a-1, c-1, z-1, z+1 that bind or do not bind ligands can be generated. The primary to quaternary structures of the protein and / or its interaction information with the ligand can be obtained through spectral matching analysis.

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