A method for detecting multiple target proteins in an MVA pathway of an engineering strain based on MRM technology

Abstract

The present application relates to a kind of rapid detection method of multiple target protein of engineering strain methylmalonic acid (MVA) pathway based on multiple reaction monitoring technology (MRM).Firstly, the MRM acquisition method is established with the target peptide segment;The target peptide segment standard is prepared into at least 5 different concentrations, and data is collected using liquid chromatography-mass spectrometry technology and MRM method, with concentration value and peak area as horizontal and vertical coordinates to draw standard curve;Engineering strain sample is obtained by using conventional proteomics sample pretreatment process Enzymolysis peptide segment solution, mass spectrometry data is collected using liquid chromatography-mass spectrometry technology and MRM method, and then external standard method is used for protein quantification.The present application is based on traditional external standard method quantification, constructs target protein detection method based on short chromatographic column, microliter liquid chromatography system, short separation gradient and MRM quantification strategy, and it is used for the rapid quantitative analysis of the five proteins of engineering strain MVA pathway.

Description

Technical Field

[0001] This invention belongs to the field of targeted protein quantitative analysis, and in particular relates to a rapid detection method for multiple target proteins in the mevalonic acid (MVA) pathway of engineered strains based on multiple reaction monitoring (MRM) technology. Background Technology

[0002] With the development of synthetic biology, the use of microbial cells as factories to produce metabolites has attracted extensive research. Among these, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) is often used as a model chassis cell due to its clear genetic background and relatively simple genetic modification. Terpenes are an important class of chemicals with certain physiological activities. Because the chemical synthesis and natural extraction of terpenes are complex processes, several studies have been conducted on the production of terpenes using yeast cells. The MVA pathway is the main pathway for the synthesis of terpenes in eukaryotes such as yeast. Modifying mevalonyl-CoA reductase (HMGR), farnesyl pyrophosphate synthase (ERG20), and mevalonate kinase (ERG12) in the MVA pathway can effectively increase the yield of terpenes. However, genetic modification results in a massive number of strain samples, and conventional strain screening methods mainly rely on the yield of chemical products, which is time-consuming and inefficient. Therefore, accurate quantification of relevant endogenous pathway proteases in the modified pathway is not only helpful for high-throughput strain screening but also has important guiding significance for the modification of cell factories.

[0003] Multiple reaction monitoring (MRM) mass spectrometry has attracted widespread attention in the field of protein quantification due to its outstanding features such as high specificity, good reproducibility, and high throughput. This invention, based on the MRM strategy, optimizes the gradient elution conditions in liquid chromatography, shortening them to 3 minutes, and establishes a rapid quantitative analysis method for strain proteins with high sensitivity and strong anti-interference ability. This method can achieve highly specific, high-throughput, and accurate quantification of MVA pathway proteins in engineered strains (such as yeast). Summary of the Invention

[0004] A method for detecting multiple target proteins of the MVA pathway in engineered strains based on MRM technology has the advantages of high sensitivity, high accuracy and high throughput detection, and can rapidly and accurately quantify MVA pathway-related proteins in engineered strains.

[0005] To achieve the above objectives, the technical solution adopted by this invention is as follows: a rapid detection method for multiple target proteins in the mevalonic acid (MVA) pathway of engineered strains based on multiple reaction monitoring (MRM) technology, comprising the following steps:

[0006] 1. Establish an MRM acquisition method based on the target peptide, wherein the target peptide is one or more of the following: AIILGAQSIK (corresponding to ERG10); NLSDDLR (corresponding to ERG12); LLYNDFR and / or ELDITNK (corresponding to ERG13); ADVLTAFLNK and / or ISQVDESR (corresponding to ERG20); WVSPNDLK (corresponding to IDI).

[0007] The targeted peptide is searched in the database. The peptide exists only in the corresponding protein and contains only one amino acid sequence.

[0008] 2. Prepare at least five solutions of the target peptide standard described in claim 1 with different concentrations (1 μM-100 pM), and use liquid chromatography-mass spectrometry (LC-MS) to acquire mass spectrometry data according to the established MRM method to obtain retention time and peak area information. Plot a standard curve with concentration value and peak area as the x and y axes, respectively.

[0009] 3. The engineered strain sample was dissolved using lysis buffer, and the sample protein was extracted. After protein sample pretreatment, trypsin-digested peptide solution was obtained. Mass spectrometry data was acquired using liquid chromatography-mass spectrometry according to the established MRM method to obtain the retention time and corresponding peak area of ​​the peptide.

[0010] 4. Take the peak area of ​​the substance with the same retention time as in step 2 from step 3 and input it into the standard curve to obtain its concentration (nM or pM) in the actual sample enzymatic hydrolysis peptide solution, thereby obtaining the concentration of the protein corresponding to the peptide. The two concentration values ​​are the same.

[0011] When using two peptides to detect the same protein, if the detection results of the two peptides corresponding to the same protein are different, the average of the two results is taken as the protein concentration value; if they are the same, either value can be used.

[0012] 5. In step 3, the specific operation method for sample pretreatment is as follows: (a) the bacterial strain samples are treated with lysis buffer, which can be one or a mixture of several of the following: 4% sodium dodecyl sulfate (SDS), 8M urea, and NP-40; (b) the disruption method can be one of ultrasonic or magnetic bead grinding; (c) denaturation, reduction, and alkylation: dithiothreitol (DTT) or tri(2-chloroethyl) phosphate (TCEP) is added to each sample to a final concentration of 0.1 mM. (d) Denaturation and reduction: Add 100-400 μL of ammonium bicarbonate solution and trypsin, and react at 30-40 °C for 12-16 h; (e) Desalting: After desalting, the peptides to be tested are lyophilized and reconstituted with 0.1% formic acid solution.

[0013] 6. In steps 2 and 3, data acquisition was performed using an AB SCIEX Triple TOF 5600+ mass spectrometer; the packing material was selected as C18 reversed-phase chromatography packing material with a diameter of 1.7 μm to 3 μm, the column inner diameter was 0.1 mm to 2.1 mm, the column length was 30 mm to 150 mm, and the liquid chromatography flow rate was 1 μL / min to 20 μL / min; mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was a 0.1% formic acid acetonitrile solution; the separation gradient was as follows: elution conditions were 0 to 3-4 min, from 0-5% B to 30-40% B (volume ratio); then within 1-2 min, from 30-40% B to 75%-85% B (volume ratio); then within 1-2 min, from 75%-85% B to 0-5% B (volume ratio), with the remaining percentages made up with mobile phase A.

[0014] 7. In step 3, the method is applicable to engineered strains such as Saccharomyces cerevisiae, Escherichia coli, or Enterococcus faecalis.

[0015] This invention constructs a target protein detection method based on a short chromatographic column, a microliter liquid chromatography system, a short separation gradient, and an MRM quantification strategy, building upon the traditional external standard method for quantification. This method is then applied to the rapid quantitative analysis of five proteins in the MVA pathway of engineered strains.

[0016] The present invention has the following advantages:

[0017] 1. The gradient separation time is short and the detection speed is fast, which can greatly improve the throughput of quantitative analysis of samples.

[0018] 2. Based on the targeting advantages of the MRM strategy, it has high quantitative sensitivity, good accuracy, and strong anti-interference ability.

[0019] 3. This invention provides a method that can be used for the quantitative detection of proteins in other metabolic pathways of engineered strains. Attached Figure Description

[0020] Figure 1 Chromatographic retention time of the target standard peptide AIILGAQSIK (corresponding to ERG10);

[0021] Figure 2 Chromatographic retention time of the target standard peptide NLSDDLR (corresponding to ERG12);

[0022] Figure 3 Chromatographic retention times of the targeted standard peptides LLYNDFR and ELDITNK (corresponding to ERG13);

[0023] Figure 4 Chromatographic retention times of the targeted standard peptides ADVLTAFLNK and ISQVDESR (corresponding to ERG20);

[0024] Figure 5 Chromatographic retention time of the target standard peptide WVSPNDLK (corresponding to IDI). Detailed Implementation

[0025] An engineered strain that constructs a salicylide synthesis pathway in Saccharomyces cerevisiae was selected, and the present invention will be further illustrated through the description of specific embodiments.

[0026] Example 1

[0027] All target peptides were selected: AIILGAQSIK (corresponding to ERG10); NLSDDLR (corresponding to ERG12); LLYNDFR and / or ELDITNK (corresponding to ERG13); ADVLTAFLNK and / or ISQVDESR (corresponding to ERG20); WVSPNDLK (corresponding to IDI). The complete protein sequence files of *Saccharomyces cerevisiae* were downloaded from Uniprot, and a mass spectrometry (MRM) acquisition method was established using Skyline software. The above-mentioned target peptide standards were prepared into solutions with concentrations of 200, 100, 50, 10, 1, and 0.2 nM, respectively. Mass spectrometry data were acquired using an ABSCIEX Triple TOF 5600+ mass spectrometer according to the established MRM method. 5 μL of each sample was injected to obtain its retention time (e.g., ...). Figure 1-5 The standard curve was plotted with concentration value and peak area as the x and y axes, respectively, as shown in the table below:

[0028]

[0029]

[0030] Saccharomyces cerevisiae strain 1 (40 g / L initial sugar, YPD fermentation, yield 64.6 mg / L) was lysed in 4% SDS lysis buffer by sonication for 10 min, followed by centrifugation at 16000 x g for 10 min. The supernatant was collected as yeast strain protein. The supernatant protein concentration was determined to be 6.32 mg / mL using the BCA method. 50 μg of protein was collected in a centrifuge tube. The strain protein was added to DTT solution to a final concentration of 10 mM, denatured and reduced at 95 °C for 5 min, cooled to room temperature, and then IAA solution was added to a final concentration of 20 mM. The alkylation reaction was carried out at room temperature in the dark for 30 min. Subsequently, 200 μL of 50 mM ammonium bicarbonate solution and 1 μg of trypsin were added, and the reaction was carried out overnight at 37 °C for 16 h. After desalting by a desalting column, the sample was lyophilized and reconstituted with 20 μL of 0.1% formic acid solution.

[0031] Mass spectrometry data were acquired using an AB SCIEX Triple TOF 5600+ mass spectrometer according to the established MRM method. A 1.9 μm diameter C18 reversed-phase chromatography packing material was selected, with a column inner diameter of 1.5 mm and a column length of 50 mm. The liquid chromatography flow rate was 10 μL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution was used under the following conditions: 0–3 min, 5–35% B (v / v); 3–4 min, 35%–80% B (v / v); 4–5 min, 80%–5% B (v / v). 5 μL of sample was injected to obtain retention time and peak area information. The peak area of ​​substances with the same retention time as the standard peptide was used to calculate the concentration of the peptide in the actual sample enzymatic hydrolysis solution using the external standard method. The concentrations of the target peptides obtained were AIILGAQSIK 32 nM; NLSDDLR 26 nM; LLYNDFR 30 nM; ELDITNK 28 nM; ADVLTAFLNK 96 nM; ISQVDESR 78 nM; WVSPNDLK 82 nM, which correspond to the protein concentrations of ERG10 32 nM; ERG12 26 nM; ERG13 29 nM; ERG20 87 nM; and IDI 82 nM.

[0032] Example 2

[0033] All target peptides were selected: AIILGAQSIK (corresponding to ERG10); NLSDDLR (corresponding to ERG12); LLYNDFR (corresponding to ERG13); ISQVDESR (corresponding to ERG20); WVSPNDLK (corresponding to IDI). The complete protein sequence files of Saccharomyces cerevisiae were downloaded from Uniprot, and the MRM acquisition method was established using Skyline software.

[0034] Saccharomyces cerevisiae strain 2 (40 g / L initial sugar, YPD fermentation, yield 23 mg / L) was lysed with 4% SDS lysis buffer by sonication for 10 min, centrifuged at 16000 x g for 10 min, and the supernatant was collected as yeast strain protein. The supernatant protein concentration was determined to be 5.86 mg / mL using the BCA method, and 50 μg of protein was collected in a centrifuge tube. The strain protein was added to DTT solution to a final concentration of 10 mM, denatured and reduced at 65 °C for 90 min, cooled to room temperature, and then IAA solution was added to a final concentration of 20 mM. Alkylation was carried out at room temperature in the dark for 30 min. Subsequently, 200 μL of 50 mM ammonium bicarbonate solution and 1 μg of trypsin were added, and the reaction was carried out overnight at 37 °C for 16 h. After desalting by a desalting column, the sample was lyophilized and reconstituted with 40 μL of 0.1% formic acid solution.

[0035] Mass spectrometry data were acquired using an AB SCIEX Triple TOF 5600+ mass spectrometer according to the established MRM method, employing the same data acquisition conditions and procedures as in Example 1. 5 μL of sample was injected to obtain retention time and peak area information. The peak area of ​​the substance with the same retention time as the standard peptide was used in the standard curve from Example 1, and its concentration in the actual sample enzymatic digestion peptide solution was calculated using the external standard method. The concentrations of the target peptides were found to be AIILGAQSIK 22 nM; NLSDDLR 16 nM; LLYNDFR 24 nM; ISQVDESR 69 nM; WVSPNDLK 66 nM, corresponding to protein concentrations of ERG 1022 nM; ERG 1216 nM; ERG 13 24 nM; ERG 2069 nM; and IDI 66 nM.

[0036] Example 3

[0037] All target peptides were selected: AIILGAQSIK (corresponding to ERG10); NLSDDLR (corresponding to ERG12); ELDITNK (corresponding to ERG13); ADVLTAFLNK (corresponding to ERG20); WVSPNDLK (corresponding to IDI). The complete protein sequence files of Saccharomyces cerevisiae were downloaded from Uniprot, and the MRM acquisition method was established using Skyline software.

[0038] Saccharomyces cerevisiae strain 3 (40 g / L initial sugar, YPD fermentation, yield 17 mg / L) was lysed with 8 M urea lysis buffer by sonication for 10 min, centrifuged at 16000 x g for 10 min, and the supernatant was collected as yeast protein. The supernatant protein concentration was determined to be 6.04 mg / mL using the BCA method, and 60 μg of protein was collected in a centrifuge tube. The strain protein was added to DTT solution to a final concentration of 10 mM, denatured and reduced at 95 °C for 5 min, cooled to room temperature, and then IAA solution was added to a final concentration of 20 mM. Alkylation was carried out at room temperature in the dark for 30 min. Subsequently, 200 μL of 50 mM ammonium bicarbonate solution and 1 μg of trypsin were added, and the reaction was carried out overnight at 37 °C for 16 h. After desalting by a desalting column, the sample was lyophilized and the peptide sample was reconstituted with 80 μL of 0.1% formic acid solution.

[0039] Mass spectrometry data were acquired using an AB SCIEX Triple TOF 5600+ mass spectrometer according to the established MRM method, employing the same data acquisition conditions and procedures as in Example 1. 5 μL of sample was injected to obtain retention time and peak area information. The peak area of ​​the substance with the same retention time as the standard peptide was used as input to the standard curve in Example 1, and its concentration in the actual sample enzymatic digestion peptide solution was calculated using the external standard method. The concentrations of the target peptides were found to be AIILGAQSIK 43 nM; NLSDDLR 37 nM; ELDITNK 42 nM; ADVLTAFLNK 133 nM; WVSPNDLK 106 nM, corresponding to protein concentrations of ERG10 43 nM; ERG12 37 nM; ERG13 42 nM; ERG20 133 nM; and IDI 106 nM.

Claims

1. A method for detecting multiple target proteins of the MVA pathway in engineered strains based on MRM technology, characterized in that, Quantification of certain proteins in the MVA pathway of engineered strains was performed using targeted peptides. Some proteins in the MVA pathway include one or more of acetyl-CoA transferase (ERG10), mevalonate kinase (ERG12), mevalonyl-CoA synthase (ERG13), farnesyl pyrophosphate synthase (ERG20), and isopentenyl diphosphate isomerase (IDI). The targeted peptides are: AIILGAQSIK; NLSDDLR; LLYNDFR and / or ELDITNK; ADVLTAFLNK and / or ISQVDESR; WVSPNDLK, one or more of these; The target peptide for ERG10 is AIILGAQSIK; the target peptide for ERG12 is NLSDDLR; the target peptides for ERG13 are LLYNDFR and / or ELDITNK; the target peptides for ERG20 are ADVLTAFLNK and / or ISQVDESR; and the target peptide for IDI is WVSPNDLK. Based on the target peptide, an MRM method was established, and the external standard method was used to quantify some proteins in the engineered strain.

2. The detection method according to claim 1, characterized in that, step include: (1) Prepare at least five solutions of different concentrations of 1mM-100pM using the target peptide standard described in claim 1, perform proteomics analysis on the peptide using liquid chromatography-mass spectrometry, acquire mass spectrometry data using the MRM method, obtain its retention time and peak area information, and plot a standard curve with concentration value and peak area as the horizontal and vertical axes, respectively. (2) The engineered strain sample was dissolved using lysis buffer, and the sample protein was extracted by breaking it up. The protein sample was pretreated to obtain a peptide solution digested by trypsin. The peptide was analyzed by proteomics using liquid chromatography-mass spectrometry (LC-MS). The sample peptide solution was collected by mass spectrometry using the MRM method to obtain the retention time and corresponding peak area of ​​the peptide. (3) Take the peak area of ​​the substance with the same retention time as in step (1) in step (2) and bring it into the standard curve to obtain its concentration (nM or pM) in the actual sample enzymatic hydrolysis peptide solution, thereby obtaining the concentration of the protein corresponding to the peptide. The two concentration values ​​are the same. When using two peptides to detect the same protein, if the detection results of the two peptides corresponding to the same protein are different, the average of the two results is taken as the protein concentration value; if they are the same, either value can be used.

3. The detection method according to claim 1, characterized in that: The targeted peptide is searched in the database. The peptide exists only in the corresponding protein and contains only one sequence of the targeted peptide.

4. The detection method according to claim 2, characterized in that: In step (1), the specific operation method of sample pretreatment is as follows: (a) the strain sample is treated with lysis buffer, which is one or a mixture of several of the following: 4% sodium dodecyl sulfate (SDS), 8M urea, and NP-40; (b) the disruption method is one of ultrasonic or magnetic bead grinding; (c) denaturation, reduction and alkylation: each sample is added with dithiothreitol (DTT) or tri(2-chloroethyl) phosphate (TCEP) to a final concentration of 0.1mM to 100mM, denatured and reduced at 65℃ to 95℃ for 2-120 min, cooled to room temperature, and iodoacetamide (IAA) is added to a final concentration of 0.2mM to 20mM, reacted at room temperature in the dark for 15-40 min; (d) enzymatic digestion: 100-400mL of ammonium bicarbonate solution and trypsin are added, and reacted at 30-40℃ for 12-16 h. h; (e) Desalting: After desalting, the peptides to be tested are lyophilized and reconstituted with a 0.1% formic acid solution.

5. The detection method according to claim 2, characterized in that: In steps (1) and (2), data acquisition was performed using an AB SCIEXTriple TOF 5600+ mass spectrometer; the packing material used was C18 reversed-phase chromatography packing material with a diameter of 1.7 mm to 3 mm, the inner diameter of the chromatographic column was 0.1 mm to 2.1 mm, the length of the chromatographic column was 30 mm to 150 mm, and the liquid chromatography flow rate was 1 mL / min to 20 mL / min; mobile phase A was a 0.1% formic acid aqueous solution and mobile phase B was a 0.1% formic acid acetonitrile solution. Separation gradient: Elution conditions are 0 to 3-4 min, from 0-5% B to 30-40% B (volume ratio); then within 1-2 min, from 30-40% B to 75%-85% B (volume ratio); then within another 1-2 min, from 75%-85% B to 0-5% B (volume ratio), with the remaining percentage made up with mobile phase A.

6. The detection method according to claim 1, characterized in that: This method is applicable to the detection of partial proteins in the MVA pathway in engineered strains of Saccharomyces cerevisiae, Escherichia coli, or Enterococcus faecalis.

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