Antibody variable region sequencing method based on middle-to-bottom proteomics technology
By integrating multi-charge selection ion scanning strategy, ultraviolet dissociation mass spectrometry technology and map integration algorithm, the antibody variable regions are sequenced, which solves the problem of insufficient antibody sequencing coverage in the prior art, and achieves efficient and accurate antibody variable region sequencing.
Patent Information
- Application Number
- CN202311756548.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-20
- Publication Date
- 2025-06-20
AI Technical Summary
The existing antibody sequencing analysis methods are difficult to achieve high coverage sequencing, especially in sequence sequencing of antibody variable regions, where there is a problem of insufficient sequence coverage.
Using a method that integrates multi-charge selective ion scanning strategy, ultraviolet dissociation mass spectrometry technology and map integration algorithm, the variable regions of the antibody are sequenced through a liquid-mass CTG. The specific steps include cleavage of antibodies using specific cleavage enzymes, performing chemical reduction and alkylation, and then detecting the retention time and charge distribution of antibody subunits through liquid chromatography tandem mass spectrometry, combining ultraviolet laser dissociation technology for secondary fragmentation, and finally obtaining high-quality antibody subunit mass spectrometry dissociation maps through a map integration algorithm.
Complete sequencing of antibody variable regions has been achieved, significantly improving sequence coverage, and accurately identifying antibody amino acid sequences and post-translation modifications, providing a novel and efficient antibody sequencing method.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to an antibody sequencing analysis method for middle-down proteomics, specifically an antibody high-coverage sequencing analysis method that integrates multiple technologies such as multi-charge selected ion scanning strategy, ultraviolet photodissociation mass spectrometry technology, and spectrum integration algorithm. Background Art
[0002] Therapeutic monoclonal antibody drugs have become the best-selling drugs in the pharmaceutical market due to their high specificity and low adverse reactions, showing unique advantages and achieving great progress in the fields of diseases such as tumors, autoimmune diseases, metabolism, and viral infections. The specific binding of an antibody to an antigen mainly depends on the variable region at the N-terminus of the antibody. There are three complementary determining regions in this region, and their amino acid sequences vary highly. Therefore, the sequencing of the antibody variable region plays a crucial role in revealing the function of the antibody and clarifying the interaction mechanism between the antibody and the antigen.
[0003] Currently, the middle-down mass spectrometry method has become a very mature method in antibody sequencing analysis, combining the advantages of the bottom-up and top-down methods and alleviating their disadvantages. A specific cleavage enzyme is used to cleave the antibody hinge region, and then through chemical reduction and alkylation capping, three subunit parts of the antibody are obtained. Compared with the intact monoclonal antibody of the top-down method, the molecular weight of this subunit fragment is more in line with the method and technical characteristics of liquid chromatography-mass spectrometry.
[0004] Ultraviolet laser dissociation has currently become a very mature technology. Compared with traditional collision dissociation, ultraviolet laser dissociation technology directly excites the polypeptide backbone, thereby causing sufficient dissociation of the protein. It can not only generate b, y fragment ions, but also generate a large number of high-abundance a, x, c, z fragment ions, and can provide extremely rich protein sequence, modification, and structural characteristic fragment ions. The ultraviolet laser dissociation technology is combined with liquid chromatography-tandem mass spectrometry to perform characterization sequencing analysis on the macromolecular antibody subunits. Summary of the Invention
[0005] The present invention relates to an antibody variable region sequencing method based on middle-down proteomics technology, integrating multiple technologies such as multi-charge selected ion scanning strategy, ultraviolet photodissociation mass spectrometry technology, and spectrum integration algorithm, and realizing the complete sequencing of the antibody variable region on a liquid chromatography-mass spectrometer.
[0006] Technical Solution of the Present Invention:
[0007] (1) Use a specific cleavage enzyme to cleave the antibody. After chemical reduction and alkylation capping, three different subunits of the antibody are obtained. The antibody subunits are detected by liquid chromatography-tandem mass spectrometry to confirm the retention time and charge distribution of each antibody subunit.
[0008] (2) After determining the retention time and charge state distribution of each subunit, set the mass spectrometry selected ion scanning mode to perform continuous secondary fragmentation within the retention time of different subunits. Combining with the ultraviolet photodissociation of the ultraviolet laser, the secondary spectra of a single charge state of all three subunits can be obtained in a single liquid chromatography-mass spectrometry (LC-MS). A high-quality spectrum with optimized signal-to-noise ratio can be obtained by averaging all the spectra of the same subunit.
[0009] (3) After multiple selected ion scans, multiple secondary spectra with different charge states and high signal-to-noise ratios are obtained for each subunit. These data are deconvoluted and integrated together for sequencing analysis, ultimately achieving the goal of complete sequencing of the antibody variable region.
[0010] In step (1), after opening the disulfide bond by chemical reduction, an alkylating agent is required for alkylation capping to prevent the reclosure of the disulfide bond.
[0011] In step (1), the molecular weight of the antibody subunit is between 1000 daltons and 200000 daltons.
[0012] In step (2), the number of spectra averaged for each charge is 2 to 10000.
[0013] In step (2), the wavelength range of the ultraviolet laser is 50 - 300 nm.
[0014] In step (3), the integrated charge range for each subunit includes 5+ to 50+.
[0015] The present invention utilizes the complementarity of fragment ions generated between different charges of each subunit, combines the advantages of ultraviolet laser dissociation, and integrates the fragment ions of multiple charges together, resulting in a significant increase in the sequence coverage rate of the antibody. This method is rapid, simple, stable, and efficient. Compared with other sequencing methods, this method can obtain a higher coverage rate, providing a new and efficient method for sequencing the antibody variable region.
[0016] The present invention integrates a multi-charge selected ion scanning strategy, ultraviolet photodissociation mass spectrometry technology, and a spectrum integration algorithm. The antibody is enzymatically digested to obtain multiple subunits, separated by liquid chromatography, ionized by electrospray ionization and introduced into the mass spectrometer, then efficiently excited and dissociated by ultraviolet laser to generate fragment ions, and finally the fragment ions are detected by the mass spectrometer. A high-quality mass spectrometry dissociation spectrum of the antibody subunit is obtained by using a spectrum integration and averaging algorithm, and finally full-sequence coverage sequencing is performed using the complementarity of fragment ions of different charge states. The method proposed by the present invention can accurately identify the amino acid sequence and post-translational modification of the antibody, significantly improve the antibody sequence coverage rate, and achieve complete sequencing of the antibody variable region. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1It is a schematic flow chart for the preparation and collection of monoclonal antibody samples.
[0018] Figure 2 It is the base peak chromatogram and charge distribution after the enzymatic digestion of trastuzumab.
[0019] Figure 3 It is a schematic diagram of the superposition effect of ion peaks with different charge states in the light chain part of trastuzumab.
[0020] Figure 4 It is the base peak chromatogram and charge distribution after the enzymatic digestion of infliximab.
[0021] Figure 5 It is a schematic diagram of the superposition effect of ion peaks with different charge states in the light chain part of infliximab. Detailed implementation manners
[0022] The present invention will be described in detail below with reference to specific embodiments.
[0023] Example 1
[0024] Sequencing analysis of trastuzumab
[0025] (1) Sample preparation: Trastuzumab was dissolved in phosphate buffered saline at a concentration of 5 μg / μl. 50 μg of monoclonal antibody sample with a volume of 10 μl was taken. The antibody hinge region was specifically cleaved using immunoglobulin G degrading enzyme (IdeS, 2.5 μl) at 37 °C for 1 hour. The chemical reducing agent tris(2-carboxyethyl)phosphine (TCEP, final concentration 5 mM) was added and reacted for 30 minutes to break the disulfide bonds. The alkylating agent iodoacetamide (IAA, final concentration 10 mM) was added and reacted for 30 minutes. After the reaction, desalting was carried out using a desalting column. Then the sample was freeze-dried using a vacuum freeze dryer and re-dissolved with 0.1% formic acid in volume concentration.
[0026] (2) Liquid chromatography - mass spectrometry analysis: The sample was diluted to 100 ng / μl with 0.1% formic acid in volume concentration, and 2 μl was injected. The packing material was a butylsilane - bonded silica gel chromatographic column (C4). The analytical column had an inner diameter of 75 μm and a packing particle size of 3 μm, and the pre - column had an inner diameter of 200 μm and a packing particle size of 5 μm. The sample was first injected onto the pre - column, then eluted through the analytical column and into the mass spectrometer. Mobile phase A was 0.1% formic acid and 99.9% water in volume concentration, and mobile phase B was 0.1% formic acid and 99.9% acetonitrile. The elution gradient was as follows: from 0 - 5 min, mobile phase B increased from 5% to 35%; from 5 - 35 min, mobile phase B increased from 35% to 65%; from 35 - 60 min, mobile phase B increased from 65% - 90%. Before collecting the second - order spectrum, the retention time and charge - state distribution of the sample were first determined by the full - scan mode. The first - order spectrum scanning range was 400 - 2000 m / z, the resolution was 15000, the voltage was 2000 V, and the ion source temperature was 305 °C. The base - peak chromatogram of trastuzumab after enzymatic digestion is as Figure 2 shown, which shows the retention times of the three subunits and the charge distribution (from 10+ to 30+). From left to right, they are the C - terminal part of the antibody heavy chain (Fc / 2), the antibody light chain (Lc), and the N - terminal part of the antibody heavy chain (Fd). For each subunit, ion peaks with different charge states were selected for isolation and second - order dissociation.
[0027] (3) Data acquisition: After determining the retention times and charge distributions of the three subunits of trastuzumab, the second - order spectra were continuously collected within the retention - time range by the selected - ion scanning mode. The second - order scanning interval for the Fc / 2 subunit was 25 - 27 min, for the Lc subunit was 28 - 30 min, and for the Fd subunit was 30 - 32 min. The scanning range was 350 - 2000 m / z, the resolution was 240000, the maximum injection time was 500 ms. For the mass spectrometer with a linear ion trap, the pulsed extreme ultraviolet laser was introduced into the linear ion trap along the axial direction of the linear ion trap. The ultraviolet laser dissociation wavelength was 300 nm and the energy was 1 mj.
[0028] (4) Analysis results: Taking the light - chain part of trastuzumab as an example, 700 second - order spectra of the 21+ charge state collected by the single - selected - ion scanning mode were averaged into 1 spectrum, which improved the signal - to - noise ratio of the spectrum and enhanced the spectrum quality. After de - convolution of the spectrum of a single charge state of the trastuzumab subunit by the TopFD software, the obtained fragment - ion information was matched with the protein sequence. In the ProSight Lite software, the detected molecular weight was compared with the theoretical molecular weight with a tolerance error of 20 ppm to confirm the composition of the generated fragment ions, and finally the antibody sequence coverage was obtained. As Figure 3As shown, the sequence coverage of a single charge state can reach over 50%. In the same way, the average spectra of 16+-30+ charge states are obtained. Through TopFD deconvolution, multiple sets of data obtained are imported into ProSight Lite for matching. The effect of superposition of different charges of the light chain part of trastuzumab is as Figure 3 shown. After adding 21+ and 23+, the coverage rate is close to 80%. By further superimposing the charge state data of 16+-30+, the sequence coverage of the light chain part reaches 97%. Continuing to perform charge state superposition analysis on the C-terminal part and N-terminal part of the heavy chain in the same way, the C-terminal part integrates 15+-29+ charges to reach a sequence coverage of 96%, while the N-terminal part integrates 20+-37+ to reach a sequence coverage of 98%. The high sequence coverage enables accurate coverage of the variable regions located at the N-terminals of the light chain and heavy chain, and at the same time post-translational modifications are identified, ultimately achieving complete sequencing of the antibody.
[0029] Example 2
[0030] Sequencing analysis of infliximab sample
[0031] (1) Sample preparation: Infliximab is dissolved in phosphate buffer at a concentration of 10 μg / μl. 50 μg of antibody sample with a volume of 5 μl is taken. Pepsin is used to specifically cleave the antibody hinge region, and the reaction is carried out at 37 °C for 30 minutes. The reducing agent tris(2-carboxyethyl)phosphine (TCEP, final concentration 5 mM) is added and reacted for 30 minutes to break the disulfide bonds. The alkylating agent iodoacetamide (IAA, final concentration 10 mM) is added and reacted for 30 minutes for alkylation capping. After the reaction, desalting is carried out using a desalting column. Then the sample is freeze-dried using a vacuum freeze dryer and redissolved with 0.1% formic acid.
[0032] (2) LC-MS analysis: The sample is diluted to 100 ng / μl and 2 μl is loaded. The analytical column has an inner diameter of 75 μm and a particle size of 3 μm, and the pre-column has an inner diameter of 200 μm and a particle size of 5 μm. The packing material is a polystyrene-divinylbenzene chromatographic column (PLRP-S). The sample is first loaded onto the pre-column and then eluted through the analytical column into the mass spectrometer. Mobile phase A is 0.1% formic acid and 99.9% water by volume, and mobile phase B is 0.1% formic acid, 80% acetonitrile and 19.9% water by volume. The elution gradient is as follows: 0-5 min, mobile phase B ranges from 5% to 45%; 5-40 min, mobile phase B ranges from 45% to 75%; 35-60 min, mobile phase B ranges from 75% to 90%. Before collecting the secondary spectrum, the retention time and charge state distribution of the sample are first determined through the full scan mode. The scanning range of the primary spectrum is 400-2000 m / z, the resolution is 15000, the voltage is 2000 V, and the ion source temperature is 305 °C. The base peak chromatogram of infliximab after enzymatic digestion is as Figure 4As shown, the retention times and charge distributions of the three subunits (from 15+ to 40+) are presented. From left to right, they are the C-terminal part of the antibody heavy chain (Fc / 2), the N-terminal part of the antibody heavy chain (Fd), and the antibody light chain part (Lc). Then, ion peaks of different charge states of each subunit are selected for isolation and tandem dissociation respectively.
[0033] (3) Data acquisition: After determining the retention times and charge distributions of the three subunits of trastuzumab, tandem spectra are continuously acquired within the elution time range by selected ion scanning mode. The tandem interval for the Fc / 2 subunit is 16 - 19 min, for the Fd subunit is 21 - 23 min, and for the Lc subunit is 23 - 25 min. The scanning range is 350 - 2000 m / z, the resolution is 240000, the maximum injection time is 500 ms, the ultraviolet laser dissociation wavelength is 50 nm, and the energy is 1.5 mj.
[0034] (4) Result analysis: Taking the light chain part of trastuzumab as an example, 500 tandem spectra of the 24+ charge state acquired by single selected ion scanning mode are averaged into 1 spectrum, significantly improving the signal-to-noise ratio. After averaging the single charge state spectra of the light chain part (Lc) of infliximab and deconvoluting them with the TopFD software, the fragment ion information obtained is matched with the protein sequence in the ProSight Lite software. The molecular weight detected is compared with the theoretical molecular weight with a tolerance error of 20 ppm to confirm the composition of the generated fragment ions, and finally the antibody sequence coverage rate is obtained. As Figure 5 shown, the sequence coverage rate of a single charge state can reach over 50%. Then, average spectra of the 18+ and 35+ charge states are obtained in the same way. After deconvoluting with TopFD, multiple groups of data obtained are imported into ProSight Lite for matching. The effect of superimposing the 10+ and 24+ charges of infliximab is as Figure 5 shown. The coverage rate is close to 80% after the two are added together. By further superimposing the charge state data of 10+ - 24+, the sequence coverage rate of the light chain part reaches 99%. Continuing to analyze the charge state superposition of the C-terminal and N-terminal parts of the heavy chain in the same way, the C-terminal part integrates the 14+ - 29+ charges to reach a sequence coverage rate of 96%, while the N-terminal part integrates the 18+ - 35+ charge data to reach a sequence coverage rate of 98%. The high sequence coverage rate enables accurate coverage of the variable regions located at the N-terminals of the light and heavy chains, and at the same time, post-translational modifications are accurately identified, finally achieving the complete sequencing of the antibody.
Claims
1. An antibody variable region sequencing method based on top-down proteomics technology, characterized in that: The intact monoclonal antibody is enzymatically digested by a specific cleavage enzyme, followed by chemical reduction and alkylation capping to obtain multiple subunits. These subunits are separated by liquid chromatography and then subjected to mass spectrometry detection. First, the retention time and charge state distribution are determined on the spectrum through a full-scan mode of mass spectrometry. Then, the selected ion scan mode is used to select different charge states for data acquisition. In the linear ion trap of the mass spectrometer, the second-order spectrum of each subunit is obtained through ultraviolet laser dissociation. The spectra of a single charge state are averaged into one spectrum as a set of data. Deconvolution software is used to obtain fragment information. Then, the fragment information of different charge states of the same subunit is added together. By utilizing the complementarity of fragment ions of different charge states and matching them with the antibody sequence, the sequence coverage of a single subunit is obtained. Using the same method for charge state superposition sequencing of other subunits, the sequence coverage of all subunits is finally obtained, achieving the complete sequencing of the antibody.
2. The method according to claim 1, characterized in that: The selected ion scan strategy isolates and continuously dissociates the selected single charge, combined with ultraviolet laser dissociation mass spectrometry technology, to obtain multiple second-order spectra within the elution time of liquid chromatography.
3. The method according to claim 1 or 2, characterized in that: The map integration algorithm is used to average the multiple maps obtained by selected ion scanning to obtain a high-quality map with a high signal-to-noise ratio. The integration quantity includes 2 to 10,000 maps.
4. The method according to claim 1, characterized in that: The superposition of fragment ions between different charges, with the number including two or more charges, and the charge selection range including 5+ to 50+.
5. The method according to claim 1, characterized in that: The analytical column packing materials used in liquid chromatography tandem mass spectrometry include, but are not limited to, one or more of C18, C4, and PLRP-S, etc.
6. The method according to claim 1, characterized in that: The types of antibodies described include all monoclonal antibodies and polyclonal antibodies. The antibody subunit is obtained by enzymatically digesting the intact monoclonal antibody, followed by chemical reduction and alkylation capping.
7. The method according to claim 6, characterized by: Each antibody can obtain multiple different subunits, with their molecular weights ranging from 1000 daltons to 200,000 daltons.
8. The method according to claim 1 or 6, characterized by: The enzymes specifically include, but are not limited to, one or two enzymes for proteolysis such as IdeS protease and papain, etc.
9. The ultraviolet laser dissociation according to claim 1, 2, characterized in that: For a mass spectrometer with a linear ion trap, a pulsed extreme ultraviolet laser is introduced into the linear ion trap along the axial direction of the linear ion trap. Its ultraviolet light wavelength is 50 - 300 nm, and the energy is 0.1 mj - 10 mj.