CN114057827B

CN114057827B - Method for marking protein

Info

Publication number: CN114057827B
Application number: CN202111461946.4A
Authority: CN
Inventors: 李子刚; 尹丰; 万川; 冯源; 王蕊; 孔凌微
Original assignee: Shenzhen Bay Laboratory Pingshan Biomedical R & D And Transformation Center; Peking University Shenzhen Graduate School; Shenzhen Bay Laboratory
Current assignee: Shenzhen Bay Laboratory Pingshan Biomedical R & D And Transformation Center; Peking University Shenzhen Graduate School; Shenzhen Bay Laboratory
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2023-10-13
Anticipated expiration: 2041-12-02
Also published as: CN114057827A

Abstract

The invention discloses a method for selecting protein to be marked, which uses functional molecules with carboxyl or thiocarboxyl as a substrate, uses unsaturated sulfonium salt as a carboxyl activating agent, and generates solid phase peptide bond formation reaction of the marked protein in alkaline buffer solution; in a buffer solution, performing an amine transesterification reaction activated by unsaturated sulfonium salt and performing a specific modification reaction on amino groups on protein; and identifying the specific modification condition by means of western blot or mass spectrum. The invention uses functional molecule containing carboxyl or thiocarboxyl as substrate, uses unsaturated sulfonium salt to act on carboxyl activator, and can mark different kinds of proteins through amine transesterification in different types of buffer solution. The invention uses safe low-toxicity sulfonium salt activated ester as a labeling molecule, can label protein with high efficiency and high selectivity, and can be used for activity-based protein analysis and labeling of medium antibody molecules of drug-coupled antibodies.

Description

Method for marking protein

Technical Field

The invention belongs to the field of biochemistry, and relates to a protein labeling method, in particular to a method for covalently labeling proteins under buffer solutions in different physiological environments by activating carboxylic acid through unsaturated sulfonium salt.

Background

Covalent labelling of native proteins by site-specific is the most common method. Compared with genetic engineering, the method has the advantages of shorter experimental period, less uncertainty factors in experiments and the like, so that the method becomes a protein labeling method favored by researchers. However, not all organic chemical reactions are suitable for protein labelling, since the environment of living organisms is relatively mild. Attention is therefore paid to the following problems: first, the chemical reaction of the protein label must be resistant to biological environmental conditions (pH 6-8, < 37 ℃ C., aqueous solvent); second, the substrate concentration during protein reaction is typically low (μM and/or nM); thirdly, most protecting group strategies widely used in organic synthesis cannot be used; fourth, high selectivity to POI (protein of interest, POI) is maintained under complex systems.

Among them, cysteine is the most active amino acid residue, and thus has been one of the most widely studied protein residue markers to date. However, since cysteines are only about 200000 out of about 20000 proteins in humans, accounting for 1.9% of the total number of residues, and disulfide bonds are formed between many cysteines, the residues of these cysteines are difficult to label by nucleophilic reaction. In contrast, there were 650000 lysine residues, accounting for 5.9% of the total residues. Meanwhile, various substrates for labeling cysteine include α -halogenated hydrocarbons, michael acceptors, disulfides, isoxazolinium, perfluoroaryl reagents, and alkynes, and the reaction between these compounds and thiols is often severe, making it difficult to regioselectively label specific sites. While the average pKa of lysine is 10.5 (the average pKa of cysteine is 8.0), this results in the vast majority of lysine residues being protonated under physiological conditions. Thus, changes in the microenvironment near the protein provide the potential for specific covalent labeling of lysine.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for marking protein, which aims to solve the problems of poor selectivity of lysine modification, low solubility of modified substrates and the like commonly used in protein marking in the prior art.

The invention provides a protein labeling method, which comprises the following steps:

1) Selecting a protein to be marked, using a functional molecule with carboxyl or thiocarboxyl as a substrate, using an unsaturated sulfonium salt as a carboxyl activating agent, and generating a solid-phase peptide bond reaction of the marked protein in the presence of alkali;

2) In a buffer solution, performing an amine transesterification reaction activated by unsaturated sulfonium salt and performing a specific modification reaction on amino groups on protein;

3) And identifying the specific modification condition by means of western blot or mass spectrum.

Further, the protein used for labeling is any one of purified protein, protein added with cell lysate or protein in a cell culture environment.

Further, the buffer solution is any one of dimethylformamide, dimethylacetamide, dichloromethane, methanol, acetonitrile or dimethyl sulfoxide.

Further, the buffer solution can be any one of boric acid-sodium hydroxide buffer solution, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, tris-hydrochloric acid buffer solution and sodium carbonate-sodium bicarbonate buffer solution.

Further, the unsaturated sulfonium salt is an alpha, beta-or beta, gamma-unsaturated substituted sulfonium salt.

Further, the functional molecule with carboxyl or thiocarboxyl is carboxylic acid or thiocarboxyl.

Further, the unsaturated sulfonium salt activator is selected from any one of vinyl sulfonium salt, ethynyl sulfonium salt, propynylsulfonium salt or acetonitrile sulfonium salt and derivatives thereof, and the anion of the sulfonium salt is selected from any one of bromide ion, iodide ion, trifluoromethane sulfonate ion or tetrafluoroborate ion.

Further, the protein used for labeling can be any protein with lysine residues or exposed N-terminal.

The invention discloses a method for labeling proteins in a biocompatible environment, which realizes specific targeting labeling of lysine in proteins obtained by separation and purification and lysine in complex cell environments (lysate and cell culture environment). The method uses functional molecules containing carboxyl or thiocarboxyl as a substrate, uses alpha, beta-and beta, gamma-unsaturated sulfonium salt to act as a carboxyl activator, and in different types of buffer solutions, generates solid-phase peptide bond generation reaction in the presence of alkali, and marks various proteins of different types through amine transesterification reaction. The invention uses sulfonium salt activated ester with low safety as a labeling molecule, can label protein with high efficiency and high selectivity, and can be used for activity-based protein analysis and labeling of medium antibody molecules of drug-coupled antibodies.

Compared with the prior art, the invention has obvious technical progress. The invention is suitable for the amidation reaction technology of polypeptide solid phase synthesis, and realizes the one-by-one formation of peptide bonds through amine transesterification reaction so as to achieve the purpose of synthesizing the polypeptide. The invention uses safe low-toxicity sulfonium salt as an activating reagent, and is suitable for laboratory polypeptide synthesis and industrial production.

Drawings

FIG. 1 shows the labelling of proteomes using sulfonium salts to activate esters.

FIG. 2 shows labeling using NHS activated ester proteomes.

Figure 3 shows toxicity studies on a549 using various activated esters.

FIG. 4 shows the localization within cells using various activated esters.

Detailed Description

The following examples serve to illustrate the invention in further detail, but the invention is by no means limited thereto.

Example 1

1. The A549 cell line was cultured in a 10cm cell culture dish until the growth density reached 80% -90%, the medium was discarded, and after three washes with 2mL PBS, the cells were carefully scraped from the dish with a cell scraper and collected in a 2mL ep tube. Cells were lysed using 40% power ultrasound for 5min at 1s stop for 1s per ultrasound.

2. After centrifugation at 13000rpm for 10min, the supernatant was collected, 1. Mu.L BSA was collected at 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, and 1. Mu.L of the supernatant was placed in a 96-well plate, 150. Mu.L of Coomassie Brilliant blue was added, and the absorbance at 490nm was measured using a multifunctional microplate reader. Then, a standard curve of the protein concentration and the absorbance is drawn, and the protein concentration in the supernatant is converted.

3. The protein concentration was diluted to 2mg/mL with boric acid buffer solution at ph=10, 1mL of the lysate was taken and added to S-dimethyl-2-propargyl sulfonium salt at a final concentration of 50 μm and 50 μm p-alkynyl propoxy (thio) benzoic acid and reacted for 4 hours. After the reaction was completed, 5mL of ice acetone pre-cooled at-80℃was used to precipitate at-20℃for 2h. Followed by centrifugation at 5000rpm for 10min in a centrifuge at 4 ℃.

4. After discarding the supernatant, the pellet was resuspended with 200. Mu.L of 1.2% SDS. The resuspended solution was diluted with double distilled water to a total volume of 1mL. Followed by addition of a premixed 55. Mu.M biotin azide UV cleavage probe, 100. Mu.M t-butyl 2, 2-trichloro-imide ester, 1mM tris (2-chloroethyl) phosphate and 1mM copper sulfate for 2 hours, thereby labeling the photocleavable biotin on the alkynyl-bearing probe. After the reaction was completed, 5mL of ice acetone pre-cooled at-80℃was used to precipitate at-20℃for 2h. Followed by centrifugation at 5000rpm for 10min in a centrifuge at 4 ℃.

5. After discarding the supernatant, the pellet was resuspended with 200. Mu.L of 1.2% SDS by volume. The resuspended solution was diluted with double distilled water to a total volume of 1mL. A wet volume of 50. Mu.L of streptavidin agarose beads was then added and incubated for 3h. After the incubation was completed, the supernatant was discarded and the streptavidin sepharose beads were resuspended with 500 μl of Tris aqueous solution containing 6M urea (ph=8.5).

6. Adding 10mM tri (2-chloroethyl) phosphate into the solution to react for 30min, so that disulfide bonds in the protein are fully reduced; followed by the addition of 20mM iodoacetamide for 20min to alkylate unlabeled cysteines in the protein.

7. Subsequently, 2. Mu.g of pancreatin in 1.5mL Tris (pH=8.5) was added and the protein was cleaved sufficiently to peptide fragments for 17 hours. After removal of the supernatant, the agarose beads were resuspended with 1mL of 70% acetonitrile and the biotin was excised at 365nm and repeated once. After desalting with a C18 column, the liquid phase was spun dry. The remaining peptide fragments were solubilized and analyzed.

8. After drying in vacuo, the obtained sample was loaded onto a Thermo Easy-Spray analytical column (75 μm inside diameter. Times.500 mm) C18 column with Easy-nLC 1200 chromatography pump. For each analysis, we dissolved the peptide fragment in 10 μl 0.1% formic acid and load 8 μl onto the column for running. Peptides were isolated on a 155 min (5-40% acetonitrile) gradient in each run. And (3) completion: the mass analyzer was in the m/z range of 350-1500 with a mass resolution of 60000 (at m/z=200) in data correlation mode, 1.6m/z isolation window. Using the HCD collision mode, the 20 strongest ions were selected for MS/MS analysis at a resolution of 15000. In vitro labeled peptides were analyzed by Orbitrap Exploris 480,480 under the same set parameters.

9. For all lysines identified in the proteomic analysis experiments, motifs (±7 amino acids) were determined using the R package, resolving UniProtKB entries for all identified proteins.

Conclusion: FIG. 1 shows that the number and specificity of lysine sites detected in borate buffer for labelling A549 cell lines using this method, was better selective and response compared to the conventional NHS-activated ester shown in FIG. 2.

FIG. 3 shows the potential toxicity of three activated ester probes and commercial activated ester sulfoNHS-Ac to intracellular protein markers developed using the present method, which was found to be non-cytotoxic within 100. Mu.M, without substantially affecting the proliferative activity of the cells.

FIG. 4 shows the localization of three activated ester probes developed using the present method within cells and their penetration. The graph shows that the PhCOSH-yne-S+ and the PhCOOH-yne-S+ have good film penetrating property compared with the C5-S+. After sulfo-NHS-Ac is added in the culture medium in advance, the fluorescence signal intensity of PhCOSH-Yne-S+ and PhCOOH-Yne-S+ is reduced. The figure shows that sulfonium salt active ester is mainly located in the nucleus and a small part in the cytoplasm.

Claims

1. A method for protein labelling, comprising the steps of:

1) Selecting a protein to be marked, wherein the protein for marking is any protein with lysine residues or exposed N-terminal; using p-alkynyl propoxy (thio) benzoic acid as a substrate, using S-dimethyl-2-propargyl sulfonium salt as a carboxyl activating agent, and carrying out an amide bond condensation reaction on the marked protein under an alkaline buffer solution;

2. The method of protein labelling according to claim 1, wherein: the buffer solution is any one of water, dimethylformamide, dimethylacetamide, dichloromethane, methanol, acetonitrile or dimethyl sulfoxide.

3. The method of protein labelling according to claim 1, wherein: the alkaline buffer solution is any one of boric acid-sodium hydroxide buffer solution, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution and sodium carbonate-sodium bicarbonate buffer solution.

4. The method of protein labelling according to claim 1, wherein: the unsaturated sulfonium salt activator is selected from any one of vinyl sulfonium salt, ethynyl sulfonium salt, propynylsulfonium salt or acetonitrile sulfonium salt, and the anion of the sulfonium salt is selected from any one of bromide ion, iodide ion, trifluoromethane sulfonate ion or tetrafluoroborate ion.