CN115128183A - High-throughput screening method of kinase inhibitor - Google Patents
High-throughput screening method of kinase inhibitor Download PDFInfo
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- CN115128183A CN115128183A CN202210688802.0A CN202210688802A CN115128183A CN 115128183 A CN115128183 A CN 115128183A CN 202210688802 A CN202210688802 A CN 202210688802A CN 115128183 A CN115128183 A CN 115128183A
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Abstract
The invention discloses a high-throughput screening method of a kinase inhibitor, which comprises the following steps: (1) inhibiting the kinase by adopting inhibitors with a plurality of concentrations, then labeling the polypeptide of the active site of the kinase by utilizing multiple equal-weight labels, and enriching the polypeptide which is not combined with the inhibitors; (2) and (3) performing data-dependent mode mass spectrometry on the kinase active site polypeptide with higher abundance, and performing selective ion monitoring mode mass spectrometry on the kinase active site polypeptide with lower abundance. The method has the advantages of high analysis flux, high detection sensitivity and less required sample amount, and greatly improves the accuracy and efficiency of screening the kinase inhibitor and determining the IC50 value.
Description
Technical Field
The invention relates to a high-throughput screening method of a kinase inhibitor.
Background
Protein kinases are key enzymes in a series of complex cellular functions and pathways, are widely involved in cell signaling, and mutation or abnormal expression of the protein kinases causes occurrence and development of many diseases (including cancers) and is an important target for drug development. The protein kinase is used as a drug target, small molecular compounds interacting with the protein kinase are searched, and further optimization for research and development of new drugs is an important direction in the research field of the current protein kinase. The existing kinase inhibitor screening method adopts independent liquid chromatography-mass spectrometry analysis of each concentration, and uses non-standard quantity to carry out relative quantitative analysis on the kinase active site polypeptide under the action of each concentration; the method has low analysis flux, low sensitivity and low accuracy. In view of the above problems, there is a need for further solutions.
Disclosure of Invention
In order to solve the above technical problems, the present invention aims to provide a method for screening kinase inhibitors with high throughput, which has high analysis throughput, high detection sensitivity, and a small amount of samples, and greatly improves the accuracy and efficiency of screening kinase inhibitors and measuring IC50 values.
In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:
a method for high throughput screening of kinase inhibitors comprising the steps of:
(1) inhibiting the kinase by adopting inhibitors with a plurality of concentrations, labeling the polypeptide of the active site of the kinase by utilizing multiple equal-weight labels, and enriching the polypeptide which is not combined with the inhibitors;
(2) and (3) performing data-dependent mode mass spectrometry on the kinase active site polypeptide with higher abundance, and performing selective ion monitoring mode mass spectrometry on the kinase active site polypeptide with lower abundance.
Further, in the step (1), after the kinase is subjected to inhibition treatment, a thiobiotin nucleotide probe is utilized to react with an uninhibited active site of the kinase; during enrichment, the agarose resin microsphere material grafted with streptavidin is used for enriching the polypeptide containing the desthiobiotin nucleotide probe.
Furthermore, before enrichment, the kinase in which the active site is reacted with the inhibitor and the nucleotide probe is subjected to reduction, alkylation and trypsin digestion in sequence.
Furthermore, the polypeptide after the enzyme digestion by the trypsin is also subjected to desalting treatment and then elution treatment.
Further, the mass spectrometry adopts a liquid mass spectrometry tandem mass spectrometer.
Compared with the prior art, the invention has the beneficial effects that:
1) the strength of the report ions generated by the polypeptide after multiple equal-weight labeling in the cascade mass spectrum is used for relative quantification, so that the method and system errors generated in liquid chromatography-mass spectrum combined analysis by the traditional non-standard quantification are effectively overcome, and the quantification accuracy is higher than that of the non-standard quantification;
2) after multiple marked samples are mixed in equal mass, only one-time liquid chromatography-mass spectrometry analysis is needed, so that the cost of instrument analysis is saved in multiples, and the analysis flux is improved in multiples;
3) the polypeptide after the equal-weight multiple labeling has peaks superimposed in the primary mass spectrum due to the equal weight, so that the detection sensitivity is improved, and the required sample amount is reduced by times.
4) The invention utilizes the reaction of the desthiobiotin nucleotide probe and the uninhibited active site of the kinase, and utilizes the agarose resin microsphere material grafted with streptavidin to enrich the polypeptide containing the desthiobiotin nucleotide probe, thereby improving the quantitative accuracy of the uninhibited kinase active site polypeptide.
The method greatly improves the accuracy and efficiency of kinase inhibitor screening and IC50 value determination, and provides a basis for relevant structure and function research.
The high-throughput screening method of the kinase inhibitor is suitable for multiple equal-weight covalent labeling reaction labels which can be distinguished by any mass spectrometric detection.
The high throughput screening method for kinase inhibitors of the present invention is applicable to all probes that selectively bind to the kinase active site and covalently label one of the amino acids in the site binding region.
The high-throughput screening method for kinase inhibitors of the present invention is applicable to all kinase inhibitors.
The high-throughput screening method of the kinase inhibitor is suitable for the combination of data-dependent mass spectrometry detection of all high-abundance polypeptides and selective ion monitoring and targeting detection of low-abundance polypeptides.
Drawings
FIG. 1 is a schematic flow chart of a method for high throughput screening of kinase inhibitors according to an embodiment of the present invention.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Examples
As shown in FIG. 1, the screening method of the present invention is described below by taking the activity of staurosporine, a commonly used kinase inhibitor, to inhibit kinases in HeLa cells as an example.
One, multiple equal-weight markers
The method adopts multiple equal-weight labels to mark the kinase active site polypeptides after the action of a plurality of concentration inhibitors, and comprises the following specific steps:
lysis and desalting of HeLa cells; HeLa cells (about 10) 7 One) was placed in 1mL of lysis solution (0.1M Tris/HCl, 4% SDS, pH 8.0) and sonicated on ice for 15 min; the mixture was centrifuged at 14000rpm and 4 ℃ for 15 minutes, and the supernatant protein mixture solution was collected. The protein mixture solution was desalted using a molecular size cut-off Membrane (MWCO) and the protein concentration was determined by BCA method. Six equal amounts of protein solution (1 mg per group) were taken and treated with inhibition by adding 0, 0.01, 0.3, 1, 3 and 10. mu.M staurosporine, respectively. The solution after the inhibition treatment is added with a desthiobiotin nucleotide probe (ATP or ADP) respectively to carry out reaction of uninhibited sites.
The protein solution of which the active site is reacted by the inhibitor and the nucleotide probe is sequentially subjected to reduction, alkylation and trypsin enzyme digestion; the respective reaction and treatment conditions were as follows:
reduction: 20mM DTT, 55 ℃, 20 minutes;
alkylation: 20mM iodoacetamide, at room temperature, in the dark for 30 minutes;
and (3) carrying out trypsin digestion: trypsin was used at 1:50(w/w), 37 ℃ for 16 hours, and quenched with 0.5% TFA;
desalting the polypeptide solution after enzyme digestion by using a C18 SPE column, and eluting by using 400 mu L of 50% ACN and 400 mu L of 80% ACN in sequence; combining the eluates, and vacuum concentrating to dryness.
Six groups of polypeptides concentrated to dryness were labeled with equal-weight multiplex tags (e.g., ThermoFisher TMT 127N, 127C, 128N, 128C, 129N and 129C) according to the manufacturer's protocol. And mixing the marked groups of polypeptides, and enriching the polypeptides containing the desthiobiotin nucleotide probe, namely the polypeptides which are not combined with the inhibitor, by using an agarose resin microsphere material grafted with streptavidin.
Second, mass spectrometry
Analysis of higher abundance kinase active site polypeptides based on data-dependent pattern mass spectrometry acquisition
And (3) carrying out mass spectrometry and database search on the enriched polypeptide mixture by using a data-dependent C18-RPLC-NanoESI-MS/MS (liquid chromatography-mass spectrometry tandem mass spectrometer), and obtaining the retention time of the kinase active site polypeptide, the valence states of precursor and fragment ions and the distribution of product ions.
First, C18 particles were packed on a Dionex Ultimate 3000RSLCnano HPLC system (Thermo Fisher Scientific) with a 20cm long analytical column (360. mu. od. times.75. mu. mid) ((R))
1.7 μm). Mobile phase a is 99.8% H 2 A mixture of O and 0.2% FA. Mobile phase B was 95.0% ACN, 4.8% H 2 A mixture of O and 0.2% FA. Elution was carried out at a constant flow rate of 300nL/min under a gradient of 1 hour: the sample is loaded with 2% of mobile phase B and 98% of mobile phase A for 5 minutes, the volume ratio of the mobile phase B is 2-40% in 45 minutes, and then the volume ratio of the mobile phase B is increased to 95% in 5 minutes and kept for 5 minutes.
The eluted polypeptides were then detected on-line using nanoESI Q exact tandem mass spectrometry. The first-stage spectral mass resolution is 70k (m/z 200), the spectral range is m/z 350-1800, and the Automatic Gain Control (AGC) target value and the maximum ion implantation time are 1e6 and 50ms respectively. The secondary spectral mass resolution was set at 35k (m/z 200) and the secondary mass spectra were obtained in data dependent mode (Top20) with high energy collision dissociation (HCD), AGC and maximum ion implantation times of 2e5 and 250ms respectively. The trapping windows and dynamic exclusion times were set at 0.7m/z and 20.0 s. The gradient collision energy was set to 30.0%. The temperature of the ion transmission tube was set to 320 ℃ and the electrospray voltage was set to 1.9 kV.
Finally, a polypeptide search engine is used for carrying out database search on the data group obtained by the liquid chromatography-mass spectrometry combined analysis; the database adopts a human whole protein database; the alkylamidomethylated carbamate (molecular weight 57.02Da) was set as a static modification on cysteine and desthiobiotin (molecular weight 196.12Da) was set as a dynamic modification on lysine. The uninhibited kinase active site polypeptides were relatively quantified at each concentration based on the intensity of the respective reporter ions of the isobaric labels.
Analysis of lower abundance kinase active site polypeptides based on selective ion monitoring mode mass spectrometry acquisition
The lower abundance kinase active site polypeptide is subjected to targeted analysis with higher sensitivity and accuracy based on mass spectrometry acquisition in a selective ion monitoring mode under the same chromatographic separation condition. And establishing a low-abundance polypeptide target monitoring list based on the obtained retention time, the mass-to-charge ratio and the characteristic fragment ions, and inputting the low-abundance polypeptide target monitoring list into an acquisition process for target detection.
The IDs and the relative strength of the polypeptides at the active site of the kinase are given based on the combination of Identification (IDs) of the polypeptides respectively obtained by data-dependent mode mass spectrometry and selective ion monitoring mode mass spectrometry; the percent inhibition of the active polypeptide was plotted against inhibitor concentration, and an IC50 value for the active site was obtained at 50% inhibition.
Based on the method of the present invention, the inhibitory effects of staurosporine on different kinases in HeLa cells were analyzed, and representative results obtained are shown in table 1; the relevant details include the gene name, AC number, short name for protein, full name for protein, active site amino acid sequence, active site, and IC50 value for staurosporine.
TABLE 1
The analysis method is based on multiple equal-weight quantitative labeling and single liquid chromatogram-tandem mass spectrum combined analysis, effectively overcomes the method and system errors generated in the liquid chromatogram-mass spectrum combined analysis by the traditional non-standard quantity, has higher quantitative accuracy than the non-standard quantity, saves the cost of instrument analysis in multiples, improves the analysis flux in multiples, improves the detection sensitivity, and reduces the required sample amount in multiples.
It is obvious to those skilled in the art that the present invention is not limited to the above embodiments, and the invention is not limited to the above embodiments, and it is within the scope of the present invention to use the method concept and technical solution of the present invention directly in other fields without any substantial modification or improvement.
Claims (5)
1. A method for high throughput screening of kinase inhibitors comprising the steps of:
(1) inhibiting the kinase by adopting inhibitors with a plurality of concentrations, labeling the polypeptide of the active site of the kinase by utilizing multiple equal-weight labels, and enriching the polypeptide which is not combined with the inhibitors;
(2) and (3) performing data-dependent mode mass spectrometry on the kinase active site polypeptide with higher abundance, and performing selective ion monitoring mode mass spectrometry on the kinase active site polypeptide with lower abundance.
2. The method for high throughput screening of kinase inhibitors according to claim 1, wherein in step (1), the kinase is subjected to an inhibition treatment, and then a desthiobiotin nucleotide probe is used to react with the uninhibited active site of the kinase; and during enrichment, the streptavidin-grafted agarose resin microsphere material is used for enriching the polypeptide containing the desthiobiotin nucleotide probe.
3. The method for high throughput screening of kinase inhibitors according to claim 1, wherein the kinase, the active site of which is reacted with the inhibitor and the nucleotide probe, is subjected to reduction, alkylation and trypsin digestion in sequence before enrichment.
4. The method for high throughput screening of kinase inhibitors according to claim 3, wherein the trypsin cleaved polypeptide is further subjected to desalting treatment and then to elution treatment.
5. The method for high throughput screening of kinase inhibitors according to claim 1, wherein the mass spectrometry is performed using a LC-MS tandem mass spectrometer.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111220690A (en) * | 2018-11-27 | 2020-06-02 | 中国科学院大连化学物理研究所 | Direct mass spectrometry detection method for low-abundance protein posttranslational modification group |
| CN111351878A (en) * | 2020-04-01 | 2020-06-30 | 上海中科新生命生物科技有限公司 | Proteome research method based on ATP probe enriched kinase technology |
| US20210318318A1 (en) * | 2018-08-31 | 2021-10-14 | Institute For Cancer Research D/B/A The Research Institute Of Fox Chase Cancer Center | Kinase Activity In Tumors |
| CN113945670A (en) * | 2021-09-30 | 2022-01-18 | 上海中科新生命生物科技有限公司 | Kinase enrichment detection method based on chemical proteomics |
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- 2022-06-17 CN CN202210688802.0A patent/CN115128183A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210318318A1 (en) * | 2018-08-31 | 2021-10-14 | Institute For Cancer Research D/B/A The Research Institute Of Fox Chase Cancer Center | Kinase Activity In Tumors |
| CN111220690A (en) * | 2018-11-27 | 2020-06-02 | 中国科学院大连化学物理研究所 | Direct mass spectrometry detection method for low-abundance protein posttranslational modification group |
| CN111351878A (en) * | 2020-04-01 | 2020-06-30 | 上海中科新生命生物科技有限公司 | Proteome research method based on ATP probe enriched kinase technology |
| CN113945670A (en) * | 2021-09-30 | 2022-01-18 | 上海中科新生命生物科技有限公司 | Kinase enrichment detection method based on chemical proteomics |
Non-Patent Citations (4)
| Title |
|---|
| MARKUS SCHIRLE 等: "Kinase Inhibitor Profifiling Using Chemoproteomics", KINASE INHIBITORS, pages 161 - 177 * |
| RYAN D. BOMGARDEN 等: "Kinase Inhibitor Profi ling of TrkA- and TrkB-Expressing Neuroblastoma Cell Lines Using Desthiobiotin Nucleotide Probes", pages 1 - 6, Retrieved from the Internet <URL:https://assets.thermofisher.cn/TFS-Assets/CMD/Application-Notes/AN-515-LC-MS-Kinase-Inhibitor-Desthiobiotin-Nucleotide-Probes-TrkA-TrkB-Neuroblastoma-AN63355-EN.pdf> * |
| SCOTT PETERMAN 等: "Targeted Kinase Inhibitor Profiling Using a Hybrid Quadrupole-Orbitrap Mass Spectrometer", Retrieved from the Internet <URL:https://assets.thermofisher.cn/TFS-Assets/CMD/Application-Notes/AN-574-LC-MS-Targeted-Kinase-Inhibitor-AN63662-EN.pdf> * |
| 石文昊 等: "基于质谱的磷酸化蛋白质组学：富集、检测、鉴定和定量", 生物化学与生物物理进展, vol. 45, no. 12, pages 1250 - 1258 * |
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