A method for enriching thiol-containing peptides based on covalent binding

Abstract

The present invention discloses a method for enriching thiol-containing peptide segments based on covalent binding, in which a chemical reagent that can react with thiol groups, i.e., 2-(2-pyridyldisulfide)ethylamine, is used, and the reagent contains two functional reactive groups, specifically, amino groups and pyridyldisulfide groups, wherein the amino groups can react with carboxyl magnetic beads to form new pyridyldisulfide magnetic bead materials, i.e., pyridyldisulfide agar magnetic beads. Pyridyldisulfide groups can directly react with thiol peptide segments to form covalent bonds, thereby achieving the enrichment of thiol peptide segments, and then by adding a reducing agent such as DTT, the thiol peptide segments can be eluted, and the method has the characteristics of low cost and good selectivity.

Description

Technical Field

[0001] The invention relates to the technical field of protein analysis, and in particular to a method for enriching thiol-containing peptide segments based on covalent binding. Background Art

[0002] Enrichment of thiol peptides is of great significance for biological activity research, drug development, scientific research and practical applications. The current method for enriching thiol peptides is mainly a non-covalent binding enrichment method, such as a method for enriching cysteine-containing peptides based on fluorine solid phase extraction and mass spectrometry analysis with application number CN201710051523.2. This method uses a solid phase extraction method, and the direct binding between the solid phase material and the thiol peptide is a non-covalent bond. This covalent binding enrichment method will have more non-specific binding, low binding efficiency, and this non-specific binding will affect the specific binding of the thiol peptide. Summary of the invention

[0003] The embodiment of the present application provides a method for enriching thiol-containing peptide segments based on covalent binding, in which a chemical reagent that can react with thiol groups is used, namely 2-(2-pyridyldisulfide)ethylamine, and the reagent contains two functional reactive groups, specifically amino and pyridyldisulfide, wherein the amino group can react with carboxyl magnetic beads to form a new pyridyldisulfide magnetic bead material, namely pyridyldisulfide agar magnetic beads. Pyridyldisulfide can directly react with thiol peptide segments to form a covalent bond, thereby achieving the enrichment of thiol peptide segments, and then by adding a reducing agent such as DTT, the thiol peptide segments can be eluted, and the method has the characteristics of low cost and good selectivity.

[0004] The present invention provides a method for enriching thiol-containing peptide segments based on covalent binding, comprising:

[0005] Step (1): preparing pyridyldisulfide agarose magnetic beads; wherein the step of preparing pyridyldisulfide agarose magnetic beads comprises: mixing carboxyl magnetic beads, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 2-(2-pyridyldisulfide)ethylamine hydrochloride, reacting in a water bath at 37-42°C for 1-3 hours, removing the supernatant after the reaction, and washing the magnetic beads with water several times to obtain pyridyldisulfide agarose magnetic beads;

[0006] In this embodiment, if the reaction time in the water bath is less than 1 hour, the reaction time is too short and the reaction is not thorough. If it is more than 3 hours, it will cause a waste of resources.

[0007] Step (2): preparing thiol peptide segments; wherein the step of preparing thiol peptide segments comprises: adding urea to the tissue and then grinding it, centrifuging it after grinding, taking the supernatant after the centrifugation, adding ammonium bicarbonate buffer and trypsin to the supernatant after the centrifugation, and then enzymatically digesting it to obtain the thiol peptide segments;

[0008] Step (3): incubating the thiol peptide segment and the pyridyldisulfide agarose magnetic beads, and washing with water;

[0009] Step (4): adding the eluent to the material washed in step (3) and incubating the mixture, retaining the supernatant peptides;

[0010] Step (5): desalting the supernatant peptide fragments and freeze-drying them for mass spectrometry detection.

[0011] In some embodiments, the volume ratio of the carboxyl magnetic beads, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 2-(2-pyridyldisulfide)ethylamine hydrochloride is 10-20:20:20, wherein the concentration of the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 0.1-1 mol / L, and the concentration of the 2-(2-pyridyldisulfide)ethylamine hydrochloride is 0.1-1 mol / L.

[0012] In some embodiments, the ratio of the tissue, urea and ammonium bicarbonate buffer is 0.25-4 mg: 200 μL: 800 μL, wherein the concentration of the urea is 6-8 mol / L, and the concentration of the ammonium bicarbonate buffer is 50 mmol / L.

[0013] In this embodiment, the role of ammonium bicarbonate is to dilute urea to facilitate subsequent enzymatic cleavage. The enzyme will denature under a higher concentration of urea, so using ammonium bicarbonate for dilution can maintain the activity of the protease.

[0014] In some embodiments, the mass ratio of the tissue to trypsin is 25-400:0.5-1.

[0015] In some embodiments, the centrifugation speed is 12000 rpm.

[0016] In some embodiments, the temperature of the enzyme cleavage is 37°; the time of the enzyme cleavage is 4-18 hours.

[0017] In some embodiments, the incubation time in step (3) is 1-3 hours. In this embodiment, the incubation time in step (3) is set to 1-3 hours, which can both fully react and save time.

[0018] In some embodiments, the eluent comprises DTT and CAA, wherein the molar ratio of DTT to CAA is 1:5.

[0019] In some embodiments, the incubation time in step (4) is 1 hour.

[0020] In some embodiments, the parameters of mass spectrometry detection are: positive ion voltage, V: 2200; ion transfer tube, ° C: 320; first-stage full scan Orbitrap resolution: 60000; mass range, m / z: 350-1500; first-stage acquisition dynamic gain, %: 300; separation window range, m / z: 1.6; HCD fragmentation energy, %: 28; second-stage Orbitrap resolution: 15000; second-stage acquisition dynamic gain, %: 75;

[0021] The chromatographic column was C18, with an inner diameter of 75um and a length of 30cm; the column temperature was 55 degrees; the flow rate was 400nL / min, 0 minutes, 5% B phase; 47 minutes, 25% B phase; 51 minutes, 31% B phase; 60 minutes, 90% B phase.

[0022] The present invention provides a method for enriching thiol-containing peptide segments based on covalent binding, in which a chemical reagent that can react with thiol groups is used, namely 2-(2-pyridyldisulfide)ethylamine, and the reagent contains two functional reactive groups, specifically amino groups and pyridyldisulfide groups, wherein the amino groups can react with carboxyl magnetic beads to form new pyridyldisulfide magnetic bead materials, namely pyridyldisulfide agar magnetic beads. Pyridyldisulfide groups can directly react with thiol peptide segments to form covalent bonds, thereby achieving the enrichment of thiol peptide segments, and then by adding a reducing agent such as DTT, the thiol peptide segments can be eluted, and the method has the characteristics of low cost and good selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram of the reaction principle of a method for enriching thiol-containing peptides based on covalent binding according to some embodiments is exemplified. DETAILED DESCRIPTION

[0024] In order to better understand the above technical solution, the technical solution of the present application is described in detail below through specific implementation methods.

[0025] In order to solve the above technical problems, the embodiment of the present application provides a method for enriching thiol-containing peptide segments based on covalent binding, in which a chemical reagent that can react with thiol groups is used, namely 2-(2-pyridyldisulfide)ethylamine, and the reagent contains two functional reactive groups, specifically amino groups and pyridyldisulfide groups, wherein the amino groups can react with carboxyl magnetic beads to form new pyridyldisulfide magnetic bead materials, namely pyridyldisulfide agar magnetic beads. Pyridyldisulfide groups can directly react with thiol peptide segments to form covalent bonds, thereby achieving the enrichment of thiol peptide segments, and then by adding a reducing agent such as DTT, the thiol peptide segments can be eluted, and the method has the characteristics of low cost and good selectivity.

[0026] Figure 1A schematic diagram of the reaction principle of a method for enriching thiol-containing peptides based on covalent binding according to some embodiments is exemplified.

[0027] The embodiment of the present application provides a method for enriching thiol-containing peptide segments based on covalent binding, in which a chemical reagent that can react with thiol groups is used, namely 2-(2-pyridyldisulfide)ethylamine, and the reagent contains two functional reactive groups, specifically amino and pyridyldisulfide, wherein the amino group can react with carboxyl magnetic beads to form a new pyridyldisulfide magnetic bead material, namely pyridyldisulfide agar magnetic beads. Pyridyldisulfide can directly react with thiol peptide segments to form a covalent bond, thereby achieving the enrichment of thiol peptide segments, and then by adding a reducing agent such as DTT, the thiol peptide segments can be eluted, and the method has the characteristics of low cost and good selectivity.

[0028] The method comprises steps (1) to (5):

[0029] Step (1): preparing pyridyldisulfide agarose magnetic beads; wherein the step of preparing pyridyldisulfide agarose magnetic beads comprises: mixing carboxyl magnetic beads, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 2-(2-pyridyldisulfide)ethylamine hydrochloride, reacting in a water bath at 37-42°C for 1-3 hours, removing the supernatant after the reaction, washing the magnetic beads with water several times, and obtaining pyridyldisulfide agarose magnetic beads. In the embodiment of the present application, the carboxyl magnetic beads are chemically modified agarose magnetic beads, such as those formed by carboxyl activation.

[0030] In one example, after the reaction, the pyridyl disulfide group has been connected to the agarose beads. The supernatant is removed by magnetic attraction of a magnetic rack, the beads are washed three times with water, and the liquid is removed to obtain pyridyl disulfide agarose beads.

[0031] In some embodiments, the volume ratio of the carboxyl magnetic beads, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 2-(2-pyridyldisulfide)ethylamine hydrochloride is 10-20:20:20, wherein the concentration of the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 0.1-1 mol / L, and the concentration of the 2-(2-pyridyldisulfide)ethylamine hydrochloride is 0.1-1 mol / L.

[0032] In one example, the amount of carboxyl magnetic beads used is 20 μL, approximately 20 mg. The concentration of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 500 mM (mmol / L) and the amount used is 20 μL. The concentration of 2-(2-pyridyldithio)ethylamine hydrochloride is 500 mM and the amount used is 20 μL.

[0033] Step (2): preparing thiol peptide segments; wherein the step of preparing thiol peptide segments comprises: adding urea to the tissue and then grinding it, centrifuging it after grinding, taking the supernatant, adding ammonium bicarbonate buffer and trypsin to the supernatant and then performing enzymatic digestion to obtain the thiol peptide segments.

[0034] In some embodiments, in some embodiments, the ratio of the tissue, urea and ammonium bicarbonate buffer is 0.25-4 mg: 200 μL: 800 μL, wherein the concentration of the urea is 6-8 mol / L, and the concentration of the ammonium bicarbonate buffer is 50 mmol / L.

[0035] In this embodiment, the concentration of urea after adding ammonium bicarbonate buffer is below 2 mol / L. Protease modification can be avoided by diluting urea.

[0036] In some embodiments, the mass ratio of the tissue to trypsin is 25-400:0.5-1.

[0037] In one example, 200 μL of urea at a concentration of 6-8 M is added to 0.25-4 mg of tissue. The concentration of ammonium bicarbonate buffer is 50 mM and the amount used is 800 μL. The amount of trypsin used is 5-10 μg.

[0038] In some embodiments, the centrifugation speed is 12,000 rpm.

[0039] In some embodiments, the temperature of the enzyme cleavage is 37°; the time of the enzyme cleavage is 4-18 hours.

[0040] Step (3): incubate the thiol peptide segment and the pyridyldisulfide agarose magnetic beads, and wash with water.

[0041] In some embodiments, the incubation time in step (3) is 1-3 hours.

[0042] Step (4): Add the elution buffer to the material washed in step (3) and incubate, retaining the supernatant peptides.

[0043] In some embodiments, the eluent includes DTT (dithiothreitol) and CAA (chloroacetamide), wherein the molar ratio of DTT to CAA is 1: 5. In one example, 200 μL of the eluent is added, the concentration of DTT is 10 mM, and the concentration of CAA is 50 mM.

[0044] In some embodiments, the incubation time in step (4) is 1 hour.

[0045] Step (5): desalting the supernatant peptide fragments and freeze-drying them for mass spectrometry detection.

[0046] Specifically, freeze drying can be performed using a freeze dryer.

[0047] In some embodiments, the parameters of mass spectrometry detection are: positive ion voltage, V: 2200; ion transfer tube, ° C: 320; first-stage full scan Orbitrap resolution: 60000; mass range, m / z: 350-1500; first-stage acquisition dynamic gain, %: 300; separation window range, m / z: 1.6; HCD fragmentation energy, %: 28; second-stage Orbitrap resolution: 15000; second-stage acquisition dynamic gain, %: 75;

[0048] The chromatographic column was C18, with an inner diameter of 75um and a length of 30cm; the column temperature was 55 degrees; the flow rate was 400nL / min, 0 minutes, 5% B phase; 47 minutes, 25% B phase; 51 minutes, 31% B phase; 60 minutes, 90% B phase.

[0049] In the embodiment of the present application, a chemical reagent that can react with thiol groups is provided, namely 2-(2-pyridyldisulfide)ethylamine, which contains two functional reactive groups, specifically amino and pyridyldisulfide, wherein the amino group can react with carboxyl magnetic beads to form a new pyridyldisulfide magnetic bead material, namely pyridyldisulfide agar magnetic beads. Pyridyldisulfide can directly react with thiol peptide segments to form covalent bonds, thereby achieving the enrichment of thiol peptide segments, and then by adding a reducing agent such as DTT, the thiol peptide segments can be eluted, and this method has the characteristics of low cost and good selectivity.

[0050] Example 1

[0051] Step (1.1): preparing pyridyldisulfide agarose magnetic beads; wherein the step of preparing pyridyldisulfide agarose magnetic beads comprises: mixing 20 μL of carboxyl magnetic beads, 20 μL of 500 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 20 μL of 500 mM 2-(2-pyridyldisulfide)ethylamine hydrochloride, reacting in a 37°C water bath for 3 hours, removing the supernatant by magnetic attraction of a magnetic stand after the reaction, washing the magnetic beads with water 3 times, removing the liquid, and obtaining pyridyldisulfide agarose magnetic beads;

[0052] Step (1.2): preparing thiol peptides; wherein the step of preparing thiol peptides comprises: adding 200 μL of 8M urea to 1 mg of tissue and grinding the tissue, centrifuging the tissue at 12,000 rpm after grinding, taking the supernatant, adding 800 μL of 50 mM ammonium bicarbonate buffer and 5 μg of trypsin to the supernatant, and performing enzyme digestion at 37°C for 18 hours to obtain thiol peptides;

[0053] Step (1.3): incubating the thiol peptide fragment and the pyridyldisulfide agarose magnetic beads for 1 hour, and washing three times with water;

[0054] Step (1.4): Add 200 μL of elution buffer to the material washed in step (1.3) and incubate for 1 h, wherein the elution buffer includes 10 mM DTT and 50 mM CAA, and retain the supernatant peptides;

[0055] Step (1.5): desalting the supernatant peptide fragments and freeze-drying them using a freeze dryer, and performing mass spectrometry detection;

[0056] The parameters of mass spectrometry detection are as follows: positive ion voltage, V: 2200; ion transfer tube, °C: 320; first-stage full scan Orbitrap resolution: 60000; mass range, m / z: 350-1500; first-stage acquisition dynamic gain, %: 300; separation window range, m / z: 1.6; HCD fragmentation energy, %: 28; second-stage Orbitrap resolution: 15000; second-stage acquisition dynamic gain, %: 75;

[0057] The chromatographic column was C18, with an inner diameter of 75um and a length of 30cm; the column temperature was 55 degrees; the flow rate was 400nL / min, 0 minutes, 5% B phase; 47 minutes, 25% B phase; 51 minutes, 31% B phase; 60 minutes, 90% B phase.

[0058] Example 2

[0059] The difference between Example 2 and Example 1 is that the thiol peptide segment and the pyridyldisulfide agarose magnetic beads are incubated for 3 hours, and the other steps remain unchanged.

[0060] Example 3

[0061] The difference between Example 3 and Example 1 is that the thiol peptide segment and the pyridyldisulfide agarose magnetic beads are incubated for 12 hours, and the other steps remain unchanged.

[0062] Example 4

[0063] The difference between Example 4 and Example 1 is that the mass of tissue used to obtain the enzyme-digested peptide fragments is 0.25 mg, and the other steps remain unchanged.

[0064] Example 5

[0065] The difference between Example 5 and Example 1 is that the mass of tissue used to obtain the enzyme-digested peptide fragments is 0.5 mg, and the other steps remain unchanged.

[0066] Example 6

[0067] The difference between Example 6 and Example 1 is that the mass of tissue used to obtain the enzyme-digested peptide fragments is 2 mg, and the other steps remain unchanged.

[0068] Example 7

[0069] The difference between Example 7 and Example 1 is that the mass of tissue used to obtain the enzyme-digested peptide fragments is 4 mg, and the other steps remain unchanged.

[0070] Comparative Example

[0071] The difference between the comparative example and Example 1 is that 2-(2-pyridyldisulfide)ethylamine is not added in the comparative example, and the other steps remain unchanged.

[0072] After mass spectrometry detection, the following results were obtained:

[0073] Example 1 obtained 6542 peptide segments containing thiol groups;

[0074] Example 2 obtained 6742 peptide segments containing thiol groups;

[0075] Example 3 obtained 5064 peptide segments containing thiol groups;

[0076] Example 4 obtained 5279 peptide segments containing thiol groups;

[0077] Example 5 obtained 5852 peptide segments containing thiol groups;

[0078] Example 6 obtained 6476 peptide segments containing thiol groups;

[0079] Example 7 obtained 6552 peptide segments containing thiol groups;

[0080] The comparative example obtained 301 peptide segments containing thiol groups.

[0081] By comparing the results obtained by mass spectrometry, it can be found that the use of 2-(2-pyridyldisulfide)ethylamine can significantly increase the number of peptides containing thiol groups; in addition, when the thiol peptides and pyridyldisulfide agarose magnetic beads are incubated for 1-3 hours, more thiol-containing peptides can be obtained.

[0082] In summary, the present invention provides a method for enriching thiol-containing peptide segments based on covalent binding, in which a chemical reagent that can react with thiol groups is used, namely 2-(2-pyridyldisulfide)ethylamine, and the reagent contains two functional reactive groups, specifically amino and pyridyldisulfide, wherein the amino group can react with carboxyl magnetic beads to form a new pyridyldisulfide magnetic bead material, namely pyridyldisulfide agar magnetic beads. Pyridyldisulfide can directly react with thiol peptide segments to form covalent bonds, thereby achieving the enrichment of thiol peptide segments, and then by adding a reducing agent such as DTT, the thiol peptide segments can be eluted, and the method has the characteristics of low cost and good selectivity.

[0083] It is easy for those skilled in the art to understand that, under the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed. The above are only preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application. The above are only preferred implementation methods of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present application, and these improvements and variations should also be regarded as the protection scope of the present application.

Claims

1. A method for enriching thiol-containing peptides based on covalent binding, characterized in that: include: Step (1): preparing pyridyldisulfide agarose magnetic beads; wherein the step of preparing pyridyldisulfide agarose magnetic beads comprises: mixing carboxyl magnetic beads, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 2-(2-pyridyldisulfide)ethylamine hydrochloride, reacting in a water bath at 37-42°C for 1-3 hours, removing the supernatant after the reaction, and washing the magnetic beads with water several times to obtain pyridyldisulfide agarose magnetic beads; Step (2): preparing thiol peptide segments; wherein the step of preparing thiol peptide segments comprises: adding urea to the tissue and then grinding it, centrifuging it after grinding, taking the supernatant after the centrifugation, adding ammonium bicarbonate buffer and trypsin to the supernatant after the centrifugation, and then enzymatically digesting it to obtain the thiol peptide segments; Step (3): incubating the thiol peptide segment and the pyridyldisulfide agarose magnetic beads, and washing with water; Step (4): adding the eluent to the material washed in step (3) and incubating the mixture, retaining the supernatant peptides; Step (5): desalting the supernatant peptide fragments and freeze-drying them for mass spectrometry detection.

2. The method according to claim 1, characterized in that The volume ratio of the carboxyl magnetic beads, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 2-(2-pyridyldisulfide)ethylamine hydrochloride is 10-20:20:20, wherein the concentration of the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 0.1-1 mol / L, and the concentration of the 2-(2-pyridyldisulfide)ethylamine hydrochloride is 0.1-1 mol / L.

3. The method according to claim 1, characterized in that The ratio of the tissue, urea and ammonium bicarbonate buffer is 0.25-4 mg: 200 μL: 800 μL, wherein the concentration of the urea is 6-8 mol / L, and the concentration of the ammonium bicarbonate buffer is 50 mmol / L.

4. The method according to claim 3, characterized in that The mass ratio of the tissue to trypsin is 25-400:0.5-1.

5. The method according to claim 1, characterized in that The rotation speed of the centrifugal treatment is 12000 rpm.

6. The method according to claim 1, characterized in that The temperature of the enzyme digestion is 37°; the time of the enzyme digestion is 4-18h.

7. The method according to claim 1, characterized in that The incubation time in step (3) is 1-3 hours.

8. The method according to claim 1, characterized in that The eluent includes DTT and CAA, wherein the molar ratio of DTT to CAA is 1:

5.

9. The method according to claim 1, characterized in that: The incubation time in step (4) is 1 h.

10. The method according to claim 1, characterized in that The parameters of the mass spectrometry detection are: positive ion voltage, V: 2200; ion transfer tube, °C: 320; first-stage full scan Orbitrap resolution: 60000; mass range, m / z: 350-1500; first-stage acquisition dynamic gain, %: 300; separation window range, m / z: 1.6; HCD fragmentation energy, %: 28; second-stage Orbitrap resolution: 15000; second-stage acquisition dynamic gain, %: 75; The chromatographic column was C18, with an inner diameter of 75um and a length of 30cm; the column temperature was 55 degrees; the flow rate was 400nL / min, 0 minutes, 5% B phase; 47 minutes, 25% B phase; 51 minutes, 31% B phase; 60 minutes, 90% B phase.

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