Method for on-line hydrogen-deuterium exchange mass spectrometry analysis of protein structure

Abstract

The invention discloses a method for on-line hydrogen deuterium exchange mass spectrometry analysis of a protein structure. A capillary electrophoresis-mass spectrometry (CE-MS) technology is used, a capillary tube is used as a microreactor for protein hydrogen-deuterium exchange reaction, and conformation-sensitive deuterated labeling of protein is completed in the electromigration process; introducing an acidic sheath fluid through a combined interface to carry out reaction quenching, and carrying out complete protein structure analysis by utilizing a Top-down analysis strategy; according to the method, the reaction, separation and detection of the protein in the capillary tube in the migration process are realized in one step, the hydrogen-deuterium exchange mass spectrometry step is simplified, the on-line hydrogen-deuterium exchange time can be accurately controlled, the reverse exchange is effectively inhibited, and the structural characteristics of the protein in a solution state are conveniently and quickly revealed by comparing the deuteration rate change of different peptide fragments of the protein.

Description

Technical Field

[0001] This invention relates to the field of bioanalytical detection technology, specifically a method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure. Background Technology

[0002] Protein structure determines function, and precise structural analysis of proteins is crucial for understanding their biological mechanisms, identifying drug targets, and developing biopharmaceuticals. Hydrogen-deuterium exchange mass spectrometry (HDEMS) is an important and rapidly developing technique for analyzing the advanced structure of proteins. Its principle is based on the fact that the exchange rate between amide hydrogens in the protein backbone and deuterium in the solvent environment is highly dependent on hydrogen bond formation and solvent accessibility. Therefore, the exchange rate of amide hydrogens forming stable hydrogen bonds in secondary structures (such as hydrogens in α-helices and β-sheets) is relatively slow, while the hydrogen exchange rate in random coil regions or flexible loop regions is faster (Konermann L., Pan J., and Liu Y.-H. Hydrogen Exchange Mass Spectrometry for Studying Protein Structure and Dynamics. Chemical Society Reviews, 2011. 40(3): p. 1224-1234). By accurately measuring the mass-to-charge ratio shift of peptides or intact proteins using mass spectrometry, the structural dynamics of different regions of the protein can be analyzed and obtained.

[0003] Currently, most hydrogen-deuterium exchange mass spectrometry (HDEMS) analyses of protein structures employ a bottom-up approach. This involves pre-mixing the protein with a deuteration buffer and incubating for a specific time, then quenching the reaction under low-temperature, acidic conditions. The protein is then enzymatically digested into peptides, which are then detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS). This method is technically mature and relatively standardized, suitable for analyzing the structure of proteins with large molecular weights. However, the enzymatic digestion process leads to the loss of some sequence and modification information, and it fails to obtain structural information on side chains. Spatial resolution is limited by the length of the peptides produced by the digestion. The top-down approach, on the other hand, directly detects the intact deuterated protein using mass spectrometry. This preserves protein modification information, and the fragmentation process precisely locates the deuteration sites to individual amino acid residues. The spatial resolution is significantly superior to the bottom-up method, clearly defining the boundaries of secondary structures such as α-helices and β-sheets.

[0004] Suppressing the back-exchange in the hydrogen-deuterium exchange reaction is one of the core challenges of hydrogen-deuterium exchange mass spectrometry (HDEMS). In bottom-up methods, back-exchange mainly occurs during enzymatic digestion and liquid chromatography separation. During liquid chromatography, deuterated peptides undergo back-exchange with protons in the mobile phase, leading to a significant loss of the deuterium signal. Therefore, back-exchange is difficult to completely suppress, affecting the accuracy of protein structure analysis. Our previous work found that controlling the protein hydrogen-deuterium exchange reaction through capillary electrophoresis, combined with top-down mass spectrometry for direct analysis of intact proteins, effectively avoids back-exchange during sample pretreatment and separation compared to bottom-up methods. Therefore, this invention combines CE-MS with gas-phase mass spectrometry dissociation to achieve one-step reaction, separation, and detection of proteins during capillary migration, facilitating rapid revelation of the spatial structural features of apoptosis-related proteins in solution. Summary of the Invention

[0005] To address the problems mentioned in the background section, this invention provides a method for online hydrogen-deuterium exchange mass spectrometry (HDE-MS) analysis of protein structure. This method utilizes capillary electrophoresis-mass spectrometry (CES-MS) to achieve direct analysis of intact proteins, precisely locating protein fragmentation sites through a top-down analysis strategy. Using ammonium acetate buffer prepared with deuteration reagents as the background electrolyte, the protein undergoes a HDE-deuterium exchange reaction within the capillary, is quenched at the interface, and then directly enters the mass spectrometer for detection. Based on the tandem mass spectra before and after deuteration, the deuteration level of the protein's amino acid backbone can be determined, which can be used to resolve the protein's spatial structure.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure, comprising the following steps; S01. Preparation of protein stock solution: Dissolve the protein powder in ultrapure water, determine the concentration using a micro-ultraviolet spectrophotometer, and obtain the protein stock solution. S02. Preparation of protein sample solution: Dilute the protein stock solution with ammonium acetate buffer to obtain the protein sample solution; S03. Capillary electrophoresis-mass spectrometry (CES-MS) detection of protein sample solutions: Using ammonium acetate buffer as the background electrolyte, the protein sample solution is introduced through the positive end of the capillary electrophoresis tube. It migrates to the coupling interface and mixes with the sheath fluid. Subsequently, electrospray ionization is used for primary mass spectrometry detection, generating multiple different charge states to obtain the primary mass spectrum of the protein before deuteration. One charge state is selected from the primary mass spectrum for tandem mass spectrometry detection to obtain the tandem mass spectrum of the protein before deuteration. Deconvolution analysis is performed on fragment ions in the tandem mass spectrum to assign peptides and determine the fragmentation sites of the protein sequence. S04. Online Hydrogen-Deuterium Exchange Mass Spectrometry Detection of Protein Sample Solutions: Using deuterated ammonium acetate buffer prepared with heavy water as the background electrolyte, the protein sample solution is injected through the positive end of capillary electrophoresis and mixed with the deuterated sheath fluid via electromigration. By changing the auxiliary pressure during the capillary electrophoresis separation process, the retention time of the protein in the capillary is controlled to obtain a higher level of protein deuteration. During the migration, the amide hydrogen of the protein backbone undergoes hydrogen-deuterium exchange. When the protein migrates to the coupling interface and mixes with the deuterated sheath fluid, the deuteration reaction is quenched. Subsequently, after electrospray ionization, primary mass spectrometry is performed to obtain the primary mass spectrum of the deuterated protein. From the primary mass spectrum, protein ions with the same charge state as before deuteration are selected for tandem mass spectrometry to obtain the tandem mass spectrum of the deuterated protein. By comparing the tandem mass spectra of protein ions with the same charge state before and after deuteration, the peptide assignment of the deuterated fragment ions is determined. S05. Calculate the main chain deuteration level of a single amino acid residue based on the tandem mass spectra of the protein before and after deuteration. Step 1: Record fragment ions based on the tandem mass spectra of the protein before and after deuteration. m / z The numerical value and charge number are used to determine the mass of fragment ions before and after deuteration; the mass of fragment ions is calculated according to formula (1). c The total deuteration number of the peptide obtained by ion assignment is calculated according to formula (2). z The total deuteration number of the peptide obtained by ion assignment; ; ; Suppose a protein is composed of X amino acids, where, The total deuteration number of the peptide segment representing the first to m amino acid residues at the N-terminus; Represents the total deuteration generation of the peptide segment at the (Xn)-Xth amino acid residue at the C-terminus; and Represent 2 H and 1 The atomic mass of H; and Represent and The mass of fragment ions after deuteration; and Represent and The mass of the fragment ions before deuteration; the subscripts m and n represent respectively c ion, z The ion's serial number; Step 2, calculate the result according to formula (3) from c The deuterium algebra number of the peptide backbone to which the ion belongs; calculated according to formula (4) zThe deuterium algebra of the peptide backbone to which the ion is assigned; ; ; in, and These represent the deuterium generation number of the peptide backbone and the number of hydrogen atoms in the side chain of the N-terminal 1-m amino acid residues, respectively; and These represent the deuterium generation number of the peptide backbone and the number of hydrogen atoms in the side chain of the (Xn)-Xth amino acid residue at the C-terminus, respectively. Step 3: Calculate the main chain deuteration level of individual amino acid residues, and calculate the level using formula (5). c The main chain deuteration level of a single amino acid residue obtained by ionization; calculated according to formula (6) z The deuteration level of the main chain of a single amino acid residue obtained by ionization; ; ; Where the subscript 'i' represents the sequence number of the protein's amino acid residue, Represents the main chain deuteration level of the i-th amino acid residue; The number of hydrogen atoms in the main chain of a peptide segment representing the first to m amino acid residues at the N-terminus; This represents the number of hydrogen atoms in the main chain of the peptide segment from amino acid residue (Xn) to X at the C-terminus.

[0007] In cases where the main chain deuteration level of individual amino acid residues cannot be obtained due to the loss of some fragment ions, c The main chain deuteration level of the ions is calculated according to formula (7). z The main chain deuteration level of the ions is calculated according to formula (8); ; ; Where j represents the number of missing amino acid residues between two adjacent fragment ions.

[0008] S06. Based on the different deuteration levels of the main chain, information on the secondary structure of the protein is obtained; a lower main chain deuteration level for a single amino acid residue indicates higher structural protection of the main chain amide hydrogen; a higher deuteration level indicates lower structural protection of the main chain amide hydrogen.

[0009] Further, in step S01, the protein is equine cytochrome c, and the concentration of the protein stock solution is 500-600 μmol / L.

[0010] Further, in step S02, the protein concentration in the protein sample solution is 5-10 μmol / L, the ammonium acetate concentration is 20-30 mmol / L, and the pH is 6.5-7.5.

[0011] Further, in step S03, the concentration of ammonium acetate in the background electrolyte is 20-30 mmol / L; the sheath fluid is a mixture of 0.3% formic acid aqueous solution and methanol, with a volume ratio of 0.3% formic acid aqueous solution to methanol of 45:55; and the flow rate of the sheath fluid is 1 μL / min.

[0012] Further, in step S04, the background electrolyte is a heavy aqueous solution of ammonium acetate at a concentration of 20-30 mmol / L, the sheath fluid is a mixture of 0.3% deuterated formic acid aqueous solution and deuterated methanol, and the volume ratio of 0.3% deuterated formic acid heavy aqueous solution to deuterated methanol is 45:55; the flow rate of the sheath fluid is 1 μL / min.

[0013] Furthermore, in steps S03 and S04, the capillary used in the capillary electrophoresis is a neutral-coated capillary coated with hydroxypropyl cellulose, with a length of 70-90 cm.

[0014] Furthermore, in steps S03 and S04, the sample introduction conditions for capillary electrophoresis are 1 psi pressure for 5 seconds, and the separation conditions for capillary electrophoresis are 28 kV positive voltage and 0.5-3 psi auxiliary pressure.

[0015] Further, in steps S03 and S04, the detection conditions for the primary mass spectrometry and tandem mass spectrometry are as follows: scanning in positive ion mode, electrospray voltage of 3000 V, ion transmission tube temperature of 200 °C, resolution of 60000, and scanning range of 150-2000. m / z The ion optical radio frequency voltage (RF value) is 60%, the maximum injection time is 100 ms, and the automatic gain control (AGC) is 100%. The tandem mass spectrometry utilizes a dissociation mode combining electron transfer-high energy collision (EThcD), where the reaction time of electron transfer dissociation (ETD) is 2-6 ms and the energy of high energy collision dissociation (HCD) is 1-10%.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention relates to a method for online hydrogen-deuterium exchange mass spectrometry (HDE-MS) analysis of protein structure, integrating the efficient separation of capillary electrophoresis with the high-sensitivity detection of mass spectrometry online. The device performs the HDE-MS reaction, using the capillary itself as a miniature reactor. By filling the capillary with a background electrolyte prepared with fully deuterated reagents, the protein undergoes conformationally sensitive deuteration labeling during migration under physiological conditions. Subsequently, the protein is mixed with a low-pH acidic sheath solution at the coupling interface, quenching the HDE-MS reaction and directly electrospray ionizing the protein for mass spectrometry detection. The use of the acidic sheath solution simultaneously achieves rapid quenching of the protein HDE-MS reaction and a significant improvement in electrospray ionization efficiency.

[0017] The method of this invention is carried out entirely in a closed capillary tube, integrating the hydrogen-deuterium exchange reaction, quenching, and separation analysis online. This significantly shortens the delay time from quenching to mass spectrometry detection and can significantly suppress reverse deuteration exchange. The capillary electrophoresis apparatus allows for precise control of the deuteration reaction time. Furthermore, it fully utilizes the microliter volume of the capillary, directly using fully deuterated reagents as the background electrolyte solvent, consuming only 20-40 μL of deuterated reagent.

[0018] This invention provides a method for efficiently and faithfully characterizing protein structures. The actual sample volume required for a single assay can be reduced to tens of nanoliters, making it particularly suitable for target samples with limited or high-cost sources. It achieves protein reaction, separation, and detection in a single step during capillary migration, simplifying the hydrogen-deuterium exchange reaction steps, allowing precise control of reaction time, and facilitating rapid and efficient separation and protein structure analysis of complex samples. Attached Figure Description

[0019] Figure 1 This is a diagram of the fragmentation sites of cytochrome c in Example 1; Figure 2 In Example 2, protein ions of 15+ were used under different auxiliary pressures. m / z (824.90, undeuterated) as reference, graph showing the relationship between deuteration reaction time and deuteration amount of cytochrome c; Figure 3 This is a diagram showing the deuteration levels of amino acid residues in the cytochrome c backbone in Example 3. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 (1) Weigh 5 mg of cytochrome c dry powder and dissolve it in ultrapure water. Use a micro-ultraviolet spectrophotometer to determine the concentration to obtain cytochrome c stock solution. Dilute the cytochrome c stock solution with 20 mM pH 6.86 ammonium acetate buffer to obtain a 5 μmol / L sample solution. Use 20 mM pH 6.86 ammonium acetate buffer as background electrolyte, rinse and fill the capillary. Apply 1 psi pressure to the positive end of the capillary for 5 s for injection. Then apply a positive voltage of 28 kV and supplement with 1 psi pressure to make cytochrome c electromigrate to the coupling interface and mix with the sheath fluid, which is 0.3% formic acid aqueous solution-methanol (volume ratio 45:55). Then enter the mass spectrometer for primary mass spectrometry detection. Select protein ions with a charge of 15+ in the primary mass spectrometer ( m / z 824.90) was used as the precursor ion for tandem mass spectrometry detection. The detection conditions for primary mass spectrometry and tandem mass spectrometry were: scanning in positive ion mode, electrospray voltage of 3000 V, ion transmission tube temperature of 200 °C, mass spectrometry resolution of 60000, and scanning range... m / z The range is 150-2000, the ion optical radio frequency voltage (RF value) is 60%, the maximum injection time is 100 ms, and the automatic gain control (AGC) is 100%. The tandem mass spectrometry uses a dissociation mode that combines electron transfer-high energy collision (EThcD), with an electron transfer dissociation reaction time of 6 ms and a high energy collision dissociation energy of 4%.

[0022] (2) Data analysis: Deconvolution analysis of the tandem mass spectra was performed using Xtract to obtain the molecular weight information of the fragment ions. The molecular weights of the fragment ions were compared with the theoretical molecular weights of the fragment ions (with an allowable error of ≤10 ppm) to determine the type of fragment ions and the fragmentation sites of cytochrome c.

[0023] As attached Figure 1 As shown, using the above-described method, tandem mass spectrometry fragmented the intact cytochrome c cells, yielding 50 fragments. c Ions, 48 z The ions showed 65 fragmentation sites with a spatial resolution of 63%. Cytochrome c exhibits an acetylated group on Gly1 (molecular weight 42.0156). The experimental results indicate characteristic fragment ions... c 5 The theoretical molecular weight is 545.2882, and the measured experimental molecular weight is 587.2955, a difference of 42.0072. The acetylation modification of cytochrome c was completely preserved, with an error of 6.47 ppm. Cytochrome c contains two cysteine ​​residues, Cys14 and Cys17, covalently linked to a heme group (molecular weight 616.1976). Characteristic fragment ions. c 24The theoretical molecular weight is 2588.3696, and the measured experimental molecular weight is 3246.5812, a difference of 658.2116. The acetylation modification of cytochrome c and the heme group are completely preserved, with an error of 6.15 ppm. This demonstrates that the method of this invention, based on a top-down strategy, fragments the protein to obtain fragmentation sites, achieves the localization of amide hydrogen, and completely preserves the protein's modification information.

[0024] Example 2 (1) Weigh 5 mg of cytochrome c dry powder and dissolve it in ultrapure water. Use a micro-ultraviolet spectrophotometer to determine the concentration to obtain cytochrome c stock solution. Dilute the cytochrome c stock solution with 20 mM pH 6.86 ammonium acetate buffer to obtain a 5 μmol / L sample solution. Use deuterated ammonium acetate buffer prepared with heavy water as the background electrolyte, rinse and fill the capillary. Apply 1 psi pressure to the positive end of the capillary for 5 seconds for injection. Then apply a positive voltage of 28 kV, supplemented with different auxiliary pressures (range 0.5-3.0 psi) to allow cytochrome c to electromigrate to the coupling interface and mix with the deuterated sheath solution. The deuterated sheath solution is prepared using deuterated methanol, deuterated formic acid, and heavy water to prepare a 0.3% formic acid aqueous solution-methanol (volume ratio 45:55). Then, perform primary mass spectrometry detection. The primary mass spectrometry detection conditions are the same as in Example 1.

[0025] (2) Data analysis: using 15+ protein ions ( m / z 824.90 (before deuterium) is for reference only, see appendix. Figure 2 This demonstrates how adjusting the auxiliary pressure controls the migration time of cytochrome c in the capillary, which is also the time for deuteration. The migration time and deuteration amount of cytochrome c in the capillary were measured (in triplicate) under different auxiliary pressure conditions.

[0026] As attached Figure 2 The results showed that the migration time gradually shortened with increasing auxiliary pressure, indicating that the amount of deuteration could be regulated by changing the auxiliary pressure. The amount of deuteration of cytochrome c increased with increasing deuteration time within the auxiliary pressure range of 1.5–3.0 psi, but remained almost constant with increasing deuteration time within the auxiliary pressure range of 0.5–1.5 psi. The deuteration reaction of cytochrome c reached equilibrium at approximately 11 min, with 15+ protein ions undergoing deuteration. m / z The value was 832.81, and the equilibrium deuteration amount was 119 Da. The above describes the method of this invention, which utilizes a capillary electrophoresis-mass spectrometry system to precisely control the deuteration reaction time of proteins, with a small RSD and good reproducibility.

[0027] Example 3 (1) Weigh 5 mg of cytochrome c dry powder and dissolve it in ultrapure water. Use a micro-ultraviolet spectrophotometer to determine the concentration to obtain cytochrome c stock solution. Dilute the cytochrome c stock solution with 20 mM pH 6.86 ammonium acetate buffer to obtain a 5 μmol / L sample solution. Use deuterated ammonium acetate buffer prepared with heavy water as the background electrolyte, rinse and fill the capillary. Apply 1 psi pressure to the positive end of the capillary for 5 s for injection. Then apply a positive voltage of 28 kV and supplement with 1.0 psi auxiliary pressure to make cytochrome c electromigrate to the coupling interface and mix with the deuterated sheath solution. The deuterated sheath solution is prepared with deuterated methanol, deuterated formic acid and heavy water to prepare 0.3% formic acid aqueous solution-methanol (volume ratio 45:55). Then enter the mass spectrometer for primary mass spectrometry detection. Select protein ions with a charge of 15+ in the primary mass spectrometer as the parent ion. m / z 832.81 (after deuteration) was detected by tandem mass spectrometry. The detection conditions for primary mass spectrometry and tandem mass spectrometry were the same as in Example 1.

[0028] (2) Data analysis: Based on the tandem mass spectra before and after deuteration in Example 1, the main chain deuteration level of a single amino acid residue was calculated using formulas (1) to (8). A lower main chain deuteration level indicates higher structural protection of the main chain amide hydrogen; a higher deuteration level indicates lower structural protection of the main chain amide hydrogen.

[0029] As attached Figure 3As shown, the main chain deuteration levels of most amino acid residues in Leu35-Thr49, Asn54-Thr58, and Ile75-Ala83 are around 1.0, indicating a relatively high deuteration level. This suggests that the structure of these regions is relatively loose, belonging to flexible, randomly coiled regions. In contrast, the main chain deuteration levels of amino acid residues in Lys60-Leu68, Asn70-Tyr74, and Lys88-Asn103 are around 0.0, significantly lower than adjacent amino acid residue segments, showing a clear boundary. This suggests that these areas are located in the internal structure of the protein (solvent-inaccessible) or contain relatively compact higher-order structural regions (such as helices). The above data indicate that information about the structure of cytochrome c can be inferred based on different deuteration levels. This is consistent with the cytochrome c structure resolved by nuclear magnetic resonance spectroscopy reported in previous literature (PDB ID: 1OCD), showing that regions with low-level deuteration (i.e., highly protected from hydrogen-deuterium exchange) correspond to structured helical segments (Qi PX, Beckman RA, and Wand AJ Solution Structure of Horse Heart Ferricytochrome C and Detection of Redox-Related Structural Changes by High-Resolution). 1 H NMR. Biochemistry, 1996. 35(38): p. 12275-12286).

[0030] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0031] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure, characterized in that, Includes the following steps; S01. Preparation of protein stock solution: Dissolve the protein powder in ultrapure water, determine the concentration using a micro-ultraviolet spectrophotometer, and obtain the protein stock solution. S02. Preparation of protein sample solution: Dilute the protein stock solution with ammonium acetate buffer to obtain the protein sample solution; S03. Capillary electrophoresis-mass spectrometry (CES-MS) detection of protein sample solutions: Using ammonium acetate buffer as the background electrolyte, the protein sample solution is introduced through the positive end of the capillary electrophoresis tube. It then migrates to the coupling interface and mixes with the sheath fluid. Subsequently, electrospray ionization is used for primary mass spectrometry detection, generating multiple different charge states to obtain the primary mass spectrum of the protein before deuteration. One charge state is selected from the primary mass spectrum for tandem mass spectrometry detection to obtain the tandem mass spectrum of the protein before deuteration. Deconvolution analysis of fragment ions in the tandem mass spectrum is performed to assign peptides and determine the fragmentation sites of the protein sequence. S04. Online hydrogen-deuterium exchange mass spectrometry detection of protein sample solutions: Deuterated ammonium acetate buffer prepared with heavy water was used as the background electrolyte. Protein sample solutions are introduced via the positive end of a capillary electrophoresis tube and then electromigrated to the coupling interface to mix with deuterated sheath fluid. Subsequently, the protein was deuterated and then detected by primary mass spectrometry. The primary mass spectrum of the protein was obtained by electrospray ionization. Protein ions with the same charge state as before deuteration were selected from the primary mass spectrum and detected by tandem mass spectrometry to obtain the tandem mass spectrum of the protein after deuteration. By comparing the tandem mass spectra of protein ions with the same charge state before and after deuteration, the peptide assignment of the deuterated fragment ions was determined. S05. Calculate the main chain deuteration level of a single amino acid residue based on the tandem mass spectra of the protein before and after deuteration. S06. Based on the different deuteration levels of the main chain, information on the secondary structure of the protein is obtained.

2. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 1, characterized in that, In step S01, the protein is equine cytochrome c, and the concentration of the protein stock solution is 500-600 μmol / L.

3. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 1, characterized in that, In step S02, the protein concentration in the protein sample solution is 5-10 μmol / L, the ammonium acetate concentration in the protein sample solution is 20-30 mmol / L, and the pH is 6.5-7.

5.

4. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 1, characterized in that, In step S03, the concentration of ammonium acetate in the background electrolyte is 20-30 mmol / L; the sheath fluid is a mixture of 0.3% formic acid aqueous solution and methanol, with a volume ratio of 0.3% formic acid aqueous solution to methanol of 45:55; and the flow rate of the sheath fluid is 1 μL / min.

5. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 1, characterized in that, In step S04, the background electrolyte is a heavy aqueous solution of ammonium acetate at a concentration of 20-30 mmol / L, and the deuterated sheath solution is a mixture of 0.3% deuterated formic acid aqueous solution and deuterated methanol, with a volume ratio of 45:55 between the 0.3% deuterated formic acid heavy aqueous solution and deuterated methanol; the flow rate of the deuterated sheath solution is 1 μL / min.

6. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 1, characterized in that, In steps S03 and S04, the capillary used for capillary electrophoresis is a neutral-coated capillary coated with hydroxypropyl cellulose, with a length of 70-90 cm.

7. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 1, characterized in that, In steps S03 and S04, the sample introduction conditions for capillary electrophoresis are 1 psi pressure for 5 s, and the separation conditions for capillary electrophoresis are 28 kV positive voltage and 0.5-3 psi auxiliary pressure.

8. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 1, characterized in that, In steps S03 and S04, the detection conditions for the primary mass spectrometry and tandem mass spectrometry are as follows: scanning in positive ion mode, electrospray voltage of 3000 V, ion transmission tube temperature of 200 °C, and resolution of 60000. m / z The scanning range is 150-2000, the ion optical radio frequency voltage is 60%, the maximum injection time is 100 ms, and the automatic gain control is 100%. The tandem mass spectrometry utilizes an electron transfer-high-energy collision dissociation mode, where the reaction time of electron transfer dissociation is 2-6 ms and the energy of high-energy collision dissociation is 1-10%.

9. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 1, characterized in that, In step S05, calculating the main chain deuteration level of a single amino acid residue based on the tandem mass spectra of the protein before and after deuteration specifically includes the following steps: Step 1: Record fragment ions based on the tandem mass spectra of the protein before and after deuteration. m / z The numerical value and charge number are used to determine the mass of fragment ions before and after deuteration; the mass of fragment ions is calculated according to formula (1). c The total deuteration number of the peptide obtained by ion assignment is calculated according to formula (2). z The total deuteration number of the peptide obtained by ion assignment; ; ； Suppose a protein is composed of X amino acids, where, The total deuteration number of the peptide segment representing the first to m amino acid residues at the N-terminus; Represents the total deuteration generation of the peptide segment at the (Xn)-Xth amino acid residue at the C-terminus; and Represent 2 H and 1 The atomic mass of H; and Represent and The mass of fragment ions after deuteration; and Represent and The mass of the fragment ions before deuteration; the subscripts m and n represent respectively c ion, z The ion's serial number; Step 2, calculate the result according to formula (3) from c The deuterium algebra number of the peptide backbone to which the ion belongs; calculated according to formula (4) z The deuterium algebra of the peptide backbone to which the ion is assigned; ； ； in, and These represent the deuterium generation number of the peptide backbone and the number of hydrogen atoms in the side chain of the N-terminal 1-m amino acid residues, respectively; and These represent the deuterium generation number of the peptide backbone and the number of hydrogen atoms in the side chain of the (Xn)-Xth amino acid residue at the C-terminus, respectively. Step 3: Calculate the main chain deuteration level of individual amino acid residues, and calculate the level using formula (5). c The main chain deuteration level of a single amino acid residue obtained by ionization; calculated according to formula (6) z The deuteration level of the main chain of a single amino acid residue obtained by ionization; ； ； Where the subscript 'i' represents the sequence number of the protein's amino acid residue, Represents the main chain deuteration level of the i-th amino acid residue; The number of hydrogen atoms in the main chain of a peptide segment representing the first to m amino acid residues at the N-terminus; This represents the number of hydrogen atoms in the main chain of the peptide segment from amino acid residue (Xn) to X at the C-terminus.

10. The method for online hydrogen-deuterium exchange mass spectrometry analysis of protein structure according to claim 9, characterized in that, Step three also includes: for cases where the main chain deuteration level of individual amino acid residues cannot be obtained due to the loss of some fragment ions, c The main chain deuteration level of the ions is calculated according to formula (7). z The main chain deuteration level of the ions is calculated according to formula (8); ； ； Where j represents the number of missing amino acid residues between two adjacent fragment ions.

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