Methane-oxidizing bacteria protein extraction and mass spectrometry pre-treatment method

Abstract

This invention discloses a method for protein extraction and mass spectrometry pretreatment from methanogenic bacteria, belonging to the field of protein extraction technology. The method involves pretreating a methanogenic bacteria enrichment culture to remove inorganic salt ions and other impurities, resulting in a pretreated culture. Then, a lysis buffer composed of sodium dodecyl sulfate, Tris-HCl buffer, and a protease inhibitor is added to the pretreated culture, followed by ultrasonic disruption to obtain a cell lysate, from which a crude protein extract solution is obtained. Finally, the protein extract is extracted from the crude protein extract solution. This invention significantly improves protein extraction efficiency and peptide purity by optimizing the extraction process and subsequent enzymatic digestion, while preserving the methanogenic activity of the extracted protein as much as possible, providing more accurate and reliable sample support for downstream protein mass spectrometry analysis.

Description

Technical Field

[0001] This invention belongs to the field of protein extraction technology, specifically relating to a method for extracting whole proteins from methanogenic bacteria and generating high-purity peptides through enzymatic hydrolysis. Based on this method, protein extraction efficiency and the purity of enzymatically hydrolyzed peptides can be significantly improved, providing high-quality samples for downstream protein mass spectrometry analysis and enhancing the accuracy and reliability of mass spectrometry analysis. Background Technology

[0002] Methane-oxidizing bacteria are a class of bacteria that can utilize methane as their sole carbon and energy source. They include two types: aerobic and anaerobic methane-oxidizing bacteria. They play a crucial role in the carbon cycle and methane emission reduction, and the extraction of their key functional proteins (such as methane monooxygenase) is of great significance for environmental pollution control, bioenergy development, and enzyme catalysis research.

[0003] Methane-dependent denitrifiers (MDs) oxidize methane while simultaneously reducing nitrate (NO3) levels. - ) or nitrite (NO2) - The reduction of nitrogen (N2) to nitrogen gas is an important component of the carbon and nitrogen cycle in natural habitats. Traditionally, it is believed that the entire denitrification process of methane-type organisms is mainly carried out by ANME-2d archaea (NO3). - →NO2 - )and Methylomirabilis Bacteria (NO2) - →N2) work synergistically. A recently discovered Methane-dependent complete denitrifying anaerobic methane-oxidizing bacterium (M-CD) Candidatus Methylomirabilis sinica ( M. sinica It can use methane as an electron donor to directly convert NO3. - Converting it into N2 is expected to achieve synergistic emission reduction of greenhouse gases N2O and CH4 in natural and artificial habitats, providing a new perspective for the sustainable management of greenhouse gases.

[0004] M. sinica It possesses unique physiological and biochemical characteristics, but its key enzymatic mechanisms remain to be elucidated. M. sinica First, NO3 is reduced by the nitrate reductase NapAB in the periplasmic space. - To NO2 - Then NO2 -Nitrite is converted to NO under the catalysis of cytochrome cd1-type nitrite reductase NirS. NO is further disproportionated by a currently unknown nitric oxide superoxide dismutase (Nod), ultimately producing N2 and O2. The intracellularly produced O2 is then used for methane oxidation by particulate methane monooxygenase (pMMO). M . sinica The structural and functional analysis of functional proteins relies on protein extraction methods and protein mass spectrometry. Due to the slow growth and long doubling time of this bacterium, the biomass obtainable within a limited timeframe is restricted. Therefore, there is an urgent need to develop an efficient protein extraction method to significantly improve the extraction rate. The development of this method can also provide a reference and be widely applied to the protein extraction of other Gram-negative bacteria with long growth cycles.

[0005] Protein mass spectrometry requires samples with high purity and stability, and necessitates the removal of impurities that may interfere with the analysis. Currently, protein extraction methods from Gram-negative bacteria typically employ non-ionic denaturing agents such as urea, leveraging its cell membrane-dissolving properties to enhance protein solubility. However, to mitigate the inhibitory effect of urea on subsequent trypsin digestion, the urea in the extraction system must be diluted before enzymatic digestion. This operation not only reduces protein concentration but also inevitably interferes with the trypsin digestion effect, further impacting peptide generation efficiency and quality. Furthermore, residual urea may trigger enzymatic side reactions, reducing the overall efficiency of protein extraction and the purity of the obtained peptides, directly leading to unsatisfactory protein mass spectrometry analysis results and limiting its application in proteomics research.

[0006] The catalytic activity of pMMO, a key membrane-bound copper enzyme in methane-oxidizing bacteria, plays a decisive role in the bio-oxidation of methane and is a core biocatalytic unit in applications such as greenhouse gas methane oxidation and bioenergy methanol production. pMMO is a transmembrane multi-subunit metalloenzyme complex, typically composed of three subunits (pmoA, pmoB, and pmoC) and containing a copper ion active center. Its unique membrane-bound properties and multi-subunit conformation make this enzyme highly dependent on the lipid bilayer environment to maintain conformational integrity and catalytic function. M. sinica Extracting and purifying pMMO from methanogenic bacteria is even more challenging. Current research on the structure and function of pMMO is based on aerobic methanogenic bacteria. However, the structure and methanogenic mechanism of anaerobic methanogenic bacteria in the NC10 phylum are more complex than those of aerobic methanogenic bacteria, so related research is currently lacking.

[0007] Given the physiological characteristics of methane-oxidizing bacteria and the technical challenges in protein extraction, there is an urgent need to develop an efficient and low-interference protein extraction and enzymatic digestion method to improve protein extraction rate and peptide purity, thereby providing a reliable guarantee for high-quality protein mass spectrometry analysis and functional protein research. Summary of the Invention

[0008] The purpose of this invention is to address the problems of low extraction efficiency, insufficient purity, and low methane oxidation activity of proteins from slowly proliferating Gram-negative bacteria. It provides a method for efficient protein extraction and pretreatment for mass spectrometry analysis from methane-oxidizing bacteria. By optimizing the extraction process and subsequent enzymatic digestion, this invention significantly improves protein extraction efficiency and peptide purity, and verifies the methane oxidation activity of the extracted proteins, providing more accurate and reliable sample support for downstream protein mass spectrometry analysis.

[0009] The specific technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a method for extracting proteins from methane-oxidizing bacteria, the steps of which are as follows: S1. The methane-oxidizing bacteria enrichment culture was centrifuged at high speed to separate the precipitate. The precipitate was then resuspended in PBS buffer, mixed, and homogenized. The precipitate was separated again by high-speed centrifugation to obtain the pretreated culture. S2. Add a lysis buffer consisting of sodium dodecyl sulfate (SDS), Tris-HCl buffer and protease inhibitor to the pretreated culture. After initial lysis by incubation on ice, disperse the cell aggregates by homogenization. Then place the culture in an ice-water bath and further lyse by sonication to obtain cell lysis buffer. After high-speed centrifugation to precipitate the incompletely lysed bacteria and some cell debris, collect the supernatant to obtain crude protein extract solution. S3. Add pre-cooled anhydrous acetone to the crude protein extraction solution, mix well, and incubate at low temperature. Then, remove the acetone solution by low-speed centrifugation to obtain protein precipitate. Repeat the process of pre-cooling anhydrous acetone resuspension followed by high-speed centrifugation to remove impurities from the protein precipitate, and finally obtain the desired protein extract.

[0010] As a preferred embodiment of the first aspect above, the lysis buffer contains sodium dodecyl sulfate (SDS) at a mass percentage of 1 wt.% to 2 wt.% and the pH of the Tris-HCl buffer is 7.8.

[0011] As a preferred embodiment of the first aspect above, the amount of lysis buffer added is 5-10 mL / g culture.

[0012] As a preferred embodiment of the first aspect above, the centrifugal force of the low-speed centrifugation is controlled at 5000~8000 g, and the centrifugal force of the high-speed centrifugation is controlled at 12000~14000 g; the incubation temperature of the low-temperature incubation is controlled at 0~-20 ℃, and the incubation time is controlled at 1~2 hours.

[0013] As a preferred embodiment of the first aspect, the ultrasonic fragmentation is performed in an alternating cycle of 2-4 seconds of ultrasonication followed by a 5-8 second pause, with the ultrasonic power controlled at 130-200 W and the total duration being 4-6 minutes.

[0014] As a preferred embodiment of the first aspect above, the methane-oxidizing bacteria are either aerobic or anaerobic methane-oxidizing bacteria.

[0015] Secondly, the present invention provides a mass spectrometry pretreatment method for a protein extract, wherein the protein extract is obtained by means of the methanogenic bacteria protein extraction method described in the first aspect above, and the method includes the following steps: S4. The protein extract is redissolved using a buffer solution, and a reducing agent is added to the protein solution and incubated at a constant temperature until the reduction reaction is complete, causing the disulfide bonds in the protein to break into thiol groups; then an alkylating agent is added and incubated at a constant temperature under light-protected conditions to alkylate the exposed thiol groups and prevent the disulfide bonds from reforming. S5. Add mass spectrometry-grade trypsin to the protein solution after reduction and alkylation treatment, and incubate at a constant temperature to enzymatically digest the protein into peptides. Then, acidify the solution with mass spectrometry-grade trifluoroacetic acid to terminate the enzymatic digestion reaction and improve the solubility and stability of the peptides. Remove the precipitate by high-speed centrifugation after acidification. Desalt and remove impurities from the supernatant containing peptides by passing it through a C18 reversed-phase chromatography column, and then use it for protein mass spectrometry analysis.

[0016] As a preferred embodiment of the second aspect above, the constant temperature incubation is maintained at 35~40 ℃, and the incubation time is 30 minutes to 1 hour.

[0017] As a preferred embodiment of the second aspect above, the buffer solution is a 50-200 mM ammonium bicarbonate solution; the reducing agent is dithiothreitol (DTT) with a final concentration of 5-20 mM, or tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) with a final concentration of 5-10 mM; and the alkylating agent is iodoacetamide with a final concentration of 10-40 mM.

[0018] As a preferred embodiment of the second aspect above, the pH value of the acidification treatment is controlled at 2 to 3.

[0019] Compared with the prior art, the present invention has the following advantages: (1) The pretreatment step provided by the present invention effectively removes inorganic salt ions and other impurities from the methane-oxidizing bacteria culture, thus providing a purer basis for subsequent protein extraction.

[0020] (2) This invention targets the cell wall characteristics of methane-oxidizing bacteria and employs an optimized protein extraction method combining urea-free lysis buffer with ultrasonic disruption, which can efficiently disrupt cells and fully release proteins. Since this protein extraction method does not involve the use of urea, it has no impact on the subsequent protein digestion process.

[0021] (3) Existing protein extraction processes often employ high-intensity mechanical shearing, which easily generates localized high heat during the disruption process. For denitrifying anaerobic methanogenic bacteria, their cell wall and potential membrane system structures differ from those of aerobic ICMs. Violent shaking may disrupt the coordination bonds between copper ions and proteins, leading to the loss of key active site information in mass spectrometry. Although this invention also uses SDS as a solubilizer, it removes urea and the metal chelating agent (EDTA). The lysis environment without chelation interference chemically avoids the passive stripping of copper ions. More importantly, this invention uses controlled low-temperature ultrasonic disruption instead of high-temperature bead milling. The cavitation effect generated by ultrasound more effectively disintegrates the cell membrane at the microscopic level. Combined with ice bath temperature control, it avoids the damage to metal coordination bonds caused by high temperatures in existing technologies, thereby maximizing the preservation of the integrity of the pMMO metal center. This is crucial for protecting the pMMO extract, whose structure is not yet clear and is extremely unstable, and can retain more conformational information.

[0022] (4) In the protein digestion step, the pre-acidification precipitation method for peptides provided by this invention not only improves the solubility and stability of peptides and increases desalting efficiency, but also helps to avoid aggregation caused by charge interactions between peptides. The trifluoroacetic acid (TFA) used in this invention is not only an acid for adjusting pH, but also a powerful ion-pairing reagent. It can bind to the positively charged groups of hydrophobic peptides, significantly increasing the retention and resolution of hydrophobic peptides on reversed-phase chromatography (C18), and preventing them from precipitating before injection. Attached Figure Description

[0023] Figure 1 The SDS-PAGE results are from Example 1; Figure 2 The SDS-PAGE results are from Comparative Example 1; Figure 3 These are the results of protein mass spectrometry. Detailed Implementation

[0024] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the present invention can be combined accordingly without mutual conflict.

[0025] In a preferred embodiment of the present invention, a method for extracting proteins from methane-oxidizing bacteria is provided, the steps of which are as follows: S1. The methane-oxidizing bacteria enrichment culture was centrifuged at high speed to separate the precipitate and resuspend it in PBS buffer. After vortexing to mix it thoroughly, it was homogenized using a homogenizer. The supernatant was removed by high-speed centrifugation again to separate the precipitate, thus obtaining the pretreated culture.

[0026] It should be noted that the methane-oxidizing bacteria enrichment culture in this invention can be in different forms, such as a culture medium enriched with methane-oxidizing bacteria, activated sludge enriched with methane-oxidizing bacteria in a reactor, etc.

[0027] It should be noted that this invention is applicable to protein extraction from methane-oxidizing bacteria in general and is not limited to specific methane-oxidizing bacterial species. The methane-oxidizing bacteria can be either aerobic or anaerobic. Table 1 lists a series of methane-oxidizing bacteria to which this invention is applicable, but it is not limited to these specific species. Furthermore, this invention is particularly suitable for methane-oxidizing bacteria with long growth cycles, such as denitrifying anaerobic methane-oxidizing bacteria. Candidatus Methylomirabilis sinica ( M. sinica ).

[0028] Table 1

[0029] In step S1 above, the optimal parameters for high-speed centrifugation are: 12000~14000 g, 4 ℃, 5~10 min. For enrichment cultures with complex components, such as activated sludge, the above-mentioned operations of PBS buffer resuspension, homogenization, and high-speed centrifugation need to be repeated multiple times to ensure the subsequent separation effect, generally at least three times.

[0030] S2. Add a lysis buffer consisting of sodium dodecyl sulfate (SDS), Tris-HCl buffer, and protease inhibitor to the pretreated culture. After initial lysis by incubation on ice for 5-10 min, disperse the cell aggregates by homogenization. Then, place the culture in an ice-water bath and sonicate to further lyse the cells, obtaining a cell lysate. Centrifuge the cell lysate at high speed to precipitate incompletely lysed bacteria and some cell debris. Collect the supernatant to obtain a crude protein extract solution.

[0031] It should be noted that the component content and the amount of lysis buffer added can be optimized based on actual results. In the embodiments of the present invention, the mass percentage of sodium dodecyl sulfate (SDS) is 1 wt.%~2 wt.%, the pH of the Tris-HCl buffer is 7.8, and the amount of protease inhibitor can be added according to the manufacturer's recommended dosage. Furthermore, the amount of lysis buffer added is 5~10 mL / g culture, that is, 5~10 mL of lysis buffer is added per 1g of culture.

[0032] In addition, in step S2 above, the ultrasonic disruption parameters can preferably be set as follows: alternating cycles of ultrasonic disruption for 2-4 seconds followed by a 5-8 second pause, with ultrasonic power controlled at 130-200 W and a total duration of 4-6 minutes. The high-speed centrifugation parameters are preferably set as follows: 12000-14000 g, 4 ℃, 5-10 minutes.

[0033] S3. Add pre-cooled anhydrous acetone to the crude protein extraction solution, mix well, and incubate at low temperature. Then, remove the acetone solution by low-speed centrifugation to obtain protein precipitate. Repeat the process of pre-cooling anhydrous acetone resuspension followed by high-speed centrifugation to remove impurities from the protein precipitate, and finally obtain the desired protein extract.

[0034] In step S3 above, multiple centrifugation separations are required. Initially, a low-speed centrifugation is performed, followed by repeated high-speed centrifugation. The preferred centrifugation parameters are as follows: for low-speed centrifugation, the centrifugal force is controlled at 5000~8000 g for 20~25 min; for high-speed centrifugation, the centrifugal force is controlled at 12000~14000 g for 5~10 min.

[0035] Furthermore, in step S3 above, the incubation temperature for low-temperature incubation is preferably controlled at 0 to -20 °C, and the incubation time is preferably controlled at 1 to 2 hours. More specifically, the incubation temperature is preferably controlled at -20 °C.

[0036] Therefore, through the pretreatment process in step S1 and the protein extraction process in step S2, inorganic salt ions and other impurities in the original culture system can be effectively removed through pretreatment. The purified whole protein is then extracted and digested into peptides through subsequent mass spectrometry pretreatment for protein mass spectrometry analysis.

[0037] Based on the same inventive concept, in another embodiment of the present invention, after obtaining the protein extract according to the methane-oxidizing bacteria protein extraction method in the above embodiment, a mass spectrometry pretreatment method for the protein extract can also be provided, the steps of which are as follows: S4. The protein extract is redissolved using a buffer solution, and a reducing agent is added to the protein solution and incubated at a constant temperature until the reduction reaction is complete, causing the disulfide bonds in the protein to break into thiol groups. Then, an alkylating agent is added and incubated at a constant temperature under light-protected conditions to alkylate the exposed thiol groups and prevent the disulfide bonds from reforming, thereby ensuring the stability of the protein structure.

[0038] In step S4 above, the specific types and amounts of buffer solution, reducing agent, and alkylating reagent can be optimized based on actual results. In this invention, the buffer solution is preferably a 50-200 mM ammonium bicarbonate solution; the reducing agent is preferably dithiothreitol (DTT) with a final concentration of 5-20 mM, or tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) with a final concentration of 5-10 mM; the alkylating reagent is preferably iodoacetamide with a final concentration of 10-40 mM. Furthermore, the isothermal incubation temperature is preferably maintained at 35-40 °C, and the incubation time is preferably 30 minutes to 1 hour to ensure the reaction proceeds fully.

[0039] S5. Add mass spectrometry-grade trypsin to the protein solution after reduction and alkylation treatment, and incubate at a constant temperature to enzymatically digest the protein into peptides. Then, acidify the solution with mass spectrometry-grade trifluoroacetic acid to terminate the enzymatic digestion reaction and improve the solubility and stability of the peptides, thereby improving the efficiency of subsequent desalting. Then, remove the precipitate by high-speed centrifugation after acidification. Pass the supernatant containing peptides through a C18 reversed-phase chromatography column to desalt and remove impurities, removing salts and other small molecule impurities that may interfere with the mass spectrometry ionization process. Finally, use the solution for protein mass spectrometry analysis.

[0040] In step S5 above, the amount of trypsin added can be based on the manufacturer's recommended amount. In the embodiments of the present invention, the actual protein content in the solution can be used as a basis, and mass spectrometry-grade trypsin can be added at a protein-to-trypsin mass ratio of 50:1. Furthermore, the isothermal incubation temperature is preferably maintained at 35-40 °C, and the incubation time is preferably 30 minutes to 1 hour to ensure that the enzymatic hydrolysis reaction proceeds fully. Additionally, when adding mass spectrometry-grade trifluoroacetic acid for acidification, its amount needs to be optimized appropriately, preferably so that the pH value after adding trifluoroacetic acid is 2-3. The centrifugal force of high-speed centrifugation is controlled at 12000-14000 g, and the time is 5-10 min.

[0041] Example 1 In this embodiment, a denitrifying anaerobic methanogenic bacterium was used. Candidatus Methylomirabilissinica ( M. sinica ) is an example of a methanogenic bacterium, for M. sinica The proteins in the enriched cultures underwent extraction and pretreatment for mass spectrometry analysis. The specific procedures are as follows: Step (1) Sample pretreatment Take activated sludge from the reactor. M. sinica The enriched culture was collected in centrifuge tubes and centrifuged at 12000 g at 4 °C for 8 min. The supernatant was removed to separate the precipitate. The precipitate was resuspended in PBS buffer (pH=7.4), vortexed to mix thoroughly, and then homogenized using a homogenizer. The mixture was then centrifuged again at 12000 g at 4 °C for 8 min, and the supernatant was removed to separate the precipitate. This process of resuspension, homogenization, and centrifugation with PBS buffer was repeated three times to ensure the removal of the original culture medium from the reactor and to obtain the pretreated culture.

[0042] Step (2), Cell disruption and protein extraction Two experimental groups with different lysis buffer formulations were set up. Group A1 consisted of 1 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. Group A2 consisted of 2 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. The protease inhibitor was Roche 04693132001-20TABLETS, in tablet form, and was added to every 50 ml of Tris-HCl buffer at a dosage of one tablet.

[0043] Take 0.5 g of the pretreated culture and add 5 mL of pre-cooled lysis buffer (for experiments A1 and A2, add the corresponding lysis buffer formulations respectively). Mix gently and incubate on ice for 8 min to complete initial lysis. Then, homogenize the lysed mixture using a glass homogenizer to disperse cell aggregates. Next, use an ultrasonic homogenizer to break up the mixture. The ultrasonic parameters are set to 195 W, with a 5-second pause after every 3 seconds of sonication, alternating cycles for a total of 8 min. The mixture must be kept in an ice-water bath throughout the sonication process to prevent overheating and protein denaturation. The sonicated cell lysate is centrifuged at 14000 g at 4 ℃ for 20 min to precipitate incompletely broken bacteria and some cell debris. Collect the supernatant to obtain the crude protein extract. The protein concentration results determined by the BCA method are shown in Table 2. SDS-PAGE gel electrophoresis was used to detect the protein solution, and the results are as follows: Figure 1 As shown in middle stripes I and II.

[0044] Table 2

[0045] For groups A1 and A2, 100 μg of crude protein was extracted and slowly mixed with 5 times its volume of pre-cooled anhydrous acetone. The mixture was then incubated at -20 °C for 1 h, followed by centrifugation at 8000 g at 4 °C for 15 min. The acetone solution was removed, and the protein precipitate was collected. Pre-cooled anhydrous acetone was then added to the protein precipitate, and the mixture was thoroughly mixed before centrifugation again at 12000 g at 4 °C for 20 min. The supernatant was discarded, and the protein precipitate was retained. This process of adding pre-cooled anhydrous acetone and centrifugation to remove the supernatant was repeated twice. The final protein precipitate was collected and air-dried in a fume hood for later use.

[0046] Step (3), protein reduction, alkylation and enzymatic hydrolysis In groups A1 and A2, 500 μL of 50 mM ammonium bicarbonate solution was added to the dried protein precipitate to redissolve it. Then, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) was added to a final concentration of 5 mM, and the mixture was incubated at 37°C for 45 min. Next, iodoacetamide was added to an alkylating agent to a final concentration of 20 mM, and the mixture was incubated in the dark for 1 h. After incubation, 2 μL of mass spectrometry-grade trypsin (1 μg / μL) was added, and the mixture was incubated overnight at 37°C to ensure complete digestion of the protein into peptides.

[0047] Step (4) Peptide impurity removal After enzymatic hydrolysis, 5 μL of 50% mass spectrometry-grade trifluoroacetic acid (TFA) was added to acidify the digestion system, thereby terminating the enzymatic digestion reaction and improving the solubility and stability of the peptides. Subsequently, the acidified system was centrifuged at 12000 g and 4 °C for 5 min to remove insoluble impurities, and the supernatant containing the peptides was collected.

[0048] To provide high-quality protein mass spectrometry samples, the supernatant containing peptides was passed through a C18 reversed-phase column to remove salts and impurities. The specific procedure was as follows: First, 200 µL of activation buffer (50% ACN) was added and centrifuged at 1500 g for 1 min to activate the resin. Next, 200 µL of equilibration buffer (0.5% TFA in 5% ACN) was added and centrifuged at 1500 g for 1 min to equilibrate the resin. After loading the sample, the column was centrifuged at 1500 g for 1 min, and the eluent was added back to the resin and centrifuged at 1500 g for 1 min, allowing the protein to be adsorbed onto the resin. The resin column was then placed in a new receiving tube, 200 µL of washing buffer (0.5% TFA in 5% ACN) was added, and the column was centrifuged at 1500 g for 1 min. The eluent was discarded, and the washing process was repeated once. Finally, place the resin column into a new receiving tube, add a total of 40 µL of elution buffer (70% ACN) in two portions, centrifuge at 1500 g for 1 min, and collect the elution buffer, which can be directly used for subsequent mass spectrometry analysis.

[0049] Step (5) Mass spectrometry setup The eluent obtained after peptide removal in step (4) was used for mass spectrometry. An ORBITRAP ECLIPSE mass spectrometer was used, equipped with the FAIMS Pro™ Interface. The compensation voltage (CV) was switched every 1 second between -45 and -65. A Nanospray Flex™ (NSI) ion source was used, with the ion spray voltage set to 2.0 kV and the ion transfer tube temperature set to 320℃. The mass spectrometry was performed in data-dependent acquisition mode, with a full scan range of m / z 350-1500. The primary mass spectrometry resolution was set to 120000 (m / z), and the AGC was 4×10⁻⁶. 5 The maximum injection time for C-trap is 50 ms; the secondary mass spectrometry detection uses the "TopSpeed" mode, with the secondary mass spectrometry resolution set to 15000 (200 m / z) and AGC of 5×10⁻⁶. 4 The maximum injection time was 22ms, the peptide fragmentation collision energy was set to 33%, and raw mass spectrometry detection data was generated.

[0050] Example 2 In this embodiment, a denitrifying anaerobic methanogenic bacterium was used. CandidatusMethylomirabilissinica ( M. sinica ) is an example of a methanogenic bacterium, for M. sinica The proteins in the enriched cultures underwent extraction and pretreatment for mass spectrometry analysis. The specific procedures are as follows: Step (1) Sample pretreatment Take activated sludge from the reactor. M. sinica The enriched culture was collected in centrifuge tubes and centrifuged at 12000 g at 4 °C for 8 min. The supernatant was removed to separate the precipitate. The precipitate was resuspended in PBS buffer (pH=7.4), vortexed to mix thoroughly, and then homogenized using a homogenizer. The mixture was then centrifuged again at 12000 g at 4 °C for 8 min, and the supernatant was removed to separate the precipitate. This process of resuspension, homogenization, and centrifugation with PBS buffer was repeated three times to ensure the removal of the original culture medium from the reactor and to obtain the pretreated culture.

[0051] Step (2), Cell disruption and protein extraction Two experimental groups with different lysis buffer formulations were set up. Group B1 consisted of 1 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. Group B2 consisted of 2 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. The protease inhibitor was Roche 04693132001-20TABLETS, in tablet form, and was added to every 50 ml of Tris-HCl buffer at a dosage of one tablet.

[0052] Take 0.5 g of the above precipitate, add 5 mL of pre-cooled lysis buffer (B1: 1% SDS, B2: 2% SDS; 50 mM Tris-HCl (pH=7.8); protease inhibitor), mix gently, and incubate on ice for 8 min. Then homogenize the lysis mixture using a glass homogenizer.

[0053] Take 0.5 g of the pretreated culture and add 5 mL of pre-cooled lysis buffer (for experiments A1 and A2, add the corresponding lysis buffer formulations respectively). Mix gently and incubate on ice for 8 min to complete initial lysis. Then, homogenize the lysed mixture using a glass homogenizer to disperse cell aggregates. Next, use an ultrasonic homogenizer to break up the mixture. The ultrasonic parameters are set to 195 W, with a 5-second pause after every 3 seconds of sonication, alternating cycles for a total of 8 min. The mixture must be kept in an ice-water bath throughout the sonication process to prevent overheating and protein denaturation. The sonicated cell lysate is centrifuged at 14000 g at 4 ℃ for 20 min to precipitate incompletely broken bacteria and some cell debris. Collect the supernatant to obtain the crude protein extract. The protein concentration results using the BCA method are shown in Table 3. The protein solution was analyzed by SDS-PAGE gel electrophoresis, and the results are as follows: Figure 1 As shown in middle stripes III and IV.

[0054] Table 3

[0055] For groups B1 and B2, 100 μg of crude protein was extracted and slowly mixed with 5 times its volume of pre-cooled anhydrous acetone. The mixture was then incubated at -20 °C for 1 h, followed by centrifugation at 8000 g at 4 °C for 15 min. The acetone solution was removed, and the protein precipitate was collected. Pre-cooled anhydrous acetone was then added to the protein precipitate, and the mixture was thoroughly mixed before centrifugation again at 12000 g at 4 °C for 20 min. The supernatant was discarded, and the protein precipitate was retained. This process of adding pre-cooled anhydrous acetone and centrifugation to remove the supernatant was repeated twice. The final protein precipitate was collected and air-dried in a fume hood for later use.

[0056] Step (3), protein reduction, alkylation and enzymatic hydrolysis For groups B1 and B2, 500 μL of 50 mM ammonium bicarbonate solution was added to the dried protein precipitate to redissolve the precipitate. Then, dithiothreitol (DTT) was added to both groups, with a final DTT concentration of 10 mM for group B1 and 20 mM for group B2; and the mixture was incubated at 37 °C for 45 min. Next, iodoacetamide was added to achieve a final concentration of 20 mM, and the mixture was incubated in the dark for 1 h. After incubation, 2 μL of mass spectrometry-grade trypsin (1 μg / μL) was added, and the mixture was incubated overnight at 37 °C to ensure complete digestion of the protein into peptides.

[0057] Step (4) Peptide impurity removal The specific procedures and parameters for peptide purification are the same as in step (4) of Example 1. Collect the final eluent, which can be used directly for subsequent mass spectrometry analysis.

[0058] Step (5) Mass spectrometry setup The eluent after removing impurities from the peptides in step (4) was used for mass spectrometry. The specific operation and parameters were the same as in step (5) of Example 1.

[0059] Comparative Example 1 This comparative example provides currently available denitrifying anaerobic methanogenic bacteria. M. sinica Commonly used protein extraction and mass spectrometry analysis pretreatment methods for cultures are as follows: Step (1) Sample pretreatment Take activated sludge from the reactor. M. sinica The enriched culture was collected in centrifuge tubes and centrifuged at 12000 g at 4 °C for 8 min. The supernatant was removed to separate the precipitate. The precipitate was resuspended in PBS buffer (pH=7.4), vortexed to mix thoroughly, and then homogenized using a homogenizer. The mixture was then centrifuged again at 12000 g at 4 °C for 8 min, and the supernatant was removed to separate the precipitate. This process of resuspension, homogenization, and centrifugation with PBS buffer was repeated three times to ensure the removal of the original culture medium from the reactor and to obtain the pretreated culture.

[0060] Step (2), Cell disruption and protein extraction Three experimental groups were set up with different lysis buffer formulations as shown in Table 4. Group C1 contained 8M urea, Tris-HCl buffer (pH=7.8), and a protease inhibitor. Group C2 contained 8M urea, 1 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. Group C3 contained 8M urea, 2 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. The protease inhibitor was Roche 04693132001-20 TABLETS, in tablet form, and was added at a dosage of one tablet per 50 ml of Tris-HCl buffer.

[0061] Table 4

[0062] Take 0.5 g of the pretreated culture and add 5 mL of pre-cooled lysis buffer (for groups C1, C2, and C3, add the corresponding formulation of lysis buffer). Gently mix and incubate on ice for 10 min to complete the initial lysis. Then, homogenize the lysed mixture using a glass homogenizer to disperse cell aggregates, and then use an ultrasonic homogenizer to break up the mixture. The ultrasonic parameters are set to 195 W, with a 5-second pause after every 3 seconds of sonication, alternating cycles for a total of 8 min. The mixture must be kept in an ice-water bath throughout the sonication process to prevent overheating and protein denaturation. The sonicated cell lysate is centrifuged at 14000 g at 4 ℃ for 20 min to precipitate incompletely broken bacteria and some cell debris. Collect the supernatant to obtain the crude protein extract. The protein concentration results determined by the BCA method are shown in Table 5. SDS-PAGE gel electrophoresis was used to detect the protein solution, and the results are as follows: Figure 2 As shown in the middle stripes I, II, and III.

[0063] Table 5

[0064] Step (3), protein reduction, alkylation and enzymatic hydrolysis Groups C1, C2, and C3 had dithiothreitol (DTT) added to the crude protein extraction solution to a final concentration of 10 mM, and were reduced at 30°C for 1 h. Iodoacetamide was then added to a final concentration of 40 mM, and the mixture was incubated at room temperature in the dark for 45 min. Subsequently, 7 volumes of ammonium bicarbonate buffer (50 mM) were added to each group to ensure the urea concentration in the system was <1 M and the pH was close to neutral. Finally, trypsin was added at a protein-to-trypsin mass ratio of 50:1 for enzymatic digestion, and the mixture was incubated overnight at 37°C for peptide breaking.

[0065] Step (4) Peptide impurity removal The enzymatically digested mixture was used to remove impurities from the peptides using a C18 reversed-phase chromatography column. The specific procedure was as follows: First, 200 µL of activation buffer (50% ACN) was added and centrifuged at 1500 g for 1 min to activate the resin. Next, 200 µL of equilibration buffer (0.5% TFA in 5% ACN) was added and centrifuged at 1500 g for 1 min to equilibrate the resin. After loading the sample, the column was centrifuged at 1500 g for 1 min, and the eluent was added back to the resin and centrifuged at 1500 g for 1 min, allowing the protein to be adsorbed onto the resin. The resin column was then placed in a new receiving tube, and 200 µL of washing buffer (0.5% TFA in 5% ACN) was added. The column was centrifuged at 1500 g for 1 min, and the eluent was discarded. This washing process was repeated once. Finally, the resin column was placed in a new receiving tube, and a total of 40 µL of eluent (70% ACN) was added in two portions. The columns were centrifuged at 1500 g for 1 min, and the eluent was collected for mass spectrometry analysis.

[0066] Step (5) Mass spectrometry setup The eluent obtained after removing impurities from the peptides in step (4) was used for mass spectrometry. The specific operation and parameters were the same as in step (5) of Example 1.

[0067] Figure 1 For the denitrifying anaerobic methanogenic bacteria in Examples 1 and 2 M. sinica After total protein extraction from the culture, the samples were subjected to SDS-PAGE electrophoresis, and the bands were obtained after Coomassie Brilliant Blue staining and destaining, where M is the indicator band used for molecular weight labeling. Figure 2 The band results for the protein extracted in Comparative Example 1 are obtained by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining-decolorization, where M is the indicator band used for molecular weight labeling. Figure 3 The results are the protein mass spectrometry results from Examples 1, 2, and Comparative Example 1.

[0068] A comparison of the experimental results in Examples 1, 2, and Comparative Example 1 shows that the method of the present invention extracts a higher protein concentration and fewer impurities in the mass spectrometry protein peaks. A detailed comparison of the specific results is provided below. 1. Comparison of protein extraction concentrations Results of Example 1: Group A1 (1% SDS, 5 mM TCEP): Protein concentration was 1.2529 μg / μL Group A2 (2% SDS, 10 mM TCEP): Protein concentration was 1.0277 μg / μL Results of Example 2: Group B1 (1% SDS, 10 mM DTT): Protein concentration was 1.1728 μg / μL Group B2 (2% SDS, 20 mM DTT): Protein concentration was 1.3362 μg / μL Results of Comparative Example 1: Group C1 (8 M urea, no SDS): protein concentration was 0.4285 μg / μL Group C2 (8 M urea, 1% SDS): protein concentration was 0.2672 μg / μL Group C3 (8 M urea, 2% SDS): protein concentration was 0.6203 μg / μL Comparative analysis of the three sets of results showed that the protein concentrations in Examples 1 and 2 were significantly higher than those in Comparative Example 1. The lowest concentration in the examples (Group A2, 1.0277 μg / μL) was still approximately 1.66 times the highest concentration in Comparative Example 1 (Group C3, 0.6203 μg / μL), while the highest concentration in the examples (Group B2, 1.3362 μg / μL) was 2.15 times the highest concentration in Comparative Example 1.

[0069] Furthermore, because urea was used as the main cleavage component in Comparative Example 1, the protein in Group C was not completely cleaved during the enzymatic digestion stage. As a result, the protein was not fully cut into peptides of the expected length, the peptide coverage decreased, and the accuracy of bottom-up protein mass spectrometry identification was reduced.

[0070] 2. Comparison of impurities in mass spectrometry protein peaks In Examples 1 and 2, SDS-PAGE gel electrophoresis was used to detect (Example 1: Figure 1 Middle stripes I and II; Example 2: Figure 1 The results of bands III and IV in the middle section and subsequent mass spectrometry analysis (ORBITRAP ECLIPSE, FAIMS Pro™ Interface optimized for ion selectivity) showed that the protein bands were clear and there were few impurity proteins. Therefore, the peptide purification step used a C18 reversed-phase column for further purification to remove insoluble impurities and salts, ensuring high purity of the protein peaks and few impurity peaks in the mass spectrometry detection.

[0071] In Comparative Example 1, SDS-PAGE detection ( Figure 2 The middle bands (I, II, III) show that the protein bands are scattered, indicating that there may be a lot of non-target proteins or impurities remaining in the extract.

[0072] Comparative analysis of the three sets of results shows that the present invention effectively reduces impurity interference by optimizing the lysis buffer composition and ultrasonic disruption process, while the C18 column desalting step, resulting in more concentrated protein peaks and a significant reduction in impurity peaks in the mass spectrometry. In contrast, Comparative Example 1 relies on a traditional urea lysis system, which results in lower protein purity during extraction and susceptibility to impurity interference during mass spectrometry detection, leading to a decrease in protein peak resolution.

[0073] In summary, this invention demonstrates significant advantages in both protein extraction concentration and mass spectrometry analysis quality, providing a superior pretreatment scheme for protein mass spectrometry studies of methanogenic bacteria.

[0074] However, it should be noted that the above embodiments and Comparative Example 1 of the present invention use denitrifying anaerobic methanogenic bacteria. Candidatus Methylomirabilis sinica ( M. sinica This invention uses an example of a methanogenic bacterium for protein extraction and pretreatment for mass spectrometry analysis in enriched cultures, as this methanogenic bacterium is a typical example of a difficult-to-extract protein. However, this invention is a general method and is not limited to this type of methanogenic bacterium; it can also be applied to other methanogenic bacteria. Another embodiment is shown below to demonstrate its versatility. Example 3 This embodiment uses aerobic methane-oxidizing bacteria as an example of methane-oxidizing bacteria. Proteins from the enriched cultures of aerobic methane-oxidizing bacteria were extracted and pretreated for mass spectrometry analysis. The specific procedures are as follows: Step (1) Sample pretreatment An aerobic methanogenic bacteria enrichment culture in the form of activated sludge was taken from the reactor and placed in a centrifuge tube. The tube was centrifuged at 12000 g at 4 °C for 8 min, and the supernatant was removed to separate the precipitate. The precipitate was then resuspended in PBS buffer (pH=7.4), vortexed to ensure thorough mixing, and homogenized using a homogenizer. The mixture was then centrifuged again at 12000 g at 4 °C for 8 min, and the supernatant was removed to separate the precipitate. This process of resuspension, homogenization, and centrifugation with PBS buffer was repeated three times to ensure the removal of the original culture medium from the reactor and to obtain the pretreated culture.

[0075] Step (2), Cell disruption and protein extraction Take 0.5 g of the pretreated culture and add 5 mL of pre-cooled lysis buffer (using the lysis buffer formulation of group A1 in the examples). Mix gently and incubate on ice for 8 min to complete the initial lysis. Then, homogenize the lysed mixture using a glass homogenizer to disperse cell aggregates. Next, use an ultrasonic homogenizer to disrupt the mixture. The ultrasonic parameters are set to 195 W, with a 5-second pause after every 3 seconds of sonication, alternating cycles for a total of 8 min. The mixture must be kept in an ice-water bath throughout the sonication process to prevent overheating and protein denaturation. The sonicated cell lysate is centrifuged at 14000 g at 4 °C for 20 min to precipitate incompletely lysed bacteria and some cell debris. Collect the supernatant to obtain the crude protein extract. Take 100 μg of crude protein extract and slowly add 5 times its volume of pre-cooled anhydrous acetone. After gently mixing, incubate at -20 °C for 1 h, then centrifuge at 8000 g at 4 °C for 15 min to remove the acetone solution and collect the protein precipitate. Next, add pre-cooled anhydrous acetone to the protein precipitate, mix thoroughly, and centrifuge again at 12000 g at 4 °C for 20 min. Discard the supernatant and retain the protein precipitate. Repeat the process of adding pre-cooled anhydrous acetone and centrifuging to remove the supernatant twice more. Collect the final protein precipitate and air dry it in a fume hood for later use.

[0076] Step (3), protein reduction, alkylation and enzymatic hydrolysis 500 μL of 50 mM ammonium bicarbonate solution was added to each of the dried protein precipitates to redissolve them. Then, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) was added to a final concentration of 5 mM, and the mixture was incubated at 37 °C for 45 min. Next, iodoacetamide was added to a final concentration of 20 mM, and the mixture was incubated in the dark for 1 h. After incubation, 2 μL of mass spectrometry-grade trypsin (1 μg / μL) was added, and the mixture was incubated overnight at 37 °C to ensure complete digestion of the protein into peptides.

[0077] Step (4) Peptide impurity removal After enzymatic hydrolysis, 5 μL of 50% mass spectrometry-grade trifluoroacetic acid (TFA) was added to acidify the digestion system, thereby terminating the enzymatic digestion reaction and improving the solubility and stability of the peptides. Subsequently, the acidified system was centrifuged at 12000 g and 4 °C for 5 min to remove insoluble impurities, and the supernatant containing the peptides was collected.

[0078] To provide high-quality protein mass spectrometry samples, the supernatant containing peptides was passed through a C18 reversed-phase column to remove salts and impurities. The specific procedure was as follows: First, 200 µL of activation buffer (50% ACN) was added and centrifuged at 1500 g for 1 min to activate the resin. Next, 200 µL of equilibration buffer (0.5% TFA in 5% ACN) was added and centrifuged at 1500 g for 1 min to equilibrate the resin. After loading the sample, the column was centrifuged at 1500 g for 1 min, and the eluent was added back to the resin and centrifuged at 1500 g for 1 min, allowing the protein to be adsorbed onto the resin. The resin column was then placed in a new receiving tube, 200 µL of washing buffer (0.5% TFA in 5% ACN) was added, and the column was centrifuged at 1500 g for 1 min. The eluent was discarded, and the washing process was repeated once. Finally, place the resin column into a new receiving tube, add a total of 40 µL of elution buffer (70% ACN) in two portions, centrifuge at 1500 g for 1 min, and collect the elution buffer. This elution buffer can be directly used for subsequent mass spectrometry analysis to generate raw mass spectrometry detection data.

[0079] Example 4 In this embodiment, a denitrifying anaerobic methanogenic bacterium was used. Candidatus Methylomirabilissinica ( M. sinica ) is an example of a methanogenic bacterium, for M. sinica Proteins were extracted from the enriched cultures, and the specific procedures were as follows: Step (1) Sample pretreatment Take activated sludge from the reactor. M. sinica The enriched culture was collected in centrifuge tubes and centrifuged at 12000 g at 4 °C for 8 min. The supernatant was removed to separate the precipitate. The precipitate was resuspended in PBS buffer (pH=7.4), vortexed to mix thoroughly, and then homogenized using a homogenizer. The mixture was then centrifuged again at 12000 g at 4 °C for 8 min, and the supernatant was removed to separate the precipitate. This process of resuspension, homogenization, and centrifugation with PBS buffer was repeated three times to ensure the removal of the original culture medium from the reactor and to obtain the pretreated culture.

[0080] Step (2) Preparation of crude protein extract Prepare the lysis buffer as follows: 1 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. The protease inhibitor is Roche 04693132001-20 TABLETS, in tablet form, and is added to every 50 ml of Tris-HCl buffer at a dosage of 1 tablet. Then, take 0.5 g of the pretreated culture, add 5 mL of pre-chilled lysis buffer, gently mix, and incubate on ice for 8 min to complete the initial lysis. Subsequently, homogenize the lysed mixture using a glass homogenizer to disperse cell aggregates, and then use an ultrasonic homogenizer to disrupt the mixture. The ultrasonic parameters are set to 200 W, with a 5-second pause after every 2 seconds of sonication, alternating cycles for a total of 6 min. The mixture must be kept in an ice-water bath throughout the sonication process to prevent overheating and protein denaturation. The cell lysate after ultrasonic treatment was centrifuged at 14000 g and 4 ℃ for 20 min to precipitate incompletely broken bacteria and some cell debris. The supernatant was collected to obtain the crude protein extract.

[0081] Step (4): Determination of protein concentration and methane oxidation activity in crude protein extract. A portion of the crude protein extract was taken, and the protein concentration was determined using the BCA method.

[0082] Additionally, the following methane oxidation activity assay was performed in an anaerobic glove box: 2 mg of the obtained crude protein extract was added to a 10 mL gas phase headspace vial (Agilent). Under ice bath conditions, 2 mL of the buffer system was added: 25 mM PIPES buffer (pH=7.2), 10 μM CuSO4. After sample addition, the vial was sealed with a butyl rubber stopper. The sealed headspace vial was then purged with an inert gas through a gas-tight dual-channel needle to completely remove the initial air; then, a mixture of oxygen and inert gas (5% O2 + 95% Ar) was introduced until the pressure inside the vial reached atmospheric pressure. The vial was placed in an ice bath, and NADH solution (final concentration 5 mM) was injected into the liquid using a gas-tight pipette. Subsequently, 1 mL of stable isotope-labeled [a specific reagent / component] was injected into the vial. 13 CH4 gas (99% purity) 13 C). The headspace vials were placed in a constant-temperature shaker (33 ℃, 150 rpm) and incubated in the dark to ensure sufficient mass transfer between the gas and liquid phases. At 0 h, 0.5 h, 1 h, 2 h, and 4 h, 50 µL of headspace gas was extracted using a gas chromatograph (Agilent). The change in methane content in the vials was monitored using gas chromatography-mass spectrometry (GC-MS, Agilent). Based on the monitoring data, the molar amount of methane oxidized per unit weight of protein per unit time was calculated to characterize the protein's methane oxidation activity.

[0083] The BCA assay results showed that the total protein concentration in the crude protein extract prepared in this example was 1.2319 μg / μL. The methane oxidation activity assay results showed that the protein methane oxidation activity in the crude protein extract prepared in this example was 0.0057 μmol·min⁻¹. -1 ·mg -1 This indicates that, in this embodiment, even with the use of SDS alone, although the protein undergoes some denaturation, weak methane oxidation activity can still be detected due to the short operation time and the fact that the entire process is carried out at low temperatures. This suggests that some copper ions remain non-covalently bound to the denatured protein peptide chains or form small copper-peptide complexes.

[0084] Step (3) Protein precipitation and extraction To further extract proteins for mass spectrometry analysis from the crude protein extract, take 100 μg of the crude protein extract, slowly add 5 volumes of pre-chilled anhydrous acetone, gently mix, and incubate at -20 °C for 1 h. Then, centrifuge at 8000 g, 4 °C for 15 min to remove the acetone solution and collect the protein precipitate. Next, add pre-chilled anhydrous acetone to the protein precipitate, mix thoroughly, and centrifuge again at 12000 g, 4 °C for 20 min. Discard the supernatant and retain the protein precipitate. Repeat the process of adding pre-chilled anhydrous acetone and centrifuging to remove the supernatant twice more. Collect the final protein precipitate and air-dry it in a fume hood for later use.

[0085] Comparative Example 2 In this comparative example, the difference from Example 4 lies only in the lysis buffer formulation; all other steps were performed in accordance with Example 4. The lysis buffer formulation for this comparative example was: 8 M urea, 1 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. The protease inhibitor type and dosage were the same as in Example 4.

[0086] The protein concentration and methane oxidation activity in the crude protein extract were determined according to Example 4. The BCA assay showed that the total protein concentration in the crude protein extract prepared in this comparative example was 1.2135 μg / μL. However, the methane oxidation activity assay showed that no methane oxidation activity was detected in the protein crude extract prepared in this comparative example. This indicates that the addition of 8 M urea to the lysis buffer in the comparative example resulted in complete protein denaturation, rendering it unable to catalyze methane oxidation.

[0087] Comparative Example 3 In this comparative example, the difference from Example 4 lies only in the lysis buffer formulation; all other steps were performed in accordance with Example 4. The lysis buffer formulation for this comparative example was: 2 mmol / L EDTA, 1 wt.% SDS, Tris-HCl buffer (pH=7.8), and a protease inhibitor. The protease inhibitor type and dosage were the same as in Example 4.

[0088] The protein concentration and methane oxidation activity in the crude protein extract were determined according to Example 4. The BCA assay showed that the total protein concentration in the crude protein extract prepared in this comparative example was 1.0121 μg / μL. However, the methane oxidation activity assay showed that almost no methane oxidation activity was detected in the crude protein extract prepared in this comparative example. This indicates that the addition of 2 mmol / L EDTA to the lysis buffer in the comparative example resulted in near-denaturation of the protein, rendering it unable to catalyze methane oxidation.

[0089] Comparative Example 4 In this comparative example, the difference from Example 4 lies in the different crushing method used for the pyrolyzed mixture; all other steps are performed in accordance with Example 4. Specifically, in step (2) of this comparative example, the crude protein extract is obtained by crushing with magnetic beads, and the preparation process is as follows: The lysis buffer formulation for this comparative example is the same as that in Example 4. After preparing the lysis buffer, 0.5 g of the pretreated culture prepared in step (1) was taken, and 5 mL of pre-cooled lysis buffer was added. After gently mixing, the mixture was incubated on ice for 8 min to complete the initial lysis. The initially lysed bacterial suspension was mixed with 1 mm magnetic beads and placed in a magnetic bead disruptor to prepare the cell lysis buffer. The parameters were set as follows: running at 6.0 m / s for 30 s, with a 60 s interval, and cooling on ice for 5 minutes between each cycle to prevent overheating. This was repeated 3 times. Subsequently, the cell lysis buffer was centrifuged at 14000 g and 4 ℃ for 20 min to precipitate the incompletely broken bacteria and some cell debris. The supernatant was collected to obtain the crude protein extract.

[0090] Similarly, referring to Example 4, the protein concentration and methane oxidation activity in the crude protein extract were determined. The BCA method showed that the total protein concentration in the crude protein extract prepared in this comparative example was 0.9719 μg / μL. However, the methane oxidation activity test results showed that no methane oxidation activity was detected in the crude protein extract prepared in this comparative example. This indicates that although the magnetic bead disruption method in the comparative example could extract total protein, the magnetic beads themselves involved a large number of collisions during rotation, resulting in instantaneous high temperatures at the impact points. Although the solution system itself could maintain a low temperature through ice cooling, the localized instantaneous high temperatures generated by the impacts and the excessive physical shear force generated by the high-speed rotation of the magnetic beads would cause the extracted protein to become inactive.

[0091] Comparative Example 5 In this comparative example, the protein extraction method used in the existing technical literature "Silvana Quitón-Tapia, Trueba-SantisoA, Garrido JM, et al. Metalloenzymes play major roles to achieve high-rate nitrogen removal in N-damo communities: Lessons from metaproteomics.[J]. Bioresource technology, 2023:, 129476. DOI:10.1016 / j.biortech.2023.129476." is reproduced. Since the description of the protein extraction process in that literature is rather brief, detailed information should be obtained from the cited reference "Kennes-Veiga DM, Alba TS, Valentina GG, et al. Sulfamethoxazole Enhances Specific Enzymatic Activities under AerobicHeterotrophic Conditions: A Metaproteomic Approach[J]. Environmental Science & Technology: ES&T, 2022." This is achieved by […]. Except for step (2), the specific implementation of the remaining steps in this comparative example is the same as in Example 4, and the implementation process of step (2) is as follows: The lysis buffer in this comparative example did not contain protease inhibitors and was formulated as follows: 1 wt.% SDS, 100 mM Tris-HCl buffer (pH=8.0). After preparing the lysis buffer, the pretreated culture prepared in step (1) was taken and gently mixed with 5 mL of pre-cooled lysis buffer per 0.5 g of culture. The mixture was then heated in a 90 ℃ water bath for 20 minutes. After cooling the initially lysed bacterial suspension, 1 mm magnetic beads were added and mixed. The mixture was then placed in a magnetic bead disruptor to prepare cell lysis buffer. The parameters were set as follows: run at 6.0 m / s for 30 s, with a 60 s interval, and cool on ice for 5 minutes between each cycle to prevent overheating. This cycle was repeated 3 times. Subsequently, the cell lysis buffer was centrifuged at 14000 g and 4 ℃ for 20 min to precipitate incompletely broken bacteria and some cell debris. The supernatant was collected to obtain the crude protein extract.

[0092] Similarly, referring to Example 4, the protein concentration and methane oxidation activity in the crude protein extract were determined. The BCA method showed that the total protein concentration in the crude protein extract prepared in this comparative example was 1.3011 μg / μL. However, the methane oxidation activity test results showed that no methane oxidation activity was detected in the crude protein extract prepared in this comparative example. This indicates that although magnetic bead disruption in the comparative example could extract total protein, the localized instantaneous high temperature generated by magnetic bead disruption and the excessive physical shear force generated by the high-speed rotation of the magnetic beads caused protein inactivation. Furthermore, the lack of protease inhibitors in the lysis buffer also led to increased protein degradation losses during the lysis process.

[0093] Comparative Examples 4 and 2-5 show that, in terms of protein extraction concentration, the embodiments of the present invention are significantly superior to Comparative Example 4 (using magnetic beads for disruption) and Comparative Example 3 (containing EDTA), demonstrating stronger cell lysis efficiency. While Comparative Examples 2 and 5 achieved similar protein concentrations, the proteins were completely inactivated due to strong denaturants or high temperatures, resulting in undetectable oxidative activity. Comparative Example 3, due to the integration of crucial copper ions, and Comparative Example 4, possibly due to localized heat generation or physical shear overload, both resulted in loss of activity. In contrast, the embodiments of the present invention, using a urea-free and EDTA-free lysis buffer formulation combined with low-temperature ultrasonic disruption, successfully extracted proteins with detectable weak methane oxidation activity under low-temperature and rapid operation throughout the entire process. This indicates that the process can maximize the preservation of the binding state of copper ions to proteins, protecting the activity of metalloenzymes that are extremely sensitive to the environment. Therefore, compared to other existing technologies, the present invention is particularly suitable for extracting pMMO, the key membrane-bound copper enzyme catalyzing the conversion of methane to methanol in methane-oxidizing bacteria.

[0094] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for extracting proteins from methane-oxidizing bacteria, characterized in that, The steps are as follows: S1. The methane-oxidizing bacteria enrichment culture was centrifuged at high speed to separate the precipitate. The precipitate was then resuspended in PBS buffer, mixed, and homogenized. The precipitate was separated again by high-speed centrifugation to obtain the pretreated culture. S2. Add a lysis buffer consisting of sodium dodecyl sulfate, Tris-HCl buffer and protease inhibitor to the pretreated culture. After initial lysis by incubation on ice, disperse the cell aggregates by homogenization. Then place the culture in an ice-water bath and further lyse by sonication to obtain cell lysis buffer. After high-speed centrifugation to precipitate the incompletely broken bacteria and some cell debris, collect the supernatant to obtain crude protein extract solution. S3. Add pre-cooled anhydrous acetone to the crude protein extraction solution, mix well, and incubate at low temperature. Then, remove the acetone solution by low-speed centrifugation to obtain protein precipitate. Repeat the process of pre-cooling anhydrous acetone resuspension followed by high-speed centrifugation to remove impurities from the protein precipitate, and finally obtain the desired protein extract.

2. The method for extracting proteins from methane-oxidizing bacteria as described in claim 1, characterized in that, The lysis buffer contains sodium dodecyl sulfate at a mass percentage of 1 wt.% to 2 wt.% and Tris-HCl buffer at a pH of 7 to 8.

3. The method for extracting proteins from methane-oxidizing bacteria as described in claim 1, characterized in that, The amount of lysis buffer added is 5-10 mL / g of culture.

4. The method for extracting proteins from methane-oxidizing bacteria as described in claim 1, characterized in that, The centrifugal force of the low-speed centrifugation is controlled at 5000~8000 g, and the centrifugal force of the high-speed centrifugation is controlled at 12000~14000 g; the incubation temperature of the low-temperature incubation is controlled at 0~-20 ℃, and the incubation time is controlled at 1~2 hours.

5. The method for extracting proteins from methane-oxidizing bacteria as described in claim 1, characterized in that, The ultrasonic fragmentation is performed in an alternating cycle of 2-4 seconds of ultrasonic stimulation followed by a 5-8 second pause, with the ultrasonic power controlled at 130-200 W and the total duration being 4-6 minutes.

6. The method for extracting proteins from methane-oxidizing bacteria as described in claim 1, characterized in that, The methane-oxidizing bacteria are either aerobic or anaerobic methane-oxidizing bacteria.

7. A mass spectrometry pretreatment method for protein extracts, characterized in that, The protein extract is obtained by the method for extracting proteins from methane-oxidizing bacteria according to claim 1, wherein the method includes the following steps: S4. The protein extract is redissolved using a buffer solution, and a reducing agent is added to the protein solution and incubated at a constant temperature until the reduction reaction is complete, causing the disulfide bonds in the protein to break into thiol groups; then an alkylating agent is added and incubated at a constant temperature under light-protected conditions to alkylate the exposed thiol groups and prevent the disulfide bonds from reforming. S5. Add mass spectrometry-grade trypsin to the protein solution after reduction and alkylation treatment, and incubate at a constant temperature to enzymatically digest the protein into peptides. Then, acidify the solution with mass spectrometry-grade trifluoroacetic acid to terminate the enzymatic digestion reaction and improve the solubility and stability of the peptides. Remove the precipitate by high-speed centrifugation after acidification. Desalt and remove impurities from the supernatant containing peptides by passing it through a C18 reversed-phase chromatography column, and then use it for protein mass spectrometry analysis.

8. The mass spectrometry pretreatment method for protein extracts as described in claim 7, characterized in that, The constant temperature incubation is maintained at 35~40 ℃, and the incubation time is 30 minutes to 1 hour.

9. The mass spectrometry pretreatment method for protein extracts as described in claim 7, characterized in that, The buffer solution is a 50-200 mM ammonium bicarbonate solution; the reducing agent is dithiothreitol with a final concentration of 5-20 mM, or tris(2-carboxyethyl)phosphonic acid hydrochloride with a final concentration of 5-10 mM; the alkylating agent is iodoacetamide with a final concentration of 10-40 mM.

10. The mass spectrometry pretreatment method for protein extracts as described in claim 7, characterized in that, The pH value of the acidification treatment is controlled at 2-3.

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