CN112924694A

CN112924694A - Discrimination-free protein thermal stability analysis method

Info

Publication number: CN112924694A
Application number: CN201911240786.3A
Authority: CN
Inventors: 叶明亮; 阮成飞
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2019-12-06
Filing date: 2019-12-06
Publication date: 2021-06-08

Abstract

The present invention relates to a discrimination-free method for analyzing thermal stability of a protein, which is used for screening a target protein having an interaction with a ligand molecule. Taking a protein sample, adding and adding ligand molecules respectively, incubating, heating, separating supernatant, performing enzymolysis and quantitative proteomics analysis, and calculating the difference of protein quantitative results of a ligand group and a blank group to screen out the ligand target protein. The invention only carries out experiments at the temperature points with great changes of the thermal stability of most proteins, omits the temperature points which have no significance for the identification of ligand molecule target proteins, directly calculates the difference of the proteins of a ligand group and a blank group without fitting a curve to calculate the delta Tm, more intuitively reflects the difference of the thermal stability of the proteins of the target proteins added into the ligand group and the blank group, also can avoid that a plurality of target proteins with irregular melting curves are omitted due to the incapability of calculating the delta Tm, is suitable for all proteins with response to heat, and improves the coverage of the identification of the target proteins.

Description

Discrimination-free protein thermal stability analysis method

Technical Field

The invention belongs to the technical field of interaction of protein and ligand molecules in the proteomic research direction, and particularly relates to a high-sensitivity high-coverage ligand target protein identification method and application thereof.

Background

The comprehensive and accurate identification of target and off-target proteins of drug molecules has always been a major challenge in the field of drug research. In recent years, with the rapid development of mass spectrometry technology, several mass spectrometry-based proteomics technologies are beginning to be applied to the identification of drug molecule target proteins. In addition to having a higher throughput, these techniques allow better identification of direct and indirect target proteins of drug molecules without requiring any modification of the drug molecule. Which comprises the following steps: drug affinity response Target stability method (DARTS) (Target identification using drug affinity reactivity stability (DARTS), Proc. Natl Acad. Sci. USA 106, 21984-21989 (2009)), restriction enzyme cleavage method (LIP) (A Map of Protein-metabolism Interactions derivatives of Chemical Communication, Cell,172,358-372.e23(2018)), Protein oxidation rate stability method (SPROX) (Thermokinetic analysis of Protein-ligand binding Interactions in complex biological chemistry using the stability of Protein from oxidation rate stability (SPROX 346, 148-161. 2013) and Protein thermal stability analysis method (TPR 5784) (Target identification using drug affinity reactivity stability of Protein stability of Protein 12542, 20147. Nat. Proc. 8, 148-161. and 2013) (TPR 5784). The main drawback of the first three methods is the need to identify and quantify specific peptide stretches, which is rather difficult due to the influence of the dynamic range of the protein and the mass spectrometric identification.

The protein thermal stability analysis method utilizes the difference of thermal stability of target protein of drug molecules before and after combining with the drug molecules, so that the amount of protein precipitated after heating at the same temperature is different, and then combines with a mass spectrometry quantitative technology to search for differential protein, namely potential target protein. Protein thermostability assays are widely used in the study of interactions between proteins and drug molecules, proteins and metabolites, proteins and nucleic acids, proteins and proteins, and the like. However, the conventional thermal protein analysis method has obvious defects in both experimental process and data processing. The experimental group and the control group are divided into different samples, and the subsequent treatment and mass spectrum quantification process are easy to bring about larger influence and the like. Several redundant temperature points were used with no significant change in protein thermostability, and Δ T was calculated only to fit an "S" -shaped melting curve when processing the data_mHowever, for many proteins without regular melting curves, it is not possible to fit an "S" shaped curve at all to calculate Δ T_mThus missing, is a method of discrimination.

In fact, when the target protein is screened, what really makes sense is the temperature range which can cause the difference between the thermal stability of the proteins of the drug adding group and the control group, and the difference can really reflect the change degree of the thermal stability of the target protein before and after the drug molecules are combined, rather than the calculated delta Tm after the curve is fitted by the traditional method.

In view of the above problems, we propose to invent a method for ligand target protein identification which is simpler and has higher identification sensitivity and coverage.

The above documents do not describe any method for analyzing the thermal stability of proteins based on non-discrimination, nor do they suggest any method.

Disclosure of Invention

The invention aims to provide a method for identifying ligand molecule target protein with high sensitivity and high coverage.

1. The method screens the ligand target protein by utilizing the direct difference of the ligand target protein in a ligand group and a blank group after heating precipitation instead of the protein melting point difference calculated by fitting a melting curve. The high sensitivity and high coverage identification of the ligand molecule interacting protein is realized by performing mass spectrum quantification on the protein in the supernatant sample after heating and centrifugation and directly analyzing the difference.

2. The invention adopts the following technical scheme:

(a) determining an optimal temperature interval for the protein mixture to precipitate;

(b) incubating the protein mixture with the ligand molecules respectively;

(c) heating in the optimum temperature interval to cause protein denaturation and precipitation;

(d) separating the heated supernatant for enzymolysis and quantitative proteomics analysis;

(e) and directly analyzing the difference of the protein quantitative results of the ligand group and the blank group, and screening the ligand molecule target protein.

3. The protein sample in the steps (a) and (b) may be a cell extract, a tissue extract, a protein mixture such as blood, or a purified protein.

4. The protein sample must retain the intact native structure of the protein and must not be denatured. Some gentle methods of protein extraction should be taken, including: a liquid nitrogen repeated freeze thawing method, a liquid nitrogen grinding method, a homogenization method and the like; the lysis solution used should also be able to maintain the structure of the protein, including Phosphate Buffered Saline (PBS) and the like.

5. The specific steps for determining the optimum temperature interval in the step (a) are as follows: the method comprises the steps of measuring protein solution in an equal amount in a plurality of new EP tubes, heating at different temperatures within the temperature range of 37-75 ℃, separating the heated supernatant, performing enzymolysis and quantitative proteomics analysis, and calculating the amount of residual protein in the supernatant at different temperatures. The temperature point at which most of the protein precipitates significantly is selected as the optimum temperature range.

6. The optimum temperature range selected by the invention is 47-59 ℃, but the temperature range is not limited to, and the temperature range can be adjusted according to specific conditions.

7. The ligand molecule of step (a) may be an active drug, a metabolite, a nucleic acid molecule, a metal ion, a peptide fragment, a protein, or any other molecule that may interact with a protein.

8. The protein solution of step (a) is divided equally into two groups, one group is added with ligand molecules with certain final concentration as ligand group, and the other group is added with blank solvent with equal volume as blank group for incubation. But not limited to two groups, the ligand group can also be added with a series of ligands with different final concentrations, and the blank group can also be molecules or blanks with similar structures.

9. The heating temperature in the step (c) comprises any two or more temperature points in the optimal temperature interval.

10. The heating induced protein precipitation method in the step (c) comprises the following specific steps: based on the number of the set heating temperature points, the protein solutions of the base group and the blank group were weighed in equal amounts in a new EP tube and heated at a set temperature.

11. The quantitative proteomics analysis method of step (d) includes label-free quantification and label quantification, and a suitable quantification method can be selected according to the number of actual samples.

12. The data processing method in the step (e) is specifically as follows: and (4) carrying out library searching on raw files obtained by mass spectrometry by using conventional proteomics analysis software, and setting a corresponding quantitative method. And (3) selecting the protein with higher quantitative result reliability from the obtained quantitative results, and carrying out normalization treatment. And (3) directly calculating the difference of the quantitative values of the proteins in the ligand group and the blank group, setting a proper threshold value, and obtaining the protein which meets the set threshold value as the potential target protein of the ligand molecules.

13. The protein with high reliability of the quantitative result comprises at least two or more than two peptide fragments and has quantitative result.

14. The difference between the protein added to the ligand group and the blank group comprises the distance or Euclidean distance between two protein melting curves, the area between the two curves and the like.

15. The threshold value can be adjusted according to different ligand molecules.

The invention has the following advantages:

1. the flux is high. Under the condition of not needing any modification to the ligand molecules, the target protein of the ligand molecules can be screened out at high flux by using a mass spectrometry quantitative method

2. The operation is simple, and the cost is saved. Compared with the traditional protein thermal stability analysis method, the method only selects the temperature points with great changes in the thermal stability of most proteins for experiment, saves redundant temperature points, simplifies experiment operation and saves reagent cost and mass spectrum time.

3. The quantitative accuracy is high. Because the ligand group and the blank group are mixed in the same sample, the experimental error caused by subsequent experimental operation and mass spectrum identification is avoided, and the reliability of the experimental result is greatly improved.

4, strong universality, wide coverage and no discrimination. The difference of the proteins of the ligand group and the control group at five temperature points is directly calculated without fitting a curve to calculate the delta Tm, so that the omission of a part of proteins due to the fact that the delta Tm cannot be calculated due to the absence of a regular melting curve is avoided, the change of the thermal stability of the ligand target protein before and after the ligand target protein is combined with ligand molecules is more intuitively reflected, and the coverage of ligand target protein identification is improved

Drawings

FIG. 1 is a graph showing the ratio distribution of the proteins remaining in the supernatant after heating at the different temperatures. The abscissa is the different heating temperatures and the ordinate is the ratio of the amount of protein remaining in the supernatant to the amount of protein at 37 ℃.

FIG. 2 is a flow chart of a novel method for non-discriminatory protein thermostability analysis.

FIG. 3 is the experimental results of the novel discrimination-free protein thermostability assay for identifying a staurosporine target protein. (a) Classification of all target proteins by threshold screening (abscissa represents classification of target protein, ordinate represents sum of distance of protein in supernatant sample and sediment sample, number represents number of such protein). (b) The Staurosporine target protein identified by the method is compared with the Overlap of the Staurosporine target protein identified by the traditional method. (c) Distance density comparison graph of staurosporine target protein identified by the method of the invention and staurosporine target protein identified by the traditional method (the abscissa is the distance between the ligand group and blank group melting curves of the protein, and the ordinate is the density size).

From the distribution in FIG. 1, it can be seen that at lower heating temperatures (<47 ℃) most of the proteins have not precipitated or have just begun to precipitate, and at higher heating temperatures (>59 ℃) most of the proteins have precipitated substantially completely, which are not sufficient to cause a significant difference in the thermostability of the ligand target proteins in the ligand and blank groups, and do not contribute to the identification of the ligand target proteins. In contrast, the vast majority of proteins only partially precipitate when heated in the intermediate temperature range (47-59 ℃), and this can vary significantly whether ligand addition causes stabilization or destabilization of the target protein. Therefore, the temperature range of 47-59 ℃ is selected as the optimum temperature range. The selection of the optimum temperature range can also be changed according to specific requirements.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

Example 1

Novel methods for the non-discriminatory protein thermostability assay were used for the target protein study of staurosporine (star cyclosporine):

(1) taking 1 dish of K562 cell sample, suspending in 600 μ L PBS buffer solution, adding 1% protease inhibitor (dimethyl sulfoxide (DMSO) solution of mixture composed of AEBSF, aprotinin, bestatin, E-64, leupeptin and pepstatin A) with final volume concentration, repeatedly freezing and thawing for three times by liquid nitrogen, centrifuging at 20000g for 10min at 4 deg.C, and collecting supernatant sample;

(2) 50 μ L of each of the above protein samples were heated in eleven new EP tubes at 37 deg.C, 41 deg.C, 44 deg.C, 47 deg.C, 50 deg.C, 53 deg.C, 56 deg.C, 59 deg.C, 62 deg.C, 65 deg.C, 68 deg.C for 3min, then cooled at room temperature for 2min, and then centrifuged at 4 deg.C, 20000g for 10 min. 30. mu.L of the supernatant from each EP tube was placed in eleven new EP tubes and Trypsin (Trypsin) was performed separately. Finally, the obtained peptide fragment solutions are respectively redissolved in 0.1% (v/v) formic acid solution for LC-MS/MS analysis. And (3) removing the residual protein amount of the supernatant corresponding to other heating temperatures by using the residual protein amount of the supernatant corresponding to 37 ℃, calculating the relative amount of the residual protein of the supernatant at different temperatures, and finally selecting 47-59 ℃ as an optimal heating temperature interval for subsequent experiments.

(3) The protein solution obtained in the step (1) is divided into two parts on average, wherein one part is added with staurosporine (the staurosporine is dissolved in DMSO) with the final concentration of 25 mu M, the other part is added with DMSO with the same volume, and the two samples are incubated for 30min at room temperature at the same time;

(4) two samples of 50. mu.L each were placed in five new 200. mu.L PCR tubes, heated at 47 ℃,50 ℃,53 ℃,56 ℃,59 ℃ for 3min, cooled at room temperature for 2min, and centrifuged at 4 ℃ and 20000g for 10min to collect the supernatant;

(5) mu.L of the supernatant from each PCR tube was placed in a new 600. mu.L EP tube, and 90. mu.L of a buffer containing 66mM TEAB (triethylamine-carbonate buffer) and 8M Gua-HCl (guanidine hydrochloride) was added;

(6) adding TCEP (tris (2-carboxyethyl) phosphine) with a final concentration of 10mM and CAA (chloroacetamide) with a final concentration of 40mM to the protein solution, and heating in a water bath at 95 ℃ for 5min to perform denaturing reductive alkylation;

(7) transferring the protein solution to an ultrafiltration tube membrane with the molecular weight cutoff of 10K, performing ultrafiltration centrifugation under 14000g of centrifugal force, washing twice with 50mM TEAB, adding 150 mu L of 50mM TEAB and 6 mu g of trypsin, and performing enzymolysis for 16h in water bath at 37 ℃;

(8) centrifuging the enzyme-hydrolyzed sample under 14000g to obtain a peptide fragment solution, washing the ultrafiltration membrane once by using 100 mu L of 50mM TEAB, and combining corresponding washing liquid and the peptide fragment solution;

(9) the concentration of the obtained peptide fragment solution was measured by Nanodrop.

(10) Adding corresponding TMT labeling reagent (TMT) into the peptide fragment sample of the step (8) according to the mass ratio of the maximum labeling reagent amount to the protein amount of 4/1¹⁰-126,TMT¹⁰-127N，TMT¹⁰-127C,TMT¹⁰-128N,TMT¹⁰-128C,TMT¹⁰-129N,TMT¹⁰-129C,TMT¹⁰-130N,TMT¹⁰-130C,TMT¹⁰The-131 samples respectively correspond to the labeling blank group at 47 ℃,50 ℃,53 ℃,56 ℃,59 ℃ and the ligand group at 47 ℃,50 ℃,53 ℃,56 ℃ and 59 ℃, and after shaking for 60min at room temperature, 1 mu L of hydroxylamine solution with the volume concentration of 5 percent is added, and the shaking is carried out for 20min at room temperature.

(11) Mixing all the ten ligand group samples and the blank group samples in the step (10) into one sample, and performing freeze-drying preservation;

(12) re-dissolving the sample of step (11) in 5mM NH₄HCO₃Respectively carrying out high-pH reverse grading in an ammonium bicarbonate solution, collecting fractions, and freeze-drying and storing;

(13) the peptide fragment mixture obtained above was reconstituted with 0.1% (v/v) formic acid and analyzed by RP LC-MS/MS.

FIG. 3 is a graph showing the results of the experiments conducted on Staurosporine target protein in the novel non-discriminatory protein thermostability assay. Staurosporine is a broad spectrum ATP competitive kinase inhibitor, binds mainly in the ATP binding pocket region of kinases, so its theoretical target protein should be some protein kinases or proteins with ATP binding pockets. As shown in fig. (a), 117 proteins having a distance D between the melting curves of the blank and the control greater than 0.17 or less than-0.4, among which 106 proteins became more stable (D >0.17), in which the number of kinases was 78, were considered as target proteins of staurosporine; 11 proteins became more unstable (D < -0.4) with a number of kinases of 3. And most of the proteins that are not kinase targets are ATP-binding proteins or regulatory subunits of kinases (fig. 3(b)), suggesting that these proteins, although not kinases, are still likely to be direct or indirect targets of staurosporine. Therefore, the method has higher reliability and accuracy.

Compared to the work published by Mikhail's in Science (FIG. 3(c)), the number of kinases identified in this example was 1.6 times greater, 60% greater, and could cover more than 70%. As can be seen from FIG. 3(b), the present invention has a better recognition effect for proteins with less change in protein thermal stability after ligand addition. In conclusion, the method has higher identification sensitivity and deeper coverage.

Claims

1. a non-discriminatory protein thermal stability analysis method is characterized in that:

This method utilizes the direct difference of the ligand target protein in the ligand group and the blank group after heating precipitation to screen the ligand target protein.

2. method according to claim 1, is characterized in that, the concrete steps of this method are:

(a) determine the optimum temperature range for the protein mixture to precipitate;

(b) The protein mixture is divided into two or more groups, one group is added with a certain final concentration of ligand molecules (the ligand molecules are dissolved in a specific solvent) as the ligand group, and the other group is added with an equal volume of blank solvent as the blank group , while incubating at room temperature;

(c) Divide the incubated ligand group and blank group into 2-5 parts respectively, and heat in the optimum temperature range to cause protein denaturation and precipitation; the ligand group and blank group are sorted according to the heating temperature from low to high, and the heating temperature is in turn The interval is 2-5 ℃, and the heating temperature of the lowest heating temperature part is 47-49 ℃, and the heating temperature of the highest heating temperature part is 58-60 ℃; the heating time is 1-5min; Serum for proteolysis and quantitative proteomic analysis;

(e) Directly analyze the difference in protein quantification results between the ligand group and the blank group, and screen the ligand molecule target protein.

3. method according to claim 2, is characterized in that:

The protein samples described in steps (a) and (b) can be one or more of cell extracts, tissue extracts, protein mixtures such as blood, or purified proteins; the protein samples must keep the protein The complete natural structure cannot be denatured; some mild protein extraction methods should be adopted, including: one or more of liquid nitrogen repeated freezing and thawing method, liquid nitrogen grinding method and homogenization method; the lysis solution used It should also be able to maintain the structure of the protein, including phosphate buffered saline (PBS), etc.

4. The method according to claim 2, wherein;

The concrete steps of determining the optimum temperature interval described in step (a) are:

Equal amount of protein solution was taken in 8-39 different EP tubes. Different EP tubes were heated at different temperatures in the temperature range of 37-75 °C. Different EP tubes were sorted according to the heating temperature from low to high. The heating temperature The interval is 1-5℃, and the heating temperature of the minimum heating temperature EP pipe is 37-38℃, and the heating temperature of the maximum heating temperature EP pipe is 72-75℃; the heating time is 1-5min;

After separation and heating, the supernatant in each EP tube was subjected to protease enzymatic hydrolysis and quantitative proteomic analysis, and the amount of remaining protein in the supernatant at different temperatures was calculated; The amount of remaining protein in the supernatant of the EP tube at the heating temperature, according to the distribution of the amount of remaining protein at each temperature, the heating temperature with a median of 0.1-0.25 was selected as the maximum temperature, and the median of 0.75-0.9 was selected The heating temperature is taken as the minimum temperature, and the temperature point where most proteins are more obviously precipitated is selected as the optimum temperature range, that is, the range from the lowest temperature to the highest temperature, usually 47-60 °C.

5. method according to claim 2, is characterized in that:

The ligand molecule described in step (a) can be one or two of active drugs, metabolites, nucleic acid molecules, metal ions, peptides, proteins and other types of molecules that may interact with proteins in the protein mixture. more than one species.

6. The method according to claim 2, wherein:

The protein solution described in step (a) is evenly divided into two groups, one of which is added with a certain final concentration of ligand molecules as a ligand group, and the other group is added with an equal volume of blank solvent as a blank group for incubation; Limited to two groups, a series of ligands with different final concentrations can also be added to the ligand group, and the blank group can also be molecules with similar structures or blanks.

7. The method according to claim 2, wherein:

The quantitative proteomic analysis method described in step (d) includes label-free quantification or separately labelled quantification, and an appropriate quantification method can be selected according to the actual number of samples.

8. The method according to claim 2 or 7, wherein:

The data processing method described in step (e) is specifically as follows: using proteomics analysis software to search the raw file obtained by mass spectrometry analysis, and setting a corresponding quantitative method; High protein, normalize it; directly calculate the difference between the quantitative value of the protein added in the ligand group and the blank group, set appropriate screening conditions (greater than 0.15 to 0.2 or less than -0.6 to -0.4), through the set The protein of the screening conditions is the potential target protein of the ligand molecule.

9. The method according to claim 8, wherein:

The said protein with higher reliability of the quantitative result refers to a protein containing at least two or more peptide segments with quantitative results.

10. The data analysis method according to claims 2 and 8. It is characterized by:

The difference between the protein added in the ligand group and the blank group includes the distance or Euclidean distance between the two protein melting curves, the area between the two curves, and the like.