Method for analyzing sequence and modification of polypeptide or protein

Abstract

The invention relates to a polypeptide and protein sequence analysis method. To-be-detected polypeptide and protein are ionized under a denaturation condition and transferred into a gas phase to form polypeptide and protein ions with multiple charge valence distribution, and sequences and modification information of the polypeptide and protein ions are kept. These polypeptides and multi-charged protein ions are then ionically dissociated with a pulsed extreme ultraviolet laser to produce fragment ions. Fragment ions comprise single-charge and multi-charge ions, and comprise polypeptide and protein sequences and site modification information. The amino acid sequence and site modification information of the protein can be obtained through molecular weight difference matching of series of sequence characteristic fragment ions. According to the method, the analysis of the extreme ultraviolet band pulse laser on the polypeptide and protein sequences is realized for the first time internationally, the dissociation efficiency of the complete polypeptide and protein can be remarkably improved, and the accurate analysis of the complete protein and long peptide fragment sequences is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomass spectrometry, and particularly relates to a method for determining the sequences of denatured polypeptides and intact proteins based on extreme ultraviolet lasers. Background Art

[0002] With the advent of the post-genomic era, more and more studies have shown that the discovery of new domains and functional sites in proteins is of great significance for in-depth understanding of biological processes and functions and providing key information on diseases. Identifying new domains and functional sites in proteins is crucial for promoting the development of application fields such as new drug research and development, synthetic biology, and personalized medicine.

[0003] For a new or uncharacterized protein, the inference of its domains and functional sites is usually based on homologous proteins with similar properties. The amino acid sequence similarity of proteins is considered the most important basis for proving homologous mapping. In addition, as the link between protein structure and coding genes, the amino acid sequence of proteins also provides a link for cell physiology and genetics. Therefore, it is extremely important to accurately obtain the sequence information of proteins.

[0004] Currently, the most commonly used protein sequencing method is the bottom-up mass spectrometry method. This method obtains the mass-to-charge ratio information of peptide fragments by fragmenting the peptide samples after enzymatic digestion through multi-stage mass spectrometry, and infers the peptide sequence through a database, connecting fragments with a specific mass difference increase at the N-terminus or C-terminus. This method can detect multiple proteins simultaneously and greatly improve the accuracy of sequence determination. However, different scans of the same peptide fragment during the chromatographic separation of complex peptide mixtures greatly increase the data redundancy and generate a large number of unassignable peptide tandem mass spectra. How to infer the protein sequence from the large amount of mass spectrometry information obtained remains a huge challenge. In addition, the complex modification information in intact proteins also increases the difficulty of protein analysis in mass spectrometry.

[0005] The top-down tandem mass spectrometry method provides a new possibility for protein sequencing. This method can directly obtain sequence and modification information from the gas-phase dissociation of intact protein ions, and achieve full coverage of the protein sequence through database search. However, traditional ion activation methods such as HCD and ETD have low dissociation efficiency, are difficult to obtain complete sequence information, and can only generate a single type of ion, with low identification credibility. How to improve the ion activation efficiency of intact protein ions, obtain more reliable sequence information, and thus achieve full coverage of the protein sequence remains a technical problem. Summary of the Invention

[0006] The present invention aims to develop a method for sequencing polypeptides and proteins based on extreme ultraviolet laser dissociation technology to address the problems of difficult identification of modification sites during mass spectrometry sequencing and low dissociation efficiency of traditional ion activation technology for long peptides, making it difficult to perform complete sequence and modification analysis. This method relies on extreme ultraviolet laser dissociation technology to transfer denatured polypeptides and proteins that retain complete sequence and polypeptide information into the gas phase through an electrospray ionization source, generating molecular weight information that includes all amino acid compositions and modification information of the polypeptides and proteins. Then, extreme ultraviolet lasers with wavelengths ranging from 60 nanometers to 180 nanometers are used to fragment the polypeptides and proteins, generating a series of fragment ions such as a-, x-, b-, y-, c-, z-. By comparing with the molecular weights of the intact polypeptides and proteins, the amino acid sequence compositions of the polypeptides and proteins can be obtained, and based on the difference between the theoretical amino acid mass and the actually detected amino acid mass, precise matching of protein modifications can be achieved.

[0007] To achieve the above object, the present invention provides, but is not limited to, the following new method for determining the sequences of polypeptides and proteins based on extreme ultraviolet lasers:

[0008] (1) For freeze-dried polypeptide and protein samples without salt, dissolve them in a methanol aqueous solution of 5% to 95% and add 1% to 10% formic acid, with the protein concentration being 5 micromolar to 500 micromolar. For polypeptide and protein samples obtained by separation and purification, replace the buffer solution with an ammonium acetate buffer solution of 10 millimolar to 200 millimolar, add 1% to 10% formic acid, and dilute the polypeptide and protein concentration to 5 micromolar to 500 micromolar with a methanol aqueous solution of 5% to 95%.

[0009] (2) Inject the sample solution into the mass spectrometer through a capillary with a frustum-shaped tip and an inner diameter at the micron level via an ion source to form polypeptide and protein ions.

[0010] (3) Connect the extreme ultraviolet laser to the mass spectrometry dissociation trap through a high-vacuum pipeline. The polypeptide and protein ions that retain complete modification and sequence information in step (2) are aggregated in the mass spectrometry dissociation trap, and are ionized and dissociated by pulsed extreme ultraviolet lasers to generate a series of fragment ions such as a-, x-, b-, y-, c-, z- that have lost 1 to multiple amino acids; the pulsed laser has a higher single-photon energy, can generate more types and quantities of fragment ions, and significantly improves the mass spectrometry fragment signal intensity and signal-to-noise ratio.

[0011] (4) Perform data matching on the mass spectrometry data obtained in step (3) to confirm the sequence and modification information of the polypeptides and intact proteins.

[0012] The present invention realizes the analysis of polypeptide and protein sequences by pulsed laser in the extreme ultraviolet band for the first time internationally, which can significantly improve the dissociation efficiency of intact polypeptides and proteins and achieve accurate analysis of the sequences of intact proteins and long peptide segments.

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

[0014] The present invention realizes the first sequence determination technology of extreme ultraviolet band laser for polypeptides and intact proteins in the world. Compared with traditional ion activation technologies, extreme ultraviolet lasers can generate more types of fragment ions and more fragment segments, so they can cover more protein sequence information and achieve almost full coverage of intact protein sequences up to 200,000 daltons. The wavelength is continuously adjustable and can excite the whole protein in the range of 60 nanometers to 180 nanometers. The single-photon energy is accurate, and it has higher repeatability and accuracy compared with other dissociation methods. Extreme ultraviolet lasers can generate a large number of peptide fragments in a short time, thus improving the detection sensitivity. Compared with the conventional bottom-up protein mass spectrometry sequencing method, this method does not require enzymatic digestion, can reflect the amino acid sequence composition information of more real polypeptides and proteins, does not require specific types of enzymes to participate, and can meet the sequencing of various types of proteins. Description of the Drawings

[0015] Figure 1 Shows the differences in the analysis of the horse myoglobin sequence between Example 1 of the present invention and Comparative Examples 1, 2, and 3.

[0016] Figure 2 Shows the dissociation of ubiquitin sequence in Example 2 of the present invention.

[0017] Figure 3 Shows the dissociation of cytochrome C sequence in Example 3 of the present invention.

[0018] Figure 4 Shows the dissociation of carbonic anhydrase sequence in Example 4 of the present invention.

[0019] Figure 5 Shows the dissociation spectrum of bovine serum albumin in Example 5 of the present invention. Detailed Description of the Invention

[0020] The following will describe the present invention in detail with specific examples. 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.

[0021] Example 1

[0022] Sequence analysis of horse myoglobin by extreme ultraviolet laser dissociation method:

[0023] 1. Sample preparation

[0024] Dissolve horse myoglobin in methanol / water / formic acid with a volume ratio of 50% / 49% / 1%, and the protein concentration is 10 micromolar concentration.

[0025] 2. Mass spectrometry detection

[0026] The protein sample is ionized by injecting it into a mass spectrometer (equipped with a quadrupole and a linear ion trap) through a capillary with a frustum-shaped tip and an inner diameter of 75 micrometers by an electrospray ionization source. The molecular weight information of the protein is determined in the positive ion mode. The denatured intact protein with all sequences and modifications is ionized by the electrospray ionization source, generating multiple different charge states, and all the amino acid sequences and modification information of the polypeptide / protein are recorded in the first-order mass spectrometry spectrum. Further select the myoglobin ion with a mass-to-charge ratio of 998.12 and 10 positive ions. Use the quadrupole to select and enrich the protein ions. The enriched protein ions are injected into the linear ion trap, and the mass- and charge-selected protein ions are ionized and dissociated by an extreme ultraviolet laser along the axial direction of the linear ion trap, generating fragment ions with rich sequence and site modification characteristics. Detect different types and valence states of fragment ions and perform data acquisition. The sequence and modification information generated by dissociation are detected and recorded by mass spectrometry.

[0027] The pulsed extreme ultraviolet laser is introduced into the linear ion trap along the axial direction of the linear ion trap. The working conditions of the extreme ultraviolet laser are: wavelength: 90 nanometers; frequency: 10 hertz; single pulse energy: 5 microjoules; pulse width: 1 picosecond. The electron beam energy is 300 megaelectron volts.

[0028] Mass spectrometry conditions: electrospray ionization source: positive ion mode; spray voltage: 1800 volts; source temperature: 275 degrees Celsius; resolution: 240,000; RF Lens: 60%; AGC: 5e6; maximum injection time: 500 milliseconds. 3. Data analysis

[0029] Use Xtract to deconvolute the detected mass spectrometry spectrum to obtain the molecular weight information of the fragment ions. Compare the detected molecular weight with the theoretical molecular weight with a tolerance error of 20 ppm to determine the type of fragment ions generated, and confirm the sequence composition of the polypeptide and protein according to the mass difference between different fragments. Use the ProSight Lite software developed by Northwestern University to determine the sequence resolution rate.

[0030] Comparative example 1

[0031] Sequence analysis of horse myoglobin by high-energy collision-induced dissociation method

[0032] The process and conditions are the same as those in Example 1. The difference between this comparative example and Example 1 lies in that in the mass spectrometry conditions in Step 2, the fragmentation mode of the second-level scan is a single high-energy collision dissociation (without pulsed extreme ultraviolet laser injection), and the fragmentation energy is 25%.

[0033] Comparative Example 2

[0034] Sequence analysis of equine myoglobin by electron transfer dissociation coupled with high-energy collision-induced dissociation method

[0035] The process and conditions are the same as those in Example 1. The difference between this comparative example and Example 1 and Comparative Example 1 lies in that in the mass spectrometry conditions in Step 2, the fragmentation mode of the second-level scan is electron transfer dissociation coupled with high-energy collision dissociation (without pulsed extreme ultraviolet laser injection). Among them, the action time of electron transfer dissociation is 10 milliseconds, and the fragmentation energy of high-energy collision dissociation is 15%.

[0036] Comparative Example 3

[0037] Sequence analysis of equine myoglobin by 193-nm ultraviolet photodissociation method

[0038] The process and conditions are the same as those in Example 1. The difference between this comparative example and Example 1, Comparative Example 2, and Comparative Example 3 lies in that in the mass spectrometry conditions in Step 2, the fragmentation mode of the second-level scan is 193-nm ultraviolet single-photon dissociation (without pulsed extreme ultraviolet laser injection). The frequency of the ultraviolet light is 10 Hz, and the single-pulse energy is 1 mJ.

[0039] Figure 1 Show the differences in the sequence analysis of equine myoglobin between Example 1 of the present invention and Comparative Examples 1, 2, and 3, where Figure 1 a The selected precursor ions undergo high-energy collision-induced dissociation fragmentation, Figure 1 b The selected precursor ions undergo electron transfer dissociation coupled with high-energy collision-induced dissociation fragmentation, Figure 1 c The selected precursor ions undergo 193-nm ultraviolet photodissociation fragmentation, Figure 1 d The selected precursor ions undergo 90-nm extreme ultraviolet laser photodissociation fragmentation. In ultraviolet photodissociation and extreme ultraviolet laser photodissociation, the laser can use a single pulse or multiple pulses. In the present invention, a single pulse is used. The sequence analysis rates of high-energy collision-induced dissociation, electron transfer dissociation coupled with high-energy collision-induced dissociation, 193-nm ultraviolet photodissociation, and 70-nm extreme ultraviolet laser photodissociation for myoglobin are 8%, 86%, 95%, and 95% respectively, and the numbers of fragments are 309, 536, 817, and 1390 respectively. The extreme ultraviolet laser can achieve complete sequence analysis of the protein and generate more fragment ions.

[0040] Example 2

[0041] Sequence analysis of ubiquitin by 80-nm, 36-µJ extreme ultraviolet free electron laser

[0042] 1. Sample Preparation

[0043] Dissolve ubiquitin in methanol / water / formic acid with a volume ratio of 50% / 49% / 1%, and the protein concentration is 10 micromolar.

[0044] 2. Mass Spectrometry Detection

[0045] The protein sample enters the ion source through a capillary with a frustum-shaped tip and an inner diameter of 75 micrometers to form protein ions. The molecular weight information of the protein is determined in the positive ion mode. Further select myoglobin ions with a mass-to-charge ratio of 1428 and 6 positive ions. Use a quadrupole to select and enrich the protein ions. The enriched protein ions are injected into a linear ion trap and ionized and dissociated with an extreme ultraviolet laser along the axial direction of the linear ion trap, and data acquisition is performed.

[0046] Operating conditions of the extreme ultraviolet laser: wavelength: 80 nanometers; frequency: 10 hertz; single pulse energy: 36 microjoules; pulse width: 1 picosecond.

[0047] Mass spectrometry conditions: electrospray ionization source: positive ion mode; spray voltage: 1000 volts; source temperature: 275 degrees Celsius; resolution: 240,000; RF Lens: 60%; AGC: 5e6; maximum injection time: 500 milliseconds.

[0048] 3. Data Analysis

[0049] Use Xtract to deconvolute the detected mass spectrometry diagram to obtain the molecular weight information of the fragment ions. Compare the detected molecular weight with the theoretical molecular weight with a tolerance error of 20 ppm to determine the type of fragment ions generated, and confirm the sequence composition of polypeptides and proteins based on the mass differences between different fragments. Obtain the modification information on amino acids according to the mass differences between theoretical and actual amino acids. For example, a modification with an N-terminal molecular weight of 42.01 is defined as acetylation modification. Use the ProSight Lite software developed by Northwestern University to determine the sequence resolution rate.

[0050] Figure 2 Show the dissociation of the human ubiquitin sequence in Example 2 of the present invention. Protein ubiquitination is a very common post-translational modification in organisms. When ubiquitin is attached to a substrate, the substrate is further modified. And this ubiquitination-mediated protein degradation plays an important role in gene transcription, DNA repair, and cell cycle regulation. The way ubiquitin is attached to the substrate can include attaching a single ubiquitin to one position of the protein, or multiple ubiquitin molecules forming a chain and then attaching to different positions of the protein. Analyzing the ubiquitin sequence has great physiological significance in regulating the cell cycle.

[0051] In this experimental example, the sequence analysis rate of the 80-nanometer, 36-microjoule extreme ultraviolet laser for ubiquitin reached 100%.

[0052] Example 3

[0053] Analysis of the sequence of horse cytochrome c by an 80-nanometer, 21-microjoule extreme ultraviolet free electron laser

[0054] The process and conditions are the same as in Example 2. The difference between this example and Example 2 lies in the sample preparation in Step 1. Ubiquitin was dissolved in methanol / water / formic acid with a volume ratio of 50% / 49% / 1%, and the protein concentration was 5 micromolar. In the mass spectrometry detection in Step 2, the selected and enriched ion was the cytochrome c ion with a mass-to-charge ratio of 1223 and 10 positive charges. The working conditions of the extreme ultraviolet laser were single-pulse energy: 21 microjoules.

[0055] Figure 3 Figure 3 shows the dissociation of the horse cytochrome c sequence in Example 3 of the present invention. Cytochrome c is a common electron transfer protein in organisms, participating in the electron transfer process in the mitochondrial respiratory chain and many biological processes such as apoptosis, with a molecular weight of 12 kDa. Therefore, studying the cytochrome c sequence helps to understand cytochrome c in other organisms and proteins with cytochrome c-like domains, and provides new ideas and methods for the treatment and prevention of related diseases.

[0056] In this experimental example, the sequence analysis rate of the 80-nanometer, 21-microjoule extreme ultraviolet laser for cytochrome c reached 96%.

[0057] Example 4

[0058] Analysis of the sequence of carbonic anhydrase by a 75-nanometer, 0.4-microjoule extreme ultraviolet free electron laser

[0059] The process and conditions are the same as in Example 2. The difference between this example and Example 2 lies in the sample preparation in Step 1. Carbonic anhydrase was dissolved in methanol / water / formic acid with a volume ratio of 50% / 49% / 1%, and the protein concentration was 10 micromolar. In the mass spectrometry detection in Step 2, the selected and enriched ion was the carbonic anhydrase ion with a mass-to-charge ratio of 907.92 and 32 positive charges. The working conditions of the extreme ultraviolet laser were wavelength: 75 nanometers; single-pulse energy: 0.4 microjoules.

[0060] Figure 4Example 4 of the present invention shows the dissociation of the carbonic anhydrase sequence. Carbonic anhydrase is a type of metalloenzyme widely present in and outside living organisms. It catalyzes the interconversion between carbon dioxide and bicarbonate and other hydrolysis reactions, which is one of the important chemical equilibrium reactions in intracellular circulation. In the human body, carbonic anhydrase is an important protein responsible for regulating important physiological processes such as maintaining acid-base balance, regulating ion exchange, and body metabolism, and its molecular weight is approximately 30,000 daltons.

[0061] In this experimental example, the sequence analysis rate of the 75-nanometer, 0.4-microjoule extreme ultraviolet laser for carbonic anhydrase reached 80%.

[0062] Example 5

[0063] Analysis of the bovine serum albumin sequence by an 80-nanometer, 31-joule extreme ultraviolet free electron laser

[0064] The process and conditions are the same as those in Example 2. The difference between this example and Example 2 lies in the sample preparation in step 1. Bovine serum albumin is dissolved in methanol / water / formic acid with a volume ratio of 50% / 49% / 1%, and the protein concentration is 5 micromolar. In the mass spectrometry detection in step 2, the selected and enriched ions are bovine serum albumin ions with a mass-to-charge ratio of 2014 and 33 positive charges. The working conditions of the extreme ultraviolet laser are wavelength: 80 nanometers; single-pulse energy: 31 microjoules.

[0065] Figure 5 Example 5 of the present invention shows the dissociation of the bovine serum albumin sequence. Albumin is a globular protein that carries and transports various exogenous or endogenous ions, fatty acids, and small molecules in the body and plays a key role in maintaining plasma pressure and nutritional balance. Bovine serum albumin, as a protein with high structural similarity to human serum albumin, has been widely studied as a protein model, and its molecular weight is approximately 67,000 daltons.

Claims

1. A method for analyzing a polypeptide or protein sequence and modification, characterized in that: It analyzes the sequences and / or modifications of denatured polypeptides and / or proteins based on pulsed extreme ultraviolet laser dissociation and mass spectrometry detection; after the denatured polypeptides and / or proteins are ionized, multiple different charge states are generated. Select the charge states corresponding to 1 to 8 target polypeptides / proteins, and perform gas-phase dissociation on the polypeptide and / or protein ions respectively by pulsed extreme ultraviolet laser in the linear ion trap of the mass spectrometer to generate fragment ions with rich sequence and site modification characteristics. Use the mass spectrometer to detect different types and valence states of fragment ions, and perform matching analysis on the fragment ion mass spectrometry data to obtain the amino acid sequence and / or site modification information of the polypeptide and / or protein.

2. The analysis method according to claim 1, wherein: Methanol and formic acid are used to destroy the spatial structure of intact polypeptides and / or proteins while retaining the sequence and modification information of the proteins; the concentration of the polypeptides and / or proteins is 2 micromolar to 500 micromolar. The specific process is as follows: Dissolve the polypeptide and / or protein in an aqueous methanol solution with a volume concentration of 5% to 95%, and add formic acid with a final volume concentration of 1% to 10%. The protein concentration is 2 to 500 micromolar; or, for the polypeptide and / or protein sample obtained by separation and purification, replace the buffer solution with an ammonium acetate buffer solution with a concentration of 10 to 200 millimolar, add formic acid with a final volume concentration of 1% to 10%, and dilute the polypeptide and protein concentration to 2 micromolar to 500 micromolar with an aqueous methanol solution with a volume concentration of 5% to 95%.

3. The analysis method according to claim 1 or 2, characterized in that: The protein source is a protein sample extracted from one or more than two biological samples such as cell tissue, body fluid, serum, urine, etc.; or, it can also be a polypeptide sample obtained by shearing the extracted protein sample by a specific enzyme or chemical method.

4. The analysis method according to claim 1, wherein: The ionization is carried out using an electrospray ion source, and the voltage for electrospray ionization is 800 to 3500 volts, and the ion source temperature is 150 to 350 degrees Celsius.

5. The analysis method according to claim 1, wherein: The pulsed extreme ultraviolet laser is introduced into the linear ion trap along the axial direction of the linear ion trap to ionize and dissociate the polypeptide and / or protein ions. The wavelength range of the pulsed laser is 60 nanometers to 180 nanometers (preferably 70 nanometers to 150 nanometers), the pulse intensity is 0.5 microjoule to 1000 microjoules (preferably 2 microjoules to 40 microjoules), the electron beam energy is 100 - 500 megaelectron volts (preferably 250 to 350 megaelectron volts), and the pulse repetition frequency is 1 hertz to 100 hertz (preferably 10 to 50 hertz).

6. The analysis method according to claim 1 or 5, characterized in that: In the process of extreme ultraviolet pulsed laser dissociation, single or multiple laser pulses can be used to perform gas-phase dissociation on polypeptides and proteins.

7. The analysis method according to claim 1, characterized in that: Extreme ultraviolet pulsed laser dissociation - mass spectrometry sequencing process: The denatured intact polypeptides and / or proteins that retain all sequences and modifications are ionized through an electrospray ion source to obtain all amino acid sequences and modification information and record them in the first-level mass spectrometry spectrum; then, after precise mass and charge selection of the protein ions in the first-level mass spectrometry, they are dissociated by extreme ultraviolet laser, and the sequence and modification information generated by the dissociation are detected and recorded by the mass spectrometry.

8. The analysis method according to claim 1, wherein: The method can be widely applied to polypeptides and proteins with molecular weights ranging from 2,000 to 200,000 Daltons.

9. The analysis method according to claim 1, wherein: The polypeptide and protein sequences can be obtained by matching the molecular weight differences of a series of fragment ions, or the polypeptide and / or protein sequences can be determined by database search and matching with the theoretical dissociation spectra of the database; During the protein sequence matching process, the chemical modifications occurring at the sites can be analyzed and determined based on the differences between the amino acid residues at the sites and the theoretical molecular weights.

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