CN-119395303-B - Lithospermum acid action target screening method based on LIP-MS technology

Abstract

The invention discloses a method for screening shikonin action targets based on LIP-MS technology, which relates to the technical field of medicines and has the technical key points that a protein sample is subjected to restricted enzymolysis by adopting broad specificity protease, the characteristic that the conformational stability of a peptide fragment in a binding area can be improved by combining a medicine and target protein is further utilized to carry out secondary enzymolysis, a peptide fingerprint spectrum is acquired by adopting a quantitative proteomic technology, the specific 'conformational retention peptide fragment' after the medicine treatment is screened out by analyzing and comparing the change of the peptide fingerprint spectrum of a control group and the medicine treatment group, and target protein information corresponding to the peptide fragment is identified by database matching.

Inventors

• LI LING
• LV LEI
• WANG HUI
• FAN FANG
• Xia Zhewei
• CHEN XIAOFEI

Assignees

• 中国人民解放军海军军医大学

Dates

Publication Date

20260508

Application Date

20241031

Claims (3)

1. 1. A method for screening shikonin acting targets based on LIP-MS technology is characterized by comprising the following steps: s1, extracting proteins, namely inoculating monoclonal candida albicans into 1mL YPD liquid culture medium, culturing for 16 hours at 30 ℃ and 200 r/min, washing cells three times by using PBS after finishing culturing, adding a total amount of 5mL protein extraction buffer solution, mixing with acidified glass beads with twice the volume of the cell volume, extracting proteins under ice bath conditions, detecting the protein concentration by using a BCA protein concentration measuring kit, diluting to 1.0mg/mL by using PBS, and storing in a refrigerator at-80 ℃ for later use; s2, limited proteolysis, namely adding a proper concentration of medicine or DMSO into 100 mug of diluted protein solution, respectively adding proteinase K into a sample according to an enzyme/substrate ratio of 1:100 to incubate the sample accurately for 3 minutes, heating to inactivate PK, then adding a sodium deoxycholate solution with a final concentration of 5%, enabling dithiothreitol with a final concentration of 12mM to enable iodoacetamide with a final concentration of 40mM to alkylate reduced cysteine residues, adding trypsin according to an enzyme/substrate ratio of wt/wt of 1:100, incubating overnight at 37 ℃, stopping digestion and precipitating sodium deoxycholate by adding 98% vol/vol formic acid to a final concentration of 2% vol/vol, and finally 16000g centrifuging for 10 minutes to remove sediment, and transferring supernatant for mass spectrometry after desalting treatment; s3, mass spectrometry, namely quantifying candida albicans proteome by a SWATH-based mass spectrometry, collecting and analyzing data on analysis 1.7 software and Protein Pilot 4.5, establishing a difference Protein information table by using MARKERVIEW software and a Uniprot database through the Uniprot database, and screening out potential targets of shikonin acting on candida albicans by comparing differences of effective peptide fragments; S4, verifying target spots, namely screening and sequencing the screened target spots according to the dependence of the drug concentration, and confirming Fructose-bisphosphate aldolase as an action target spot and adopting corresponding verification by comprehensively analyzing the drug structure and the target spot function.
2. 2. The method for screening shikonin target spot based on LIP-MS technology as claimed in claim 1, wherein Fructose-bisphosphate aldolase target spot and shikonin directly act on antifungal.
3. 3. The method for screening shikonin target spot based on LIP-MS technology according to claim 1, wherein the mass spectrometry is performed by using an ultra-high performance liquid chromatography-mass spectrometry combined system nano-UPLC-MS, model number Ekspert nano LC and Triple-TOF 5600+, AB Sciex, the system separates peptide fragments on a ChromXP C chromatographic column at a flow rate of 5 μl/min, and the elution of the peptide fragments is performed by using an acetonitrile-water gradient for 90 minutes.

Description

Lithospermum acid action target screening method based on LIP-MS technology Technical Field The invention relates to the technical field of medicines, in particular to a method for screening shikonin action targets based on LIP-MS technology. Background Shikonin (Shikonin, SK) is a fat-soluble naphthoquinone compound extracted from radix Arnebiae, and has antiinflammatory, antioxidant, antiviral, and antifungal effects. The research shows that shikonin has strong anti-candida albicans activity, and especially has more obvious antifungal effect on fluconazole resistant bacteria. The combination of shikonin, azoles and polyenes can reduce the dosage of the medicines and restore the antibacterial effect of the medicines on drug-resistant fungi, and is a good antifungal medicine synergist, however, shikonin can directly act on target spots in candida albicans cells, and the upstream and downstream genes or paths are regulated and controlled by a mechanism, so that the antifungal effect of the shikonin is exerted, and the problem is still not solved well. Drug targets are the key biological basis for drug activity, and drug target discovery and identification have been research hotspots in various fields of pharmacy. In recent years, various labeled and unlabeled drug target analysis methods have been developed, and in contrast, unlabeled drug target identification has been carried out without carrying out structure-activity relationship and label modification studies on small molecules, so that the time for target analysis has been greatly shortened, and thus the method has been widely used, and the unlabeled drug target identification methods mainly comprise DARTS (drug affinity response target stability analysis), SPROX (oxidation rate-based protein stability assessment), CETSA (cell thermal shift analysis) and the like. These methods identify drug targets by observing changes in biophysical properties (such as stability, oxidation rate, or thermal stability) that result after binding of the drug to the target protein. However, these methods also have certain limitations. First, the DARTS technology has limited ability to detect low abundance targets, and the hydrolytic sensitivity of the target protein and the specificity of the protease chosen may affect the results. Second, SPROX technology is limited by the methionine content of the protein and is currently mainly applicable to analysis of cell lysates and cannot be directly applied to living cell systems. In addition, CETSA technology requires higher drug concentrations, is time consuming and costly, and lacks information about binding of the drug to specific domains of the target protein. Therefore, the present invention aims to provide a method for screening shikonin target spots based on LIP-MS technology, so as to solve the above problems. Disclosure of Invention The invention aims to solve the problems, and provides a method for screening shikonin action targets based on LIP-MS technology, which adopts a non-labeled drug target analysis method based on peptide fingerprint spectrum to extract total proteins of candida albicans cells under the intervention of lithospermum, uses protease K to carry out limited enzymolysis, compares the change of peptide fingerprint spectrum of a control group and a drug treatment group, screens out specific 'conformation maintaining peptide segments' in the shikonin treatment group, and identifies target proteins of shikonin for inhibiting candida albicans biofilm through database matching. In order to achieve the above purpose, the technical scheme of the invention is as follows: The invention provides a method for screening shikonin action targets based on LIP-MS technology, which comprises the following steps: S1, extracting proteins, namely inoculating monoclonal candida albicans into 1mL YPD liquid culture medium, culturing for 16 hours at 30 ℃ and 200 r/min, washing cells three times by using PBS after finishing culturing, adding 5mL of protein extraction buffer solution in total, mixing with two volumes of acidified glass beads, extracting proteins under ice bath conditions, detecting the protein concentration by using a BCA protein concentration measuring kit, diluting to 1.0mg/mL by using PBS, and storing in a refrigerator at-80 ℃ for later use; S2, limited proteolysis, namely adding a proper concentration of medicine or DMSO into 100 mug of diluted protein solution, respectively adding proteinase K into a sample according to an enzyme/substrate ratio of 1:100 to incubate the sample accurately for 3 minutes, heating to inactivate PK, then adding a sodium deoxycholate solution with a final concentration of 5%, enabling dithiothreitol with a final concentration of 12mM to enable iodoacetamide with a final concentration of 40mM to alkylate reduced cysteine residues, adding trypsin according to an enzyme/substrate ratio of wt/wt of 1:100, incubating overnight at 37 ℃, stopping digestion and precipitating sodium de