CN111777696B - Method for specific reversible enrichment of nascent protein - Google Patents
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Abstract
The invention belongs to the technical field of protein enrichment, and relates to a method for specific reversible enrichment of nascent protein. Marking cells by AHA, and extracting holoprotein; taking cell holoprotein and alkynyl resin to connect through click reaction; washing the non-specifically adsorbed proteins with different buffers to remove non-specifically adsorbed proteins; digesting and releasing a target peptide segment by using trypsin; samples were collected for mass spectrometry. Compared with the prior art, the invention realizes high-selectivity enrichment of newly synthesized low-abundance protein by biomarkers, selective covalent bonding, strict elution conditions and selective dissociation, can realize large-scale analysis and identification of protein by combining nano-LC-MS/MS, and can further identify the newly generated protein according to the added mass label. The method provides an efficient method for efficient enrichment and accurate identification of the nascent protein, and is widely applied to the research field of low-abundance nascent proteomics.
Description
Technical Field
The invention belongs to the technical field of protein enrichment, and particularly relates to a method for specific reversible enrichment of nascent protein.
Background
Under the condition of different pathological states or drug interference, the discovery of the nascent protein has extremely important significance in disclosing the disease occurrence mechanism and finding the explanation of the action process of the drug. However, in a complex biological sample, the nascent protein is not labeled, and it is difficult to distinguish the nascent protein from the existing protein, and the abundance of the nascent protein is extremely low and is difficult to directly detect by conventional methods.
At present, the BONCAT (PNAS,2006,103: 9482-87; Nature Protocols,2007,2: 532-. The methods are all based on the affinity enrichment of biotin (biotin) and streptavidin (avidin), and the main steps are that biotin is connected to the nascent protein containing AHA through click reaction, and the nascent protein is enriched by being labeled with biotin. In the field of biomedicine, enrichment by utilizing the affinity interaction of biotin and streptavidin is widely applied. However, this technique has problems: (1) due to the non-covalent interaction between biotin and streptavidin, washing can not be carried out under a relatively strong condition, so that the interference of non-specific adsorbed protein is severe, and the recovery rate is low if the elution condition is severe; (2) the enriched peptide segment has a biotin group, and biotin molecules can influence chromatographic separation and reduce the ionization efficiency of the peptide segment, thereby seriously influencing the identification of protein.
Disclosure of Invention
The invention provides a novel method for specific reversible enrichment of nascent proteins based on the defects of BONCAT, QuaNCAT and HILAQ methods in the background art.
The invention relates to a reversible enrichment method based on bioorthogonal reaction for detecting new synthesized protein in cells.
The alkynyl resin is prepared by bonding the designed and synthesized alkynyl peptide segment to a resin material, and has the advantages of stable structure, high enrichment capacity and convenient and reliable separation. The alkynyl resin is used for enriching AHA marked nascent protein, and the technology enables the research of the nascent protein to be more convenient and faster and has higher flux.
The method disclosed by the invention is efficient, simple and convenient, can be used for reversible enrichment, and can be widely applied to enrichment and identification of the nascent protein.
The purpose of the invention can be realized by the following technical scheme:
the invention firstly provides a preparation method of alkynyl resin, which specifically comprises the following steps: and connecting the alkynyl peptide segment with aldehyde-based resin through a reduction alkylation reaction to synthesize alkynyl resin, wherein the alkynyl resin is expressed as alkyne-resin.
Further, the alkynyl peptide fragment is GLGAGLLAR(aPra), and the amino acid sequence thereof is GLAGLAGLLAR (L-Pra-NH)2)。
Wherein, L-propargyl glycine is abbreviated as L-Pra, and the carboxyl terminal of the L-Pra is aminated to protect the active carboxyl at the tail end of the peptide segment, so that the potential side reaction of the carboxyl terminal can be avoided. Warp beamAmidated L-propargylglycine is denoted L-Pra-NH2Abbreviated as aPra, is a monolithic structure and is therefore denoted in parentheses as (aPra), i.e. the structure contains alkynyl groups for subsequent bio-orthogonal reactions.
In the invention, the alkynyl peptide fragment is designed to have two important functions: firstly, amidation protection is carried out on carboxyl of L-Pra to form aPra, and only alkynyl is left as an active group; secondly, the sequence of the peptide segment is designed to be GLAGLAGLLAR, and the sequence can be identified by trypsin and is subjected to rapid enzymolysis (Nature Methods, 2014, 11: 220-222), so that the peptide segment can be used for efficiently releasing a nascent peptide segment.
In one embodiment of the present invention, the aldehyde-based resin is an aldehyde-functionalized resin material (Pierce) that can be linked by reductive amination, and can be selected from commercial aldehyde-based resins (I)
Coupling Resin)。
In one embodiment of the present invention, the method for preparing the alkynyl resin specifically includes the steps of: coupling of alkynyl peptide fragment and aldehyde resin: reacting the alkynyl peptide segment, aldehyde resin, a coupling reagent and a reducing reagent at room temperature, and washing for multiple times by using a washing reagent;
blocking the remaining reactive groups: adding a terminating reagent, and reacting at room temperature;
washing the alkynyl resin: washing for multiple times to obtain alkynyl resin, and storing for later use.
Wherein the coupling agent is selected from the group consisting of 0.1M Na3PO40.15M NaCL, PBS pH 7.2.
Wherein the reducing agent is selected from 5M NaCNBH3。
Wherein the termination reagent is selected from 1M Trist-HCl (pH7.4).
Wherein the washing reagent is 1M NaCl.
The ratio relation of the alkynyl peptide fragment, the aldehyde resin, the coupling reagent, the reducing reagent and the termination reagent is as follows: 5-20 ug: 10 uL: 200 uL: 20 uL: 200uL, wherein the dosage of the aldehyde resin can be proportionally increased according to needs.
In one embodiment of the present invention, the preparation method of the alkynyl resin is: coupling 10. mu.g of peptide fragment with 10. mu.L of aldehyde resin: adding 200 mu L of coupling reagent and 5 mu L of reducing reagent, reacting for 6h at room temperature, and washing for 3 times by using the coupling reagent; blocking the remaining reactive groups: adding 200 mu L of termination reagent, and reacting at room temperature for 30min to obtain alkynyl resin; washing the alkynyl resin: washing 3 times with 200 μ L each time, and storing at 4 deg.C for use.
The invention further provides the alkynyl resin obtained by the preparation method. The alkynyl resin has stable structure, high enrichment capacity and convenient and reliable separation.
The invention provides a method for specific reversible enrichment of nascent protein, which comprises the following specific steps:
step 4, digesting and releasing the target peptide segment by trypsin;
and 5, collecting a sample for mass spectrometry.
In one embodiment of the present invention, the azide-labeled protein sample is obtained by labeling the cells with AHA by the method comprising:
firstly, adding dialysis serum into a cell to be detected in a methionine-free culture medium (also called methionine deficiency culture medium), and carrying out starvation culture for 30 min;
then, adding 1mM of azido alanine (AHA) into the methionine deficiency culture medium to obtain an AHA marking culture medium, adding dialyzed serum, continuously culturing the cells for 1-24 h, adjusting according to the experiment requirement, and collecting the cells;
adding cell lysis solution, ultrasonic lysis, centrifuging and taking supernatant; the BAC kit quantifies proteins and is stored at-80 ℃ for further use.
In one embodiment of the invention, the cell culture is DMEM, which is composed of Gibco 10566. The methionine deletion culture medium refers to a culture medium for removing methionine in DMEM, and specifically is a DMEM culture medium (Gibco 21013024) with deletion of methionine, cysteine and glutamine, and cysteine and glutamine are supplemented to obtain a self-made methionine deletion culture medium.
In one embodiment of the invention, the cell lysate is selected from 0.5% SDS cell lysate.
In one embodiment of the invention, the cell lysate consists of 0.5% SDS, 1M NaCl, 200mM Tris (pH 8.4).
In one embodiment of the invention, the cell lysate is added in an amount of 8 times the volume of the cell pellet.
In one embodiment of the present invention, the method for linking the cell holoprotein and the alkynyl resin through click reaction is copper-catalyzed bio-orthogonal reaction, also called CuAAC reaction, under the following reaction conditions: mixing the cell holoprotein and the alkynyl resin, adding the catalytic mixed solution, adding PBS, and oscillating for 1h at room temperature in a dark place.
Wherein the adding amount of the cell holoprotein, the alkynyl resin, the catalytic mixed solution and the PBS is 500 mu g-2 mg: 10 μ L of: 2 μ L: and 8 mu L of the catalyst, wherein the using amount of the alkynyl resin can be increased proportionally according to needs, and the reaction amount can be amplified according to the proportion according to needs during specific operation.
In one embodiment of the invention, the catalytic mixed solution consists of ascorbic acid, BTTP and copper sulfate, wherein the molar ratio of ascorbic acid to BTTP to copper sulfate is 10: 6: 1.
in one embodiment of the invention, the catalytic mixture is 10X catalyst mixture prepared from 10mM sodium ascorbate, 1mM copper sulfate and 6mM BTTP.
In one embodiment of the present invention, the method for washing the nonspecifically adsorbed proteins on the alkynyl resin with different buffers is: and (2) sequentially washing by using SDS, urea and acetonitrile, wherein the concentration of washing liquid is as follows: 1% SDS, 8M urea, 50% acetonitrile/water (v/v).
In one embodiment of the present invention, the specific operation method for washing the non-specifically adsorbed protein with different buffers is as follows: the resin material was washed with 200. mu.L of 1% SDS, centrifuged at 10000g for 3min, repeated three times, and then washed with 1% SDS, 8M urea, PBS and 50% acetonitrile in that order to remove non-specifically adsorbed proteins and other contaminants. Wherein the non-specifically adsorbed proteins on the resin material are removed by 1% SDS, the resin material is washed out by 1% SDS using 8M urea, the resin material is washed out by 8M urea using PBS, and the remaining peptide fragments are washed out by 50% acetonitrile. Because the enriched linkage is a chemical bond formed by a bioorthogonal reaction, it can be washed under harsh conditions, ensuring high selectivity of the enrichment.
In one embodiment of the present invention, the reaction conditions for digesting and releasing the target peptide fragment with trypsin are as follows: at 50mM NH4HCO3In the solution, the alkynyl resin washed with the buffer solution was digested with trypsin at 37 ℃ overnight.
In one embodiment of the present invention, when the target peptide fragment is released by trypsin digestion, the ratio of the mass of the added trypsin to the mass of the peptide fragment remaining on the washed alkynyl resin is 1: 10 (wherein, the residual peptide fragment on the washed alkynyl resin is estimated according to the standard peptide fragment, 10ug standard peptide fragment/10 uLresin). The enzyme may be cleaved twice to improve release efficiency. The new protein containing AHA is released by enzymatic dissociation, and the (aPra) group is carried on AHA.
The peptide fragment is released while the peptide fragment is cut by enzyme, the high release efficiency is ensured by fully utilizing the high cutting efficiency, and the selectivity of enrichment release is further improved by the selectivity of the enzymolysis site; compared with the traditional Biotin-streptavidin release mode, the method disclosed by the invention can not only tolerate strong elution solvent to eliminate non-specific interference, but also avoid a mode of boiling and releasing SDS (sodium dodecyl sulfate) with extreme concentration, and meanwhile, the enzymolysis condition is compatible with the chromatographic mass spectrum, so that the ionization efficiency of the mass spectrum is not influenced, the identification result is improved, and the sample loss is greatly simplified and reduced on the operation flow.
In one embodiment of the present invention, the method of collecting a sample for mass spectrometry comprises: the nano-LC-MS/MS analysis shows that the peptide segment contains (aPra) -AHA to identify the corresponding protein as the new protein.
In one embodiment of the invention, there is a lyophilization and desalting step prior to collecting the sample for mass spectrometry.
The method of the invention selects azidoalanine (AHA) to replace methionine, so that the newly generated protein in the cell has unnatural azido groups. The method is characterized in that the bio-orthogonal reaction is utilized to enrich the nascent protein with the azide amino acid, the high-selectivity enrichment is realized by virtue of the orthogonality of the reaction, the high-specificity enrichment is realized by virtue of the stability of chemical bonding, the efficient release can be realized by virtue of enzyme digestion, and finally the efficient reversible enrichment of the nascent protein in the cell is realized.
The alkynyl resin material is designed and synthesized, the enrichment connection and dissociation process ensures high selectivity and high efficiency, the enriched new peptide segment has a specific mass label (small molecular weight amino acid), the label peptide segment can be used for identifying protein, more importantly, the new protein can be directly identified by the mass increase of the label, and the problem that the mass spectrum identifies the new protein during identification is solved. According to the method, the covalent bond action replaces the affinity action to enrich the new protein, the high-efficiency enzyme digestion release replaces the SDS dissociation, the high-specificity enrichment of the new protein is realized, the mass tag mass spectrum is compatible, the identification and the identification of the new protein are facilitated, and the whole enrichment process is greatly simplified, and the sample requirement is reduced.
In the method designed by the invention, the alkynyl on the material is connected with the azide of the nascent protein through bioorthogonal, the reaction has high selectivity, the specificity of the enrichment of the nascent protein in vivo is ensured, and the high reaction efficiency of click reaction ensures that the enrichment technology has low detection limit; meanwhile, the enrichment process inevitably has non-specific adsorption interference, and a covalent bond is formed by click reaction, so that strong washing conditions are allowed to be used, and the non-specific adsorption can be reduced to the maximum extent; and finally, the material is released and digested by trypsin, so that not only is the efficient release efficiency ensured, but also the system where the obtained peptide fragment is located is compatible with the chromatographic mass spectrometry. The whole process is greatly simplified in operation on the basis of ensuring the enrichment selectivity.
The invention combines a high-selectivity enrichment method with a labeling technology and a chromatographic mass spectrometry analysis technology, and provides possibility for identifying new synthetic protein.
According to the invention, high selective enrichment of newly synthesized low-abundance protein is realized through biomarkers, selective covalent bonding, strict elution conditions and selective dissociation, large-scale analysis and identification of protein can be realized by combining nano-LC-MS/MS, and newly generated protein can be further identified according to an increased mass label. The method provides an efficient method for efficient enrichment and accurate identification of the nascent protein, and is widely applied to the research field of low-abundance nascent proteomics.
Compared with the prior art, the method has the following advantages:
1. based on the high reaction efficiency of the bioorthogonal reaction and the enzyme digestion efficiency of trypsin, the enrichment recovery rate is high and can reach 84%;
2. the high selectivity of the bioorthogonal reaction and the firm combination of covalent bonding allow the harsh washing, the enrichment specificity is high, and the highest protein selectivity of the current literature reaches more than 95 percent;
3. the method integrates elution and release into one by designing the sequence of the peptide segment, thereby simplifying the flow and reducing the consumption of samples. 4335 nascent proteins can be identified and confirmed in a single experiment with a minimum of 500ug of starting sample, which is the largest amount identified at present, and 3151 nascent proteins at the largest scale identified by the HILAQ method.
Drawings
FIG. 1 is a schematic diagram of the enrichment principle of reversible tags (CBOT).
The GLAGLLAR (aPra) peptide segment contains three reaction sites, wherein a site 1 shown in the figure is a C-terminal alkynyl group of the peptide segment used for click reaction to combine with the azido peptide segment, a sequence and a site (site 2) of TEV enzyme cutting characteristics are used for releasing the target peptide segment, and an N-terminal amino group of the peptide segment is fixed to aldehyde-based resin (resin-CHO) through reductive amination. The CBOT-enriched peptide fragment, with one (aPra), had mass gain 112.14 for mass spectrometric identification and peptide fragment assignment.
FIG. 2 is a MALDI spectrum for evaluating the alkynyl capacity of the functionalized resin material (resin-alkyne).
The alkynylpeptide fragment glaglagllar (apra) (m/z 864, marked with an "-"); the internal standard peptide is marked with "#" (m/z 1048). Taking 1 μ g (a),5 μ g (b),10 μ g (c) and 20 μ g (d) of alkynyl peptide fragment, respectively, coupling with 10 μ L of aldehyde resin, and detecting residue of alkynyl peptide fragment in supernatant by MALDI.
FIG. 3 is a comparison of reducing agents and ligands in copper catalyzed bioorthogonal (CuAAC) reactions. The products were detected by MALDI with the reducing agents ascorbic acid and tcep (a), respectively, and the ligands compared BTTP, BTTAA, BTTPs and thpta (b), respectively.
FIG. 4 shows the recovery rate of the enriched azide peptide fragments from CBOT in example 1.
MALDI spectrogram of a product peptide section obtained by enzyme digestion before (a) enriching the azide peptide section and (b) after enriching. The azide peptide (AZA) ALELFR (m/z 860) was labeled with "#", the product (aPra) (AZA) ALELFR (m/z 972) was labeled with "#", and the internal standard peptide (m/z1048) was labeled with "diamondsolid".
FIG. 5 is an experimental procedure for the enrichment of nascent protein in CBOT-enriched cells.
The specific process includes that AHA is marked to enable the new-born protein to have an azide label, the protein is subjected to enzymolysis to obtain a peptide segment, the peptide segment is connected through click, strongly washed and enzyme-digested to release an enriched marked peptide segment, and finally the new-born protein is identified through chromatography-mass spectrometry.
FIG. 6 shows the results of the identification of nascent proteins in HEK293T cells in example 2.
The number of nascent proteins (a) and AHA peptide stretches (b) identified after CBOT enrichment, and the capping events in three technical replicates.
FIG. 7 is a graph of the efficiency of enrichment of the CBOT process evaluated on a protein and peptide fragment level in example 2.
Panel (a) shows a histogram of the proteins and peptide fragments identified before and after enrichment, with the specific numbers listed in the table of panel (b). Wherein column A, C represents nascent protein or peptide fragment with AHA marker, column B, D represents AHA-free protein or peptide fragment, dark color (A, B) represents protein, and light color (C, D) represents peptide fragment.
Figure 8 is the omics results of the CBOT method used to quantify nascent proteins in cells in example 3.
The number of AHA-containing proteins (a) and AHA peptide fragments (b) and cross-cap conditions in three technical replicates were determined for the quantification of nascent proteins by CBOT enrichment in HEK293T cells labeled with the light and heavy isotopes AHA/hHA.
FIG. 9 is a histogram of the correlation of the quantification results and the data distribution of the CBOT-quantified nascent protein of example 3.
FIG. 10 is a graph of the identification of nascent proteins (b) quantified during rapamycin activated autophagy (a) and autophagy in accordance with the CBOT method of example 4.
FIG. 11 is the biological pathway for rapamycin activated autophagy of example 4.
Known autophagy-related proteins in the autophagy pathway highlighted in grey are nascent proteins identified after CBOT enrichment.
FIG. 12 is a Western blot demonstrating the expression of LC3 and p62 after rapamycin induced activation of the autophagy pathway in example 4.
Rapa represents rapamycin activating autophagy, Baf/word is a control group inhibiting the autophagy pathway, where ACTB is the reference for the loading.
Detailed Description
The invention realizes the enrichment of new cell synthetic protein by utilizing a specific reversible enrichment method, and the invention is explained in detail by combining the attached drawings and specific examples.
Example 1: the method established based on the cleavable bioorthogonal reversible tag (CBOT) enriches the synthesized azido peptide fragments, the enrichment principle of the CBOT tag is shown in figure 1, and the specific experimental flow is as follows:
(1) taking 10 mu L of commercial aldehyde resin, adding 10 mu g of synthesized alkynyl peptide fragment GLAGGLLAR (aPra), and adding reducing agent NaCNBH3 into PBS (phosphate buffer solution) with pH of 7.4 to perform reductive alkylation reaction to obtain alkynyl functionalized resin material, namely alkynyl resin for short;
(2) taking the synthesized alkynyl resin, adding 10 mu g of azide peptide segment (m/z 860), and covalently bonding CuAAC on the alkynyl resin in a catalytic manner;
(3) trypsin enzymolysis, adding aPra group to the released target peptide fragment, and detecting by MALDI, wherein the molecular weight is increased 112.12(m/z 972).
FIG. 2 is a MALDI spectrum for evaluating the volume of alkynyl groups on the functionalized resin material. After coupling of different volumes (20, 10, 5, 1. mu.L) of resin and 10. mu.g of alkynyl peptide, the supernatant was examined by MALDI to evaluate the capacity of the resin material to 10. mu.g of peptide/10. mu.L of resin material.
FIG. 3 shows that alkynyl nonapeptide (864m/z) was subjected to click reaction with azidododecapeptide (1196m/z) and the product was detected by MALDI (2060 m/z). Click ligation efficiency was evaluated based on the reduction of the azide peptide.
FIG. 4 shows the recovery rate of the enriched azide peptide fragments from CBOT in example 1. MALDI spectrograms of the peptide sections before and after enrichment are used for obtaining that the CBOT enrichment recovery rate is 84 percent through the correction calculation of the internal standard peptide section.
Example 2: the cleavable tag based on bio-orthogonal reaction combined with pancreatin enzymolysis in example 1 is used for the enrichment identification of the nascent protein in 293T cells, and the established enrichment identification process of the nascent protein is shown in FIG. 5, and the specific experimental process is as follows:
(1) AHA-labeled 293T cells, whole sample extraction: culturing 1mM AHA for 4h, and collecting cell lysis extract protein;
(2) 10 μ L of an alkynyl resin was prepared according to the step (1) in example 1;
(3) replacing 10 μ g of azido peptide fragment with 500 μ g of AHA-labeled proteolytic peptide fragment of holoprotein according to the step (2) in example 1 to complete CuAAC catalytic ligation;
(4) the non-specifically adsorbed protein is removed under the strong elution condition. The elution reagent was washed sequentially with 200 volumes of 1% SDS, 8M urea, PBS and 50% acetonitrile, respectively.
(5) Carrying out enzymolysis by trypsin, wherein the ratio of protein to enzyme is 10: 1, the peptide fragment of the released nascent protein is separated and identified by the nanoLC-MS/MS. Adding variable modification in the setting of mass spectrum library searching: AHA (Met), mass change +107.07 was set as a variable modification for identification and assignment of proteins after Met was replaced by AHA and an enrichment tag was added.
And (3) analysis results: 2213 proteins were identified from 500. mu.g samples by a reversible enrichment procedure, with a protein selectivity of 95.3%.
Fig. 6 is a three-time repeated data cross-over map of the enriched identification result of the nascent protein sample in the HEK293T cell, which shows that the enrichment process is stable and has good repeatability, and the proportion of two-time repeated identification reaches more than 70%.
Figure 7 the enrichment efficiency of the CBOT method was evaluated at the protein and peptide level. The marked peptide segment in the identification result before enrichment is only 1.6 percent, the AHA marked peptide segment in the identification result after enrichment is 83.6 percent, and the identification sensitivity is improved by 52 times. The nascent proteins identified after enrichment account for more than 95% of the total protein.
Example 3: the procedure for CBOT enrichment of nascent proteins of example 2 was used in quantitative studies of nascent proteins in HEK293T cells in combination with the light and heavy isotope AHA/hAHA. The specific experimental procedures are as follows:
(1) respectively labeling 293T cells with AHA and hHA, incubating for 4h, and respectively collecting cell lysis;
(2) protein was extracted and quantified, respectively, 1: 1 mixing two samples with equal mass;
(3) the nascent protein was enriched according to the procedure in protein example 2;
(4) adding variable modification in the setting of mass spectrum library searching: aha (met) and haha (met) set the mass changes +107.07 and +113.17 as variable modifications for identification and assignment of proteins. And simultaneously, carrying out quantitative analysis on the protein according to the AHA peptide fragment and the corresponding hHA peptide fragment.
Figure 8 is the omics results of the CBOT method used to quantify nascent proteins in cells in example 3. The light and heavy isotope AHA/hHA-labeled HEK293T cell can quantify 2453 new proteins by CBOT enrichment, wherein 1722 proteins obtain quantitative information in at least two technical repetitions.
FIG. 9 is a histogram of the data distribution and correlation of the quantification results for the CBOT-quantified nascent proteins analyzed in example 3. Results correlation coefficients for both technical replicates exceeded 0.5, and more than 80% protein with less than 2-fold CV was obtained from the data distribution histogram. The quantitative result is proved to have good repeatability and accuracy.
Example 4: the CBOT enrichment quantification protocol of example 3 was further used to study changes in nascent proteins during autophagy. The content of this section is designed to activate autophagy with rapamycin, the experimental design is shown in fig. 10(a), and the specific experimental flow is as follows:
(1) 293T cells are respectively marked by AHA and hHA for 24 h;
(2) removing the marked culture medium, replacing the marked culture medium with a complete culture medium, simultaneously stimulating and inducing autophagy of the AHA marked sample by rapamycin, using an autophagy inhibitor Baf and word for inhibiting autophagy of the hHA marked cell as a control sample, and collecting the cell after 6 days;
(3) protein was extracted and quantified, respectively, 1: 1 mixing two samples with equal mass;
(4) the nascent protein was enriched according to the procedure in protein example 2 and the protein was quantified according to the settings in example 3;
(5) differentially expressed proteins were analyzed using bioinformatic techniques.
The change of the protein during rapamycin activation was studied by CBOT enrichment technique combined with quantification technique, and the results in fig. 10(b) show that more than 95% of all proteins identified in three technical replicates were nascent proteins and more than 93.4% of the proteins had quantitative information.
FIG. 11 is a biological pathway for rapamycin activated autophagy. The autophagy pathway highlighted in grey is the nascent protein identified after CBOT enrichment.
Figure 11 is a protein of a known autophagy pathway in which CBOT was enriched and more than 20 proteins were identified, highlighted in grey.
FIG. 12 is a graph showing the expression levels of LC3 and p62 after induction of autophagy by rapamycin by Western blotting. LC3 is a marker molecule for autophagy activation, and the up-regulation of LC3-II indicates that the autophagy pathway is activated; p62 is thought to be a substrate protein for autophagy, and its degradation can indicate the occurrence of autophagy flow and be used to monitor the cycle of autophagy degradation in different cells.
Example 4 demonstrates that CBOT technology can be used to study the dynamic changes of proteins during autophagy, and can provide changes in nascent proteins within a short time stimulation window, helping to elucidate the biological mechanisms of autophagy degradation.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (14)
1. A preparation method of alkynyl resin is characterized in that alkynyl peptide segments are connected with aldehyde-based resin through reduction alkylation reaction to synthesize alkynyl resin;
the alkynyl peptide segment is GLAGLAGLLAR (aPra), and the amino acid sequence thereof is GLAGGLLAR- (L-Pra-NH2)。
2. The method for preparing alkynyl Resin as claimed in claim 1, wherein said aldehyde Resin is selected from commercial aldehyde Resin Thermo-amino Link Coupling Resin.
3. The method for producing an alkynyl resin according to claim 1, wherein the method for producing an alkynyl resin specifically comprises the steps of:
coupling of alkynyl peptide fragment and aldehyde resin: reacting the alkynyl peptide segment, aldehyde resin, a coupling reagent and a reducing reagent at room temperature, and washing for multiple times by using a washing reagent;
blocking the remaining reactive groups: adding a terminating reagent, and reacting at room temperature;
washing the alkynyl resin: washing for multiple times to obtain alkynyl resin, and storing for later use.
4. The method for preparing alkynyl resin according to claim 3, wherein said coupling agent is selected from the group consisting of Na containing 0.1M3PO40.15M NaCl, PBS pH 7.2;
the reducing agent is selected from NaCNBH with the final concentration of 50mM3；
The termination reagent is selected from 1M Trist-HCl, pH7.4;
the washing reagent is 1M NaCl;
the ratio relation of the alkynyl peptide fragment, the aldehyde resin, the coupling reagent, the reducing reagent and the termination reagent is as follows: 5-20 ug: 10 uL: 200 uL: 20 uL: 200 uL.
5. An alkynyl resin obtained by the production method according to any one of claims 1 to 4.
6. A method for specific reversible enrichment of nascent proteins, comprising the steps of:
step 1, marking cells by AHA, and extracting holoprotein;
step 2, taking cell whole protein and alkynyl resin as the claim 5 to connect through click reaction;
step 3, washing the proteins nonspecifically adsorbed by the alkynyl resin by using different buffer solutions to remove the nonspecifically adsorbed proteins;
step 4, digesting and releasing the target peptide segment by trypsin;
and 5, collecting a sample for mass spectrometry.
7. The method for specific reversible enrichment of nascent proteins as claimed in claim 6, wherein the cells are labeled with AHA to obtain an azide-labeled protein sample by the following steps:
firstly, adding dialysis serum into cells to be detected in a culture medium without methionine, and carrying out starvation culture for 30 min;
then, changing to an AHA marking culture medium, adding dialyzate serum, continuing to culture the cells, and collecting the cells, wherein the AHA marking culture medium is obtained by adding 1mM azido alanine into a methionine-deficient culture medium;
adding cell lysis solution, ultrasonic lysis, centrifuging and taking supernatant; the BAC kit quantifies proteins and is stored at-80 ℃ for further use.
8. The method for the specific reversible enrichment of nascent proteins as claimed in claim 6, wherein the method of linking cellular whole proteins and alkynyl resins by click reaction is copper-catalyzed bioorthogonal reaction under the following conditions: mixing the cell holoprotein and the alkynyl resin, adding the catalytic mixed solution, adding PBS, and oscillating at room temperature in a dark place for reaction.
9. The method for specific reversible enrichment of nascent protein according to claim 8, characterized in that the addition amount of cell whole protein, alkynyl resin, catalytic mixed liquor and PBS is 500 μ g-2 mg: 10 muL: 2 muL: 8 muL.
10. The method for specific reversible enrichment of nascent protein according to claim 8, wherein the catalytic mixture consists of ascorbic acid, BTTP, copper sulfate in a molar ratio of 10: 6: 1.
11. the method for specific reversible enrichment of nascent proteins as claimed in claim 6, wherein the non-specific adsorbed proteins on alkynyl resin are washed with different buffers by: and (2) sequentially washing by using SDS, urea and acetonitrile, wherein the concentration of washing liquid is as follows: 1% SDS, 8M urea, 50% acetonitrile/water.
12. The method for specific reversible enrichment of nascent proteins as claimed in claim 6, wherein the reaction conditions for trypsin cleavage to release the peptide fragment of interest are: at 50mM NH4HCO3In the solution, the alkynyl resin washed with the buffer solution was digested with trypsin at 37 ℃ overnight.
13. The method for specific reversible enrichment of nascent protein according to claim 6, wherein the mass ratio of the added mass of trypsin to the mass of peptide fragment remaining on the washed alkynyl resin when the target peptide fragment is released by trypsin digestion is 1: 10.
14. the method for specific reversible enrichment of nascent protein according to claim 6, wherein the collection of samples for mass spectrometry is performed by: and (4) nano-LC-MS/MS analysis, wherein the peptide fragment contains (aPra) -AHA which can indicate that the corresponding protein is the new protein.
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