Methane oxidizing bacteria protein extraction and mass spectrum pretreatment method

Abstract

The invention discloses a methane-oxidizing bacteria protein extraction and mass spectrum pretreatment method, and belongs to the technical field of protein extraction. The method comprises the following steps: removing inorganic salt ions and other impurity components from a methane-oxidizing bacteria enrichment culture through a pretreatment step to obtain a pretreated culture; then adding a lysis solution composed of lauryl sodium sulfate, a Tris-HCl buffer solution and a protease inhibitor into the pretreated culture, carrying out ultrasonication to obtain a cell lysis solution, and separating to obtain a protein crude extraction solution; and finally extracting the protein extract from the protein crude extraction solution. By optimizing the extraction process and the subsequent enzymolysis digestion process, the protein extraction efficiency and the peptide fragment purity are remarkably improved, and more accurate and reliable sample support is provided for downstream protein mass spectrometry.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein extraction, and specifically relates to a method for extracting whole protein from methane-oxidizing bacteria and generating high-purity peptides through enzymatic hydrolysis. Based on this method, the protein extraction efficiency and the purity of enzymatic peptides can be significantly improved, high-quality samples can be provided for downstream protein mass spectrometry analysis, and the accuracy and reliability of mass spectrometry analysis can be improved. Background Art

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

[0003] Methane-dependent denitrifiers (MDs) can convert nitrate (NO) into 3 - ) or nitrite (NO 2 - ) is reduced to nitrogen (N 2 ), is an important component of the carbon and nitrogen cycle in natural habitats. The traditional view is that the methane-type denitrification process is mainly caused by ANME-2d archaea (NO 3 - →NO 2 - ) and Methylomirabilis bacteria (NO 2 - →N 2 The recently discovered anaerobic methane-dependent complete denitrifier (M-CD) Candidatus Methylomirabilis sinica (M. sinica) can use methane as an electron donor to directly convert NO 3 - Convert to N 2 , which is expected to achieve greenhouse gas N in natural and artificial habitats 2 O and CH 4 The coordinated emission reduction provides a new perspective for the sustainable management of greenhouse gases.

[0004] M. sinica has unique physiological and biochemical characteristics, but its key enzymatic mechanism remains to be clarified. M. sinica first reduces NO through the nitrate reductase NapAB in the periplasm. 3- to NO 2 - , followed by NO 2 - In cytochrome cd 1 Nitrite is converted into NO by the catalysis of NirS, and NO is further dismutated by the currently unknown nitric oxide dismutase (Nod) to finally generate N 2 and O 2 Intracellular O 2 The particulate methane monooxygenase (pMMO) is used for the oxidation of methane. The structural and functional analysis of M. sinica functional proteins depends on protein extraction methods and protein mass spectrometry technology. Since the bacteria grow slowly and have a long doubling time, the biomass that can be obtained in a limited time is limited. Therefore, it is urgent to develop an efficient protein extraction method to significantly improve the protein extraction rate. The development of this method can also provide reference and promotion significance for protein extraction of other Gram-negative bacteria with longer growth cycles.

[0005] Protein mass spectrometry requires samples to have high purity and high stability, and it is necessary to remove impurities that may interfere with the analysis. At present, the protein extraction method of Gram-negative bacteria usually uses non-ionic denaturants such as urea, which can improve the solubility of proteins by dissolving cell membranes. However, before enzymatic hydrolysis, in order to reduce the inhibitory effect of urea on the subsequent trypsin enzymatic activity, the urea in the extraction system needs to be diluted. This operation not only leads to a decrease in protein concentration, but also inevitably interferes with the enzymatic effect of trypsin, further affecting the efficiency and quality of peptide generation. In addition, the residual urea may also trigger enzymatic side reactions, reducing the overall efficiency of protein extraction and the purity of the obtained peptides, which directly leads to unsatisfactory results of protein mass spectrometry analysis and limits its application in proteomics research. In view of the physiological characteristics of methane-oxidizing bacteria and the technical difficulties in protein extraction, it is urgent to develop an efficient and low-interference protein extraction and enzymatic hydrolysis method to improve the protein extraction rate and the purity of peptides, so as to provide reliable guarantees for high-quality protein mass spectrometry analysis and functional protein research. Summary of the invention

[0006] The present invention aims to solve the current problems of low extraction efficiency and insufficient purity of slowly proliferating Gram-negative bacteria proteins, and provides a method for efficient extraction of methane-oxidizing bacteria proteins and pre-treatment of mass spectrometry analysis. By optimizing the extraction process and subsequent enzymatic digestion process, the present invention significantly improves the protein extraction efficiency and the purity of peptide segments, and provides more accurate and reliable sample support for downstream protein mass spectrometry analysis.

[0007] The technical solutions specifically adopted in the present invention are as follows:

[0008] In a first aspect, the present invention provides a method for extracting protein from methane-oxidizing bacteria, the steps of which are as follows:

[0009] S1, centrifuging the enriched culture of methane-oxidizing bacteria at high speed, separating the precipitate, resuspending and mixing with PBS buffer, and then homogenizing, and centrifuging at high speed again to separate the precipitate, thereby obtaining a pretreated culture;

[0010] S2. Adding a lysis solution composed of sodium dodecyl sulfate (SDS), Tris-HCl buffer and protease inhibitors to the pretreated culture, incubating on ice for initial lysis, dispersing the cell aggregates by homogenization, and then placing in an ice water bath for further lysis by ultrasonic disruption to obtain a cell lysis solution, precipitating incompletely disrupted bacteria and some cell fragments by high-speed centrifugation, and collecting the supernatant to obtain a crude protein extraction solution;

[0011] S3. Add pre-cooled anhydrous acetone to the crude protein extract solution, mix well, incubate at low temperature, remove the acetone solution by low-speed centrifugation to obtain a protein precipitate, repeat the impurity removal operation of resuspending the protein precipitate in pre-cooled anhydrous acetone and then separating the precipitate by high-speed centrifugation for multiple rounds, and finally obtain the desired protein extract.

[0012] As a preferred embodiment of the first aspect, in the lysate, the mass percentage of sodium dodecyl sulfate (SDS) is 1 wt.% to 2 wt.%, and the pH of the Tris-HCl buffer is 7.8.

[0013] As a preferred embodiment of the first aspect, the amount of the lysate added is 5-10 mL / g culture.

[0014] As a preferred embodiment of the first aspect, the centrifugal force of the low-speed centrifugation is controlled to be 5000~8000 g, and the centrifugal force of the high-speed centrifugation is controlled to be 12000~14000 g; the incubation temperature of the low-temperature incubation is controlled to be 0~-20°C, and the incubation time is controlled to be 1~2 hours.

[0015] As a preferred embodiment of the first aspect, the ultrasonic fragmentation is alternately cycled in a mode of ultrasonication for 2 to 4 s followed by a pause for 5 to 8 s, the ultrasonic power is controlled to be 130 to 200 W, and the total duration is 4 to 6 min.

[0016] As a preference of the first aspect above, the methane oxidizing bacteria are aerobic methane oxidizing bacteria or anaerobic methane oxidizing bacteria.

[0017] In a second aspect, the present invention provides a mass spectrometry pretreatment method for a protein extract, wherein the protein extract is extracted according to the methane oxidizing bacteria protein extraction method described in the first aspect, and the method steps include:

[0018] S4, re-dissolving the protein extract with a buffer, adding a reducing agent to the protein solution and incubating at a constant temperature until the reduction reaction is completed, so that the disulfide bonds in the protein are broken into sulfhydryl groups; then adding an alkylating agent and continuing to incubate at a constant temperature under light-proof conditions, so as to alkylate the exposed sulfhydryl groups and prevent the disulfide bonds from reforming;

[0019] S5. Add mass spectrometry grade trypsin to the protein solution after reduction and alkylation, hydrolyze the protein into peptides by constant temperature incubation, and then use mass spectrometry grade trifluoroacetic acid for acidification to terminate the enzymatic cleavage reaction and improve the solubility and stability of the peptides. The acidified system is subjected to high-speed centrifugation to remove the precipitate, and the supernatant containing the peptides is desalted and impurized by passing through a C18 reverse phase chromatography column before being used for protein mass spectrometry analysis.

[0020] As a preferred embodiment of the second aspect, the constant temperature incubation is maintained at 35-40° C., and the incubation time is 30 minutes to 1 hour.

[0021] As a preferred embodiment of the second aspect, the buffer 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)phosphine hydrochloride (TCEP) with a final concentration of 5-10 mM; and the alkylating agent is iodoacetamide with a final concentration of 10-40 mM.

[0022] As a preferred embodiment of the second aspect, the pH value of the acidification treatment is controlled at 2-3.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The pretreatment step provided by the present invention effectively removes inorganic salt ions and other impurities in the methane-oxidizing bacterial culture, thereby providing a purer basis for subsequent protein extraction.

[0025] (2) The present invention adopts an optimized protein extraction method that combines a urea-free lysis solution with ultrasonic disruption to target the cell wall characteristics of methane-oxidizing bacteria, which can efficiently disrupt cells and fully release proteins. Since the protein extraction method does not involve the use of urea, it has no effect on the subsequent protein digestion process.

[0026] (3) In the protein digestion step, the method of acidification and precipitation of peptide segments before desalting provided by the present invention can not only improve the solubility and stability of the peptide segments and improve the desalting efficiency, but also help avoid aggregation caused by charge interactions between peptide segments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1It is the SDS-PAGE result in the embodiment;

[0028] Figure 2 It is the SDS-PAGE result in the comparative example;

[0029] Figure 3 Protein mass spectrometry results. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0031] In a preferred embodiment of the present invention, a method for extracting protein from methane-oxidizing bacteria is provided, the steps of which are as follows:

[0032] S1. The methane-oxidizing bacteria enriched culture is centrifuged at high speed, the precipitate is separated and resuspended with PBS buffer, vortexed to fully mix, and then homogenized with a homogenizer, centrifuged at high speed again to remove the supernatant, and the precipitate is separated to obtain the pretreated culture.

[0033] It should be noted that the methane-oxidizing bacteria enriched culture in the present invention may be in different forms, such as a culture medium enriched with methane-oxidizing bacteria, activated sludge enriched with methane-oxidizing bacteria in a reactor, and the like.

[0034] It should be noted that the present invention is a kind of protein extraction that can be generally applied to methane-oxidizing bacteria, and is not limited to specific methane-oxidizing bacteria species. The methane-oxidizing bacteria targeted can be aerobic methane-oxidizing bacteria or anaerobic methane-oxidizing bacteria. Table 1 lists a series of methane-oxidizing bacteria that the present invention is applicable to, but is not limited to these specific species. Moreover, the present invention is particularly suitable for methane-oxidizing bacteria with a long growth cycle, such as the fully denitrifying anaerobic methane-oxidizing bacteria Candidatus Methylomirabilis sinica (M. sinica).

[0035] Table 1

[0036]

[0037] In the above step S1, the parameters of high-speed centrifugation are preferably set to: 12000~14000 g, 4°C, 5~10 min. For enrichment cultures with complex components such as activated sludge, the above PBS buffer resuspension, homogenization and high-speed centrifugation separation operations need to be repeated multiple times to ensure the subsequent separation effect, generally at least three times.

[0038] S2. Add a lysis solution consisting of sodium dodecyl sulfate (SDS), Tris-HCl buffer and protease inhibitors to the pretreated culture, incubate on ice for 5 to 10 minutes for preliminary lysis, disperse the cell aggregates by homogenization, and then place in an ice water bath for ultrasonic disruption using an ultrasonic disruptor to further lyse to obtain a cell lysate. After the cell lysate is centrifuged at high speed to precipitate the incompletely disrupted bacteria and some cell fragments, the supernatant is collected to obtain a crude protein solution.

[0039] It should be noted that the content of the components in the lysate and the amount of lysate added can be optimized according to the actual effect. In an embodiment of the present invention, the mass percentage of the above-mentioned 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 amount of the reagent itself. In addition, the amount of lysate added is 5~10mL / g culture, that is, 5~10mL of lysate is added for every 1g of culture.

[0040] In addition, in the above step S2, the parameters of ultrasonic disruption can be preferably set as follows: alternating cycles of ultrasonication for 2 to 4 seconds followed by a pause of 5 to 8 seconds, with the ultrasonic power controlled at 130 to 200 W and a total duration of 4 to 6 minutes. The parameters of high-speed centrifugation are preferably set as: 12,000 to 14,000 g, 4°C, 5 to 10 minutes.

[0041] S3. Add pre-cooled anhydrous acetone to the crude protein extract solution, mix well, incubate at low temperature, remove the acetone solution by low-speed centrifugation to obtain a protein precipitate, repeat the impurity removal operation of resuspending the protein precipitate in pre-cooled anhydrous acetone and then separating the precipitate by high-speed centrifugation for multiple rounds, and finally obtain the desired protein extract.

[0042] In the above step S3, multiple centrifugation separations are required, and generally, the initial low-speed centrifugation is performed at a low speed, and then the high-speed centrifugation is repeated at a high speed. The centrifugation parameters are preferably controlled as follows: the centrifugal force of the low-speed centrifugation is controlled to be 5000-8000 g, and the duration is 20-25 min; the centrifugal force of the high-speed centrifugation is controlled to be 12000-14000 g, and the duration is 5-10 min.

[0043] In addition, in the above step S3, the incubation temperature of the low-temperature incubation is preferably controlled to be 0 to -20°C, and the incubation time is preferably controlled to be 1 to 2 hours. Furthermore, the incubation temperature is preferably controlled to be -20°C.

[0044] Therefore, through the pretreatment process of step S1 and the protein extraction process of step S2, the inorganic salt ions and other impurities in the original culture system of the culture can be effectively removed through pretreatment, and then the purified whole protein is extracted therefrom, and then digested into peptides through subsequent mass spectrometry pretreatment for protein mass spectrometry analysis.

[0045] Based on the same inventive concept, in another embodiment of the present invention, after extracting a 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, and the method steps are as follows:

[0046] S4. Re-dissolve the protein extract using a buffer solution, then add a reducing agent to the protein solution and incubate at a constant temperature until the reduction reaction is completed, so that the disulfide bonds in the protein are broken into thiol groups; then add an alkylating agent and continue to incubate at a constant temperature under light-proof conditions, so as to alkylate the exposed thiol groups to prevent the disulfide bonds from reforming, thereby ensuring the stability of the protein structure.

[0047] In the above step S4, the specific types and addition amounts of the buffer, reducing agent, and alkylating agent can be optimized according to the actual effect. In the present invention, the buffer preferably uses a 50-200 mM ammonium bicarbonate solution; the reducing agent preferably uses dithiothreitol (DTT) and the final concentration after addition is 5-20 mM, or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and the final concentration after addition is 5-10 mM; the alkylating agent preferably uses iodoacetamide and the final concentration is 10-40 mM. In addition, the temperature of the above-mentioned constant temperature incubation is preferably maintained at 35-40 ° C, and the incubation time is preferably 30 minutes to 1 hour to ensure that the reaction is fully carried out.

[0048] S5. Add mass spectrometry grade trypsin to the protein solution after reduction and alkylation, hydrolyze the protein into peptides by constant temperature incubation, and then use mass spectrometry grade trifluoroacetic acid for acidification to terminate the enzymatic cleavage reaction while improving the solubility and stability of the peptides, thereby improving the efficiency of subsequent desalting. The acidified system is then subjected to high-speed centrifugation to remove the precipitate, and the supernatant containing the peptides is desalted and impurized by passing it through a C18 reverse phase chromatography column to remove salts and other small molecule impurities that may interfere with the mass spectrometry ionization process, and finally used for protein mass spectrometry analysis.

[0049] In the above step S5, the amount of trypsin added can refer to the manufacturer's recommended amount of the reagent. In an embodiment of the present invention, the protein actually contained in the solution can be used as a benchmark, and mass spectrometry grade trypsin is added in an amount of 50:1 of protein and pancreatic enzyme mass ratio. In addition, the temperature of constant temperature incubation is preferably maintained at 35~40 ° C, and the incubation time is preferably 30 minutes to 1 hour to ensure that the enzymatic reaction is fully carried out. In addition, when mass spectrometry grade trifluoroacetic acid is added for acidification, its amount needs to be reasonably optimized, preferably so that the pH value after adding trifluoroacetic acid is preferably 2~3. The centrifugal force of high-speed centrifugation is controlled to 12000~14000 g, and the duration is 5~10 min.

[0050] Example 1

[0051] In this example, the fully denitrifying anaerobic methane-oxidizing bacteria Candidatus Methylomirabilissinica (M. sinica) were used as an exemplary methane-oxidizing bacterium, and proteins from the enriched culture of M. sinica were extracted and pre-processed for mass spectrometry analysis. The specific operation of the method is as follows:

[0052] Step (1): Sample pretreatment

[0053] Take the M. sinica enriched culture in the form of activated sludge from the reactor into a centrifuge tube, centrifuge at 12000 g, 4°C for 8 minutes, remove the supernatant and separate the precipitate. Then add PBS buffer (pH=7.4) to resuspend the precipitate, vortex to fully mix it, and then use a homogenizer to homogenize it. Centrifuge at 12000 g, 4°C for 8 minutes again, remove the supernatant and separate the precipitate. Repeat the above PBS buffer resuspension, homogenization and high-speed centrifugation for 3 times to ensure that the original culture medium in the reactor is removed and the pretreated culture is obtained.

[0054] Step (2): Cell disruption and protein extraction

[0055] Two experimental groups with different lysis buffer formulas were set up, where the lysis buffer formula of group A1 was: 1 wt.% SDS, Tris-HCl buffer (pH=7.8) and protease inhibitors, and the lysis buffer formula of group A2 was: 2 wt.% SDS, Tris-HCl buffer (pH=7.8) and protease inhibitors. Among them, the protease inhibitor model was Roche 04693132001-20TABLETS, in the form of tablets, and the dosage was 1 tablet per 50 ml Tris-HCl buffer.

[0056] Take 0.5 g of the above-mentioned pretreated culture, add 5 mL of pre-cooled lysis solution (the A1 and A2 groups of experiments added the corresponding lysis solution respectively), mix gently and evenly, and incubate on ice for 8 min to complete the initial lysis. Subsequently, use a glass homogenizer to homogenize the lysed mixture to disperse the cell aggregates, and then use an ultrasonic disruptor to disrupt the mixture. The ultrasonic parameters are set to 195 W, and the ultrasonic is paused for 5 s after each 3 s of ultrasound. The alternating cycle lasts for 8 min. The mixture must be kept in an ice water bath environment during ultrasound to prevent overheating and protein denaturation. The cell lysate after ultrasonic treatment was centrifuged at 14000 g and 4 °C for 20 min to precipitate the incompletely broken bacteria and some cell fragments, collect the supernatant, and obtain a crude protein extract. The results of protein concentration determination by BCA method are shown in Table 2. The protein solution was detected by SDS-PAGE gel electrophoresis, and the results are shown in Figure 1 Shown in the middle strips Ⅰ and Ⅱ.

[0057] Table 2

[0058]

[0059] 100 μg crude protein was taken from each of the A1 and A2 groups, and 5 volumes of pre-cooled anhydrous acetone were slowly added. After gentle mixing, the mixture was incubated at -20 °C for 1 h, followed by low-speed centrifugation at 8000 g, 4 °C for 15 min, the acetone solution was removed, and the protein precipitate was collected. Then, pre-cooled anhydrous acetone was added to the protein precipitate, and after thorough mixing, the mixture was centrifuged again at 12000 g, 4 °C for 20 min, and the supernatant was discarded to retain the protein precipitate. After repeating the process of adding pre-cooled anhydrous acetone and high-speed centrifugation to remove the supernatant twice, the final protein precipitate was collected and naturally dried in a fume hood for later use.

[0060] Step (3): Protein reduction, alkylation and enzymatic hydrolysis

[0061] Groups A1 and A2 added 500 μL of 50 mM ammonium bicarbonate solution to the dried protein precipitate to redissolve the precipitate. Subsequently, the reducing agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to a final concentration of 5 mM and incubated at 37°C for 45 min. Then, the alkylating agent iodoacetamide was added to a final concentration of 20 mM and incubated for 1 h in the dark. After the incubation, 2 μL of mass spectrometry-grade trypsin (at a concentration of 1 μg / μL) was added and incubated overnight at 37°C to ensure that the protein was completely digested into peptides.

[0062] Step (4): Peptide removal

[0063] After enzymatic hydrolysis, 5 μL of 50% mass spectrometry grade trifluoroacetic acid (TFA) was added to acidify the digestion system to terminate the enzymatic reaction and improve the solubility and stability of the peptides. The acidified system was then centrifuged at 12,000 g, 4 °C for 5 min to remove insoluble impurities and collect the supernatant containing the peptides.

[0064] In order to provide high-quality protein mass spectrometry samples, the supernatant containing peptides was passed through a C18 reverse phase column to remove salt and impurities from the peptides. The specific steps were as follows: first, 200 µL of activation solution (50% ACN) was added and centrifuged at 1500 g for 1 min to activate the resin; then, 200 µL of equilibration solution (0.5% TFA in 5% ACN) was added and centrifuged at 1500 g for 1 min to equilibrate the resin. After loading, the sample was centrifuged at 1500 g for 1 min, the effluent was added back to the resin, and the protein was adsorbed on the resin by centrifugation at 1500 g for 1 min. The resin column was placed in a new receiving tube, 200 µL of washing solution (0.5% TFA in 5% ACN) was added, and the column was centrifuged at 1500 g for 1 min. The effluent was discarded and the washing was repeated once. Finally, the resin column was placed in a new receiving tube, and 40 µL of eluent (70% ACN) was added twice. The column was centrifuged at 1500 g for 1 min, and the eluent was collected. The eluate can be directly used for subsequent mass spectrometry analysis.

[0065] Step (5): Mass spectrometry

[0066] The eluate after peptide impurity removal in step (4) was used for mass spectrometry. The ORBITRAP ECLIPSE mass spectrometer was used with the optional FAIMS Pro™ Interface. The compensation voltage CV was switched between -45 and -65 every 1 s. The Nanospray Flex™ (NSI) ion source was used. The ion spray voltage was set to 2.0 kV, the ion transfer tube temperature was set to 320°C, the mass spectrometer was in data-dependent acquisition mode, the full scan range of the mass spectrometer was m / z 350-1500, the primary mass spectrometer resolution was set to 120000 (m / z), and the AGC was 4×10 5 The maximum injection time of C-trap was 50 ms; the secondary mass spectrometry detection adopted the “TopSpeed” mode, the secondary mass spectrometry resolution was set to 15000 (200 m / z), and the AGC was 5×10 4 , the maximum injection time was 22 ms, and the peptide fragmentation collision energy was set to 33% to generate raw data for mass spectrometry detection.

[0067] Example 2

[0068] In this example, the fully denitrifying anaerobic methane-oxidizing bacteria Candidatus Methylomirabilissinica (M. sinica) were used as an exemplary methane-oxidizing bacterium, and proteins from the enriched culture of M. sinica were extracted and pre-processed for mass spectrometry analysis. The specific operation of the method is as follows:

[0069] Step (1): Sample pretreatment

[0070] Take the M. sinica enriched culture in the form of activated sludge from the reactor into a centrifuge tube, centrifuge at 12000 g, 4°C for 8 minutes, remove the supernatant and separate the precipitate. Then add PBS buffer (pH=7.4) to resuspend the precipitate, vortex to fully mix it, and then use a homogenizer to homogenize it. Centrifuge at 12000 g, 4°C for 8 minutes again, remove the supernatant and separate the precipitate. Repeat the above PBS buffer resuspension, homogenization and high-speed centrifugation for 3 times to ensure that the original culture medium in the reactor is removed and the pretreated culture is obtained.

[0071] Step (2): Cell disruption and protein extraction

[0072] Two experimental groups with different lysis buffer formulas were set up, where the lysis buffer formula of group B1 was: 1 wt.% SDS, Tris-HCl buffer (pH=7.8) and protease inhibitors, and the lysis buffer formula of group B2 was: 2 wt.% SDS, Tris-HCl buffer (pH=7.8) and protease inhibitors. Among them, the protease inhibitor model was Roche 04693132001-20TABLETS, in the form of tablets, and the dosage was 1 tablet per 50 ml Tris-HCl buffer.

[0073] 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 inhibitors), mix gently, and incubate on ice for 8 min. Then use a glass homogenizer to homogenize the lysis mixture.

[0074] Take 0.5 g of the above-mentioned pretreated culture, add 5 mL of pre-cooled lysis solution (the A1 and A2 groups of experiments added the corresponding lysis solution respectively), mix gently and evenly, and incubate on ice for 8 min to complete the initial lysis. Subsequently, use a glass homogenizer to homogenize the lysed mixture to disperse the cell aggregates, and then use an ultrasonic disruptor to disrupt the mixture. The ultrasonic parameters are set to 195 W, and the ultrasonic is paused for 5 s after each 3 s of ultrasound. The alternating cycle lasts for 8 min. The mixture must be kept in an ice water bath environment during ultrasound to prevent overheating and protein denaturation. The cell lysate after ultrasonic treatment was centrifuged at 14000 g and 4 °C for 20 min to precipitate the incompletely broken bacteria and some cell fragments, collect the supernatant, and obtain a crude protein extract. The results of protein concentration determination by BCA method are shown in Table 3. The protein solution was detected by SDS-PAGE gel electrophoresis, and the results are shown in Figure 1 Shown in the middle strips III and IV.

[0075] Table 3

[0076]

[0077] 100 μg crude protein was taken from each of the B1 and B2 groups, and 5 volumes of pre-cooled anhydrous acetone were slowly added. After gentle mixing, the mixture was incubated at -20 °C for 1 h, and then centrifuged at 8000 g, 4 °C for 15 min, the acetone solution was removed, and the protein precipitate was collected. Then, pre-cooled anhydrous acetone was added to the protein precipitate, and after thorough mixing, the mixture was centrifuged at 12000 g, 4 °C for 20 min, and the supernatant was discarded to retain the protein precipitate. After repeating the process of adding pre-cooled anhydrous acetone and removing the supernatant by high-speed centrifugation twice, the final protein precipitate was collected and dried naturally in a fume hood for later use.

[0078] Step (3): Protein reduction, alkylation and enzymatic hydrolysis

[0079] Groups B1 and B2 added 500 μL of 50 mM ammonium bicarbonate solution to the dried protein precipitate to redissolve the precipitate. Subsequently, the reducing agent dithiothreitol (DTT) was added to the two groups, with the final concentration of DTT in group B1 being 10 mM and that in group B2 being 20 mM; and incubated at 37 °C for 45 min. Then, the alkylating agent iodoacetamide was added to make its final concentration 20 mM, and incubated for 1 h in the dark. After the incubation, 2 μL of mass spectrometry-grade trypsin (at a concentration of 1 μg / μL) was added and incubated overnight at 37 °C to ensure that the protein was completely digested into peptides.

[0080] Step (4): Peptide removal

[0081] The specific operation and parameters of peptide segment impurity removal are the same as those in step (4) of Example 1. The final eluate is collected and can be directly used for subsequent mass spectrometry analysis.

[0082] Step (5): Mass spectrometry

[0083] The eluate after peptide impurity removal in step (4) is used for mass spectrometry, and the specific operation and parameters are the same as those in step (5) in Example 1.

[0084] Comparative Example

[0085] This comparative example provides a commonly used protein extraction and mass spectrometry pre-treatment method for the culture of the fully denitrifying anaerobic methane-oxidizing bacteria M. sinica. The specific operation is as follows:

[0086] Step (1): Sample pretreatment

[0087] Take the M. sinica enriched culture in the form of activated sludge from the reactor into a centrifuge tube, centrifuge at 12000 g, 4°C for 8 minutes, remove the supernatant and separate the precipitate. Then add PBS buffer (pH=7.4) to resuspend the precipitate, vortex to fully mix it, and then use a homogenizer to homogenize it. Centrifuge at 12000 g, 4°C for 8 minutes again, remove the supernatant and separate the precipitate. Repeat the above PBS buffer resuspension, homogenization and high-speed centrifugation for 3 times to ensure that the original culture medium in the reactor is removed and the pretreated culture is obtained.

[0088] Step (2): Cell disruption and protein extraction

[0089] Three experimental groups with different lysis solution formulas as shown in Table 4 were set up respectively, wherein the lysis solution formula of group C1 was: 8 M urea, Tris-HCl buffer (pH=7.8) and protease inhibitors, the lysis solution formula of group C2 was: 8 M urea, 1 wt.% SDS, Tris-HCl buffer (pH=7.8) and protease inhibitors, and the lysis solution formula of group C3 was: 8 M urea, 2 wt.% SDS, Tris-HCl buffer (pH=7.8) and protease inhibitors. Among them, the protease inhibitor model was Roche04693132001-20 TABLETS, in the form of tablets, and the dosage was 1 tablet per 50 ml Tris-HCl buffer.

[0090] Table 4

[0091]

[0092] Take 0.5 g of the above-mentioned pretreated culture, add 5 mL of pre-cooled lysis solution (the three groups of experiments C1, C2 and C3 added the corresponding lysis solution respectively), mix gently and evenly, and incubate on ice for 10 min to complete the initial lysis. Subsequently, the lysed mixture was homogenized with a glass homogenizer to disperse the cell aggregates, and then the mixture was broken with an ultrasonic disruptor. The ultrasonic parameters were set to 195 W, and the ultrasonic was paused for 5 s after each 3 s of ultrasound. The alternating cycle lasted for 8 min. The mixture must be kept in an ice water bath environment during ultrasound to prevent overheating and protein denaturation. The cell lysate after ultrasonic treatment was centrifuged at 14000 g and 4 °C for 20 min to precipitate the incompletely broken bacteria and some cell fragments, collect the supernatant, and obtain a crude protein extract. The results of protein concentration determination by BCA method are shown in Table 5. The protein solution was detected by SDS-PAGE gel electrophoresis, and the results are shown in Figure 2 Shown in the middle strips Ⅰ, Ⅱ, and Ⅲ.

[0093] Table 5

[0094]

[0095] Step (3): Protein reduction, alkylation and enzymatic hydrolysis

[0096] In groups C1, C2, and C3, dithiothreitol (DTT) was added to the crude protein extract solution to a final concentration of 10 mM, and the solution was reduced at 30°C for 1 h, followed by the addition of iodoacetamide to a final concentration of 40 mM, and incubated at room temperature in the dark for 45 min. Subsequently, 7 volumes of ammonium bicarbonate buffer (50 mM) were added to each group, so that the urea concentration in the system was less than 1 M and the pH tended to be neutral. Finally, trypsin was added at a mass ratio of 50:1 between protein and trypsin for enzymatic hydrolysis, and the solution was incubated at 37°C overnight to break peptides.

[0097] Step (4): Peptide removal

[0098] The mixture after enzymatic digestion was subjected to a C18 reverse phase column to remove impurities from the peptides. The specific steps were as follows: First, 200 μL of activation solution (50% ACN) was added and centrifuged at 1500 g for 1 min to activate the resin; then, 200 μL of equilibration solution (0.5% TFA in 5% ACN) was added and centrifuged at 1500 g for 1 min to equilibrate the resin. After loading, the mixture was centrifuged at 1500 g for 1 min, and the effluent was added back to the resin and centrifuged at 1500 g for 1 min. The protein was adsorbed on the resin. The resin column was placed in a new receiving tube, 200 μL of washing solution (0.5% TFA in 5% ACN) was added, centrifuged at 1500 g for 1 min, the effluent was discarded, and the washing was repeated once. Finally, the resin column was placed in a new receiving tube, 40 μL of eluent (70% ACN) was added twice, centrifuged at 1500 g for 1 min, and the eluent was collected for mass spectrometry analysis.

[0099] Step (5): Mass spectrometry

[0100] The eluate after peptide segment impurity removal in step (4) is used for mass spectrometry. The specific operation and parameters are the same as step (5) in Example 1.

[0101] Figure 1 The bands are the results of SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining-decolorization treatment after the whole protein extraction of the culture of the fully denitrifying anaerobic methane-oxidizing bacteria M. sinica in Example 1 and Example 2, wherein M is an indicator band for molecular weight annotation. Figure 2 The band results after SDS-PAGE electrophoresis and Coomassie brilliant blue staining-decolorization of the protein extracted in the comparative example, wherein M is the indicator band for molecular weight annotation. Figure 3 The protein mass spectrometry results in Example 1, Example 2 and the comparative example.

[0102] Comparison of the experimental results in the above-mentioned Example 1, Example 2 and the comparative example shows that the method of the present invention has the advantages of higher protein concentration extracted and fewer protein peaks in mass spectrometry. The specific results are compared and explained in detail below.

[0103] 1. Comparison of protein extraction concentration

[0104] Example 1 Results:

[0105] Group A1 (1% SDS, 5 mM TCEP): protein concentration 1.2529 μg / μL

[0106] Group A2 (2% SDS, 10 mM TCEP): protein concentration 1.0277 μg / μL

[0107] Example 2 Results:

[0108] Group B1 (1% SDS, 10 mM DTT): protein concentration 1.1728 μg / μL

[0109] Group B2 (2% SDS, 20 mM DTT): protein concentration was 1.3362 μg / μL

[0110] Comparative results:

[0111] Group C1 (8 M urea, no SDS): protein concentration was 0.4285 μg / μL

[0112] Group C2 (8 M urea, 1% SDS): protein concentration was 0.2672 μg / μL

[0113] Group C3 (8 M urea, 2% SDS): protein concentration was 0.6203 μg / μL

[0114] Comparison of the three groups of results shows that the protein concentrations of Examples 1 and 2 are significantly higher than those of the comparative example. The lowest concentration in the examples (Group A2, 1.0277 μg / μL) is still about 1.66 times the highest concentration in the comparative example (Group C3, 0.6203 μg / μL), while the highest concentration in the examples (Group B2, 1.3362 μg / μL) is 2.15 times the highest concentration in the comparative example.

[0115] Moreover, since urea was used as the main cleavage component in the comparative example, the enzymatic hydrolysis of group C proteins was incomplete during the enzymatic hydrolysis stage, resulting in the proteins not being fully cut into peptides of the expected length, the peptide coverage decreased, and the accuracy of bottom-up protein mass spectrometry identification decreased.

[0116] 2. Comparison of protein peaks and impurities in mass spectrometry

[0117] In Example 1 and Example 2, SDS-PAGE gel electrophoresis detection (Example 1: Figure 1 Middle strips Ⅰ, Ⅱ; Example 2: Figure 1 The results of subsequent mass spectrometry analysis (ORBITRAP ECLIPSE, FAIMS Pro™ Interface optimized ion selectivity) showed that the protein bands were clear and there were few impurity proteins. Therefore, the peptide segment impurity removal step used a C18 reverse phase column for further purification to remove insoluble impurities and salts, ensuring high purity of the protein peak in mass spectrometry detection and few impurity peaks.

[0118] In the comparative example, SDS-PAGE detection ( Figure 2Bands Ⅰ, Ⅱ, and Ⅲ in the middle showed that the distribution of protein bands was scattered, indicating that there might be more non-target proteins or impurities remaining in the extract.

[0119] Comparison and analysis of the three groups of results show that the present invention optimizes the lysate composition and ultrasonic crushing process, and the C18 column desalting step effectively reduces impurity interference, making the protein peak in the mass spectrum more concentrated and significantly reducing impurity peaks. The comparative example relies on the traditional urea lysis system, the protein purity is low during the extraction process, and it is easily interfered by impurities during mass spectrometry detection, resulting in a decrease in protein peak resolution.

[0120] In summary, the present invention shows significant advantages in protein extraction concentration and mass spectrometry analysis quality, providing a better pre-treatment solution for protein mass spectrometry research of methanotrophic bacteria.

[0121] However, it should be noted that the above-mentioned embodiments and comparative examples of the present invention use the fully denitrifying anaerobic methane-oxidizing bacteria Candidatus Methylomirabilis sinica (M. sinica) as an exemplary methane-oxidizing bacterium to extract proteins from the enriched culture and perform pre-mass spectrometry analysis, because this methane-oxidizing bacterium is a typical representative of the difficulty in protein extraction. However, the present invention is a general method and is not limited to this type of methane-oxidizing bacteria, but can also be applied to other methane-oxidizing bacteria. The following is another example to demonstrate its versatility

[0122] Example 3

[0123] In this example, aerobic methane-oxidizing bacteria were used as exemplary methane-oxidizing bacteria, and proteins from aerobic methane-oxidizing bacteria enrichment culture were extracted and pre-processed for mass spectrometry analysis. The specific operation of the method is as follows:

[0124] Step (1): Sample pretreatment

[0125] Take the aerobic methane-oxidizing bacteria enrichment culture in the form of activated sludge from the reactor into a centrifuge tube, centrifuge at 12000 g, 4 ° C for 8 minutes, remove the supernatant and separate the precipitate. Then add PBS buffer (pH = 7.4) to resuspend the precipitate, vortex and shake to mix it thoroughly, and then use a homogenizer to homogenize it. Centrifuge at 12000 g, 4 ° C for 8 minutes again, remove the supernatant and separate the precipitate. Repeat the above PBS buffer resuspension, homogenization and high-speed centrifugation for 3 times to ensure that the original culture medium in the reactor is removed and the pretreated culture is obtained.

[0126] Step (2): Cell disruption and protein extraction

[0127] Take 0.5 g of the above pretreated culture, add 5 mL of pre-cooled lysis solution (using the lysis solution formula of group A1 in the embodiment), mix gently and evenly, and incubate on ice for 8 min to complete the initial lysis. Then use a glass homogenizer to homogenize the lysed mixture to disperse the cell aggregates, and then use an ultrasonic disruptor to disrupt the mixture. The ultrasonic parameters are set to 195 W, and the ultrasound is paused for 5 s after 3 s, and the alternating cycle lasts for 8 min. The mixture needs to be kept in an ice water bath environment during the ultrasound to prevent overheating and protein denaturation. The cell lysate after ultrasonic treatment is centrifuged at 14000 g and 4 ° C for 20 min to precipitate the incompletely broken bacteria and some cell fragments, and the supernatant is collected to obtain a crude protein extract. Take 100 μg of crude protein, slowly add 5 times the volume of pre-cooled anhydrous acetone, gently mix and incubate at -20 °C for 1 h, then centrifuge at 8000 g, 4 °C for 15 min, remove the acetone solution, and collect the protein precipitate. Then add pre-cooled 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-cooled anhydrous acetone and high-speed centrifugation to remove the supernatant twice, collect the final protein precipitate, and dry it naturally in the fume hood for later use.

[0128] Step (3): Protein reduction, alkylation and enzymatic hydrolysis

[0129] Add 500 μL of 50 mM ammonium bicarbonate solution to the dried protein precipitates to redissolve the precipitates. Subsequently, add the reducing agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to a final concentration of 5 mM and incubate at 37 °C for 45 min. Then add the alkylating agent iodoacetamide to a final concentration of 20 mM and incubate for 1 h in the dark. After the incubation, add 2 μL of mass spectrometry grade trypsin (concentration of 1 μg / μL) and incubate overnight at 37 °C to ensure that the protein is completely digested into peptides.

[0130] Step (4): Peptide removal

[0131] After enzymatic hydrolysis, 5 μL of 50% mass spectrometry grade trifluoroacetic acid (TFA) was added to acidify the digestion system to terminate the enzymatic reaction and improve the solubility and stability of the peptides. The acidified system was then centrifuged at 12,000 g, 4 °C for 5 min to remove insoluble impurities and collect the supernatant containing the peptides.

[0132] In order to provide high-quality protein mass spectrometry samples, the supernatant containing peptides was passed through a C18 reverse phase column to remove salt and impurities from the peptides. The specific steps were as follows: first, 200 µL of activation solution (50% ACN) was added and centrifuged at 1500 g for 1 min to activate the resin; then, 200 µL of equilibration solution (0.5% TFA in 5% ACN) was added and centrifuged at 1500 g for 1 min to equilibrate the resin. After loading, the sample was centrifuged at 1500 g for 1 min, the effluent was added back to the resin, and the protein was adsorbed on the resin by centrifugation at 1500 g for 1 min. The resin column was placed in a new receiving tube, 200 µL of washing solution (0.5% TFA in 5% ACN) was added, and the column was centrifuged at 1500 g for 1 min. The effluent was discarded and the washing was repeated once. Finally, the resin column was placed in a new receiving tube, and 40 µL of eluent (70% ACN) was added twice. The column was centrifuged at 1500 g for 1 min, and the eluent was collected. The eluent can be directly used for subsequent mass spectrometry analysis to generate raw mass spectrometry data.

[0133] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for extracting protein from methane-oxidizing bacteria, characterized in that: Here are the steps: S1, centrifuging the enriched culture of methane-oxidizing bacteria at high speed, separating the precipitate, resuspending and mixing with PBS buffer, and then homogenizing, and centrifuging at high speed again to separate the precipitate, thereby obtaining a pretreated culture; S2, adding a lysis solution composed of sodium dodecyl sulfate, Tris-HCl buffer and protease inhibitors to the pretreated culture, incubating on ice for initial lysis, dispersing cell aggregates by homogenization, placing in an ice water bath and further lysing by ultrasonication to obtain a cell lysate, precipitating incompletely broken bacteria and some cell fragments by high-speed centrifugation, and collecting the supernatant to obtain a crude protein extraction solution; S3. Add pre-cooled anhydrous acetone to the crude protein extract solution, mix well, incubate at low temperature, remove the acetone solution by low-speed centrifugation to obtain a protein precipitate, repeat the impurity removal operation of resuspending the protein precipitate in pre-cooled anhydrous acetone and then separating the precipitate by high-speed centrifugation for multiple rounds, and finally obtain the desired protein extract.

2. The method for extracting protein from methane-oxidizing bacteria according to claim 1, wherein: In the lysate, the mass percentage of sodium dodecyl sulfate is 1 wt.% to 2 wt.%, and the pH of the Tris-HCl buffer is 7 to 8.

3. The method for extracting protein from methane-oxidizing bacteria according to claim 1, wherein: The amount of the lysate added is 5-10 mL / g culture.

4. The method for extracting protein from methane-oxidizing bacteria according to claim 1, wherein: The centrifugal force of the low-speed centrifugation is controlled to be 5000-8000 g, and the centrifugal force of the high-speed centrifugation is controlled to be 12000-14000 g; the incubation temperature of the low-temperature incubation is controlled to be 0-20°C, and the incubation time is controlled to be 1-2 hours.

5. The method for extracting protein from methane-oxidizing bacteria according to claim 1, wherein: The ultrasonic disruption is performed in an alternating cycle of 2-4 s ultrasonication followed by a 5-8 s pause, the ultrasonic power is controlled at 130-200 W, and the total duration is 4-6 min.

6. The method for extracting protein from methane-oxidizing bacteria according to claim 1, wherein: The methane oxidizing bacteria are aerobic methane oxidizing bacteria or anaerobic methane oxidizing bacteria.

7. A mass spectrometry pretreatment method for a protein extract, characterized in that: The protein extract is obtained by extracting the protein from methane-oxidizing bacteria according to claim 1, and the method comprises the following steps: S4, re-dissolving the protein extract with a buffer, adding a reducing agent to the protein solution and incubating at a constant temperature until the reduction reaction is completed, so that the disulfide bonds in the protein are broken into sulfhydryl groups; then adding an alkylating agent and continuing to incubate at a constant temperature under light-proof conditions, so as to alkylate the exposed sulfhydryl groups and prevent the disulfide bonds from reforming; S5. Add mass spectrometry grade trypsin to the protein solution after reduction and alkylation, hydrolyze the protein into peptides by constant temperature incubation, and then use mass spectrometry grade trifluoroacetic acid for acidification to terminate the enzymatic cleavage reaction and improve the solubility and stability of the peptides. The acidified system is subjected to high-speed centrifugation to remove the precipitate, and the supernatant containing the peptides is desalted and impurized by passing through a C18 reverse phase chromatography column before being used for protein mass spectrometry analysis.

8. The mass spectrometry pretreatment method of protein extract according to claim 7, characterized in that: The constant temperature incubation is maintained at 35-40°C, and the incubation time is 30 minutes to 1 hour.

9. The mass spectrometry pretreatment method of protein extract according to claim 7, characterized in that: The buffer 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)phosphine 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 of protein extract according to claim 7, characterized in that: The pH value of the acidification treatment is controlled at 2-3.

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