Method for on-line screening of protein ligands and evaluation of binding capacity of protein ligands and application

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for screening protein ligands on line and evaluating the binding capacity of the protein ligands and application of the protein ligands. The method comprises the following steps: firstly, screening potential protein ligands in a complex system by combining a liquid chromatography-high resolution mass spectrometry (LC-HRMS) in a mode of injecting protein after a column, and then further collecting a mass spectrum splitting curve of precursor ions to free protein ions of a protein-ligand compound by utilizing a high resolution multi-reaction monitoring mode (MRMHR); the optimal collision energy (OCE) parameter is fitted, and the binding capacity between different ligands and the protein is evaluated according to the OCE. By adopting the method disclosed by the invention, rapid screening of the protein ligand and rapid preliminary judgment of the binding capacity of the protein ligand can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and specifically relates to a method and application for online screening of protein ligands and evaluation of their binding abilities by post-column protein injection combined with liquid chromatography-energy resolved affinity mass spectrometry (PCI-LC-ER-AMS). Background Art

[0002] Natural products are widely distributed in nature and are one of the important sources for drug development. Screening of active natural products is a prerequisite for developing natural product drugs. However, rapid screening and identification of bioactive components from natural products is still a great challenge. Traditional bioactivity-oriented separation methods require repeated separation steps and bioactivity tests before obtaining single bioactive compounds. This method is usually time-consuming, labor-intensive, expensive and inefficient. Therefore, it is urgent to develop simple, efficient and high-throughput screening methods for screening bioactive natural products in complex systems. Affinity-based methods have the advantages of good selectivity and high throughput, and have great application prospects in natural product screening, such as ultrafiltration, dialysis, magnetic beads, etc. These methods can be combined with LC-MS to identify the structure of the bioactive molecules obtained by screening. However, since this strategy is mainly carried out offline, it has disadvantages such as being time-consuming and labor-intensive. Post-column chemical derivatization combined with LC-MS analysis strategy has been widely used in compound structure identification. It is mainly achieved by introducing a derivatization reagent after the chromatographic column to react with the analyte for further MS detection.

[0003] Proteins and ligands mainly rely on covalent or non-covalent interactions to exert their efficacy, among which non-covalent interactions are dominant. To date, there are many methods for identifying and quantifying protein-ligand interactions, such as isothermal titration calorimetry (ITC), surface plasmon resonance spectroscopy (SPR), nuclear magnetic resonance spectroscopy (NMR), native mass spectrometry (native MS), etc. Among them, native MS uses the mildness of the electrospray ion source (ESI) to transfer proteins and their complexes from volatile buffer aqueous solutions to the gas phase. Under the controlled operating conditions of the mass spectrometer, biological molecules can maintain a near-native structure and can retain non-covalent interactions. By calculating the relationship between the ratio of complex and protein spectral signals and the dose, the protein-ligand binding constant can be calculated. At present, it has been widely used in the study of protein-ligand, protein-protein, protein-DNA and other interactions. However, existing studies mainly screen in the form of ligand monomers, which cannot achieve high-throughput analysis. Online energy-resolved mass spectrometry (online ER-MS) is to study the change pattern of ion abundance of collected precursor ions and corresponding fragment ions under a series of collision energies (CE). Since the non-covalent interactions between proteins and ligands mainly include hydrogen bonding, van der Waals forces, π-π interactions, electrostatic forces, etc., the greater these interaction forces are, the stronger the binding ability between the protein and the ligand is, and the greater the CE required to form free proteins and ligand small molecules through collision-induced dissociation (CID) in mass spectrometry.

[0004] Inspired by the methods of post-column derivatization and native MS, replacing the derivatization reagent with a protein solution for post-column injection may theoretically enable online screening and qualitative analysis of bioactive molecules, thereby achieving high-throughput protein ligand screening in complex systems. Based on this, the present invention constructs post-column protein injection liquid chromatography-energy-resolved affinity mass spectrometry (PCI-LC-ER-AMS) combined with "four-in-one" monitoring, which achieves rapid screening, structural identification, and affinity assessment of protein ligands in a mixture through 1) reduced response of potential ligands; 2) reduced response of proteins; 3) the appearance of ligand-protein complex ions; and 4) ligand-protein binding strength. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method and application for online screening of protein ligands and evaluation of their binding ability. The method described in the present invention can achieve rapid screening of protein ligands and rapid preliminary judgment of their binding ability.

[0006] Specifically, the present invention is realized through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for online screening of protein ligands and evaluating their binding ability, the method comprising the following steps:

[0008] Step 1: Screening potential protein ligands in complex systems by post-column protein injection combined with liquid chromatography-high-resolution mass spectrometry (LC-HRMS);

[0009] Step 2: Use high-resolution multiple reaction monitoring (MRM) HR ) Collect the mass spectrometry fragmentation curve of protein-ligand complex precursor ions → free protein ions, fit its optimal collision energy (OCE) parameters, and evaluate the binding ability between different ligands and proteins based on OCE.

[0010] As an optional manner, in the above method, in step 1, a post-column protein injection system is first constructed, the chromatographic column and the front end of the ion source are connected by a PEEK pipeline using a tee, and the remaining position is connected to an external injection pump using a PEEK pipeline.

[0011] As an optional manner, in the above method, in step one, during sample analysis, the potential protein ligand is separated by a chromatographic column of a liquid phase system, and at the same time, a syringe pump continuously injects the protein, and the liquid phase flow path and the syringe pump flow path merge at a tee and then flow into a mass spectrometry ion source through a PEEK pipeline for subsequent mass spectrometry acquisition.

[0012] As an optional manner, in the above method, in step 1, the flow rate of the protein injected by the syringe pump is 10 μL / min, and the length of the PEEK tubing is about 80 cm.

[0013] As an optional manner, in the above method, in step 1, the mobile phase, chromatographic column model, flow rate, column temperature, elution program and injection volume can be adjusted according to different samples to be tested.

[0014] As an optional manner, in the above method, in step 1, the mass spectrometry acquisition adopts ESI positive ionization mode and the data dependent acquisition (DDA) mode is used to acquire MS 1 and MS 2 data.

[0015] Preferably, during the mass spectrometry acquisition process, the parameters of the ion source are set as follows: positive ion mode acquisition; nebulizer, heater, curtain gas or collision gas are all N2; curtain gas (CUR): 30psi; nebulizer gas (GS1): 55psi; heater gas (GS2): 55psi; ion spray needle voltage: 5500V; heater gas temperature: 550℃; collision activated dissociation (CAD) gas: 9psi; declustering voltage (DP): 50V.

[0016] In addition, preferably, during the mass spectrum acquisition process, MS 1 and MS 2 The scanning ranges were m / z 100-2000 and m / z 50-2000, respectively; collision energy (CE): 30 eV; collision energy extension value (CES): ±15 eV. SCIEX OS 3.1 software (SCIEX, USA) was used for data acquisition and analysis.

[0017] As an optional manner, in the above method, in step one, the free protein and ligand extracted ion chromatograms (EIC) of single protein ligand, single post-column injected protein and potential protein ligand post-column injected protein samples are compared, and rapid screening of protein ligands can be achieved by detecting 1) a decrease in the response of the potential ligand; 2) a decrease in the response of the protein; and 3) the appearance of ligand-protein complex ions at the same retention time.

[0018] As an optional method, in the above method, in step 2, high resolution multiple reaction monitoring (MRM) is used HR ) mode, set a set of progressive CE values ​​(5-55 eV) for each ion pair, and the step was set to 2 eV. With the peak area as the ordinate and the progressive collision energy as the abscissa, Gaussian curve fitting was further performed through GraphPad Prism to obtain the optimal collision energy (OCE) value of the target ion pair.

[0019] As an optional method, in the above method, in step 2, the experimental OCE (E OCE ) value is converted to N according to the following formula OCE For comparison, N OCE The larger the value, the stronger the binding ability of the potential protein ligand:

[0020] N OCE =E OCE ×1000÷M Prec Formula 1

[0021] Among them, M Prec represents the m / z value of the precursor ion.

[0022] As an option, in the above method, the potential protein ligand is a small molecule sample.

[0023] Preferably, the small molecule samples include traditional Chinese medicine, natural medicine, single reference solution, mixed reference solution, etc.

[0024] In a second aspect, the present invention provides the application of the method described in the first aspect for rapid screening of protein ligands and rapid preliminary determination of protein ligand binding ability.

[0025] As an optional mode, in the above application, the protein ligand is a small molecule sample.

[0026] Preferably, the small molecule samples include traditional Chinese medicine, natural medicine, single reference solution, mixed reference solution, etc.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The method for rapid online screening of protein ligands by post-column protein injection provided by the present invention introduces an injection pump to inject protein after the chromatographic column to achieve protein-ligand binding and further detect it by mass spectrometry. Rapid screening of protein ligands can be achieved by identifying 1) a decrease in the response of potential ligands; 2) a decrease in the response of proteins; and 3) the appearance of ligand-protein complex ions at the same retention time.

[0029] (2) The present invention uses MRM HR The experimental OCE values ​​of protein-ligand complex ion pairs were obtained in scanning mode and converted into N OCE , and then compare N OCE The size of the value can be used to quickly determine the ligand-protein binding ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0031] In the attached picture:

[0032] Figure 1 :[M+H] of mixed reference substance (A), Cyt C (B), mixed reference substance injection combined with Cyt C post-column injection (C) sample 14 reference substances + and [M+nH] of free Cyt C n+ Typical extracted ion current (EIC) plots for (n=8, 9, 10, etc.).

[0033] Figure 2 : Representative MS images of SSA (A), free Cyt C (B), and SSA-Cyt C complex (C) at 11.15 min. 1 Spectrum; Typical EIC graph of ligand-Cyt C complex with 15+ charge (D).

[0034] Figure 3 (A) MS of Cyt C+SSA (m / z 876.94, z=15) 2 Spectrum; (B) m / z 876.94→824.97 ([Cyt C+ligand+nH]n+ →[Cyt C+nH] n+ ) Responses and Gaussian fitting curves under different CE.

[0035] Figure 4 : Mass spectrometric fragmentation curves of free proteins generated by protein-ligand complex precursor ions with charges of 19+ (A), 18+ (B), 17+ (C), 16+ (D), 15+ (E), 14+ (F), and 13+ (G) generated by 12 positive controls.

[0036] Figure 5 :12 known ligand-Cyt C complexes in different charge states [Cyt C+ligand+nH] n+ →[Cyt C+nH] n+ N OCE Distribution and violin plots. DETAILED DESCRIPTION

[0037] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0038] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0040] Example 1: Preparation of experimental materials and samples

[0041] 1.1 Experimental Materials

[0042] Reference substances catechin (Cat), epicatechin (EC), epigallocatechin gallate (EGCG), epigallocatechin gallate (GCG), belamcandarin (Tec), hesperidin (Hes), ginsenoside Re (Re), ginsenoside Rb2 (Rb2), ginsenoside Rb3 (Rb3), saikosaponin A (SSA), saikosaponin C (SSC), benzoylneaconitine (BMA), benzoylhypaconitine (BHA) were purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd. The purity of the above reference substances was greater than 98% as detected by high performance liquid chromatography (HPLC). Cytochrome C (Cyt C) was purchased from SigmaAldrich, USA. Mass spectrometry grade methanol (MeOH), acetonitrile (ACN), formic acid (FA), and ammonium acetate were purchased from Thermo Fisher, USA; dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, USA; and deionized water was laboratory-made Milli-Q ultrapure water (18.2 MΩ·cm).

[0043] Nexera series ultra-high performance liquid chromatograph (including binary high-pressure gradient pump, vacuum degasser, autosampler, column oven and controller, Shimadzu Corporation, Japan); ACQUITYUPLC HSS T3 chromatographic column (100 mm × 2.1 mm, 1.8 μm, Waters Corporation, USA); ZenoTOF 7600 mass spectrometer (Sciex Corporation, USA); Milli-Q ultrapure water purification system (Millipore Corporation, USA); ME204 electronic analytical balance (Mettler Toledo Corporation, Switzerland, with an accuracy of 0.1 mg).

[0044] 1.2 Preparation of reference samples

[0045] Accurately weigh an appropriate amount of the above reference substances and dissolve them in DMSO to prepare a molar concentration of 10 mmol / L -1 The reference substance stock solution was diluted 100 times with 20% methanol to prepare a mixed reference substance solution containing 14 reference substances. Weigh an appropriate amount of Cyt C and use 10 mmol·L -1 The ammonium acetate aqueous solution was prepared to 0.5 mmol·L -1 The protein stock solution was further diluted to 10 mmol·L -1 The ammonium acetate aqueous solution was diluted to 25 μmol·L -1 spare.

[0046] 1.3 Protein ligand screening and establishment of calculation method for protein-ligand complex stability

[0047] A post-column protein injection combined with online energy-resolved mass spectrometry was established for protein ligand screening. The protein CytC and small molecules Cat, EC, EGCG, GCG, Tec, Hes, Re, Rb2, Rb3, SSA, SSC, BMA and BHA were used as examples to confirm the effectiveness and advancement of the method described in the present invention.

[0048] 1.3.1 Establishment of online screening method for protein ligands

[0049] Chromatographic column: AcquityUPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters, USA); mobile phase: 0.1% (v / v) formic acid water (A) and acetonitrile (B); elution program: 0-3 min, 14% B; 3-5 min, 14-38% B; 5-9 min, 38% B; 9-10 min, 38-95% B; 10-10.1 min, 95-14% B; 10.1-15 min; 14% B; flow rate: 0.22 mL / min; column oven: 40 °C; injection volume: 2 μL. In addition, a syringe pump was introduced into the rear end of the chromatographic column through a three-way connection and the flow rate was 10 μL min -1 The CytC solution was continuously injected at a flow rate of 1.5 %. The ligand and protein analyte in the LC column post-flow path and the injection pump flow path were combined in a PEEK pipeline of about 80 cm in length through a three-way connection, so that they were fully combined in the pipeline and then entered the Zeno TOF 7660 mass spectrometer for analysis.

[0050] The mass spectrometry parameters were set as follows: positive ion mode acquisition; nebulizer, heater, curtain gas or collision gas were all N2; curtain gas (CUR): 30psi; nebulizer gas (GS1): 55psi; heater gas (GS2): 55psi; ion spray needle voltage: 5500V; heater gas temperature: 550℃; collision activated dissociation (CAD) gas: 9psi; declustering voltage (DP): 50V. MS 1 and MS 2 The scanning ranges were m / z 100-2000 and m / z 50-2000, respectively; collision energy (CE): 30 eV; collision energy extension value (CES): ±15 eV. SCIEX OS 3.1 software (SCIEX, USA) was used for data acquisition and analysis. 1.3.2 Establishment of online energy-resolved mass spectrometry (online ER-MS)

[0051] MS of protein-ligand complexes obtained based on the above protein ligand online screening 1 and MS 2 The target ion pairs were constructed and further analyzed by high-resolution multiple reaction monitoring (MRM HR) mode to optimize the parameters of all target ion pairs. R =11.15min) as an example, the complex after binding with Cyt C was detected in MS 1 The mass spectrum shows multiple multi-charged mass spectrometry signals. The complex signals formed by Cyt C binding to one molecule of SSA are mainly m / z 692.68 (z=19), 730.99 (z=18), 773.94 (z=17), 822.24 (z=16), 876.93 (z=15), 939.56 (z=14), 1011.76 (z=13), 1095.99 (z=12), 1195.34 (z=11), 1314.89 (z=10), and 1460.75 (z=9). The parent ions of each complex [CytC+ligand+nH] n+ (i.e., the signal of a complex with n charges) The signal generated by the MS 2 The fragments mainly include [Cyt C+(n+1)H] n+1 (i.e. protein signal with n+1 charges), [Cyt C+nH] n+ (i.e., protein signal with n charges) and the [M+H] of the ligand + To simplify the analysis process, the present invention uses [Cyt C+ligand+nH] n+ →[Cyt C+nH] n+ Construct ion pairs and schedule MRM based on compound retention time HR Method (Table 1). Taking the ion pair m / z 876.93→824.97 as an example, in MRM HR In this mode, the precursor ion was set to m / z 876.93, the product ion scanning range was m / z 750-900, the accumulation time was 5 ms, the retention time and retention time extension range were 11.15±0.5 min, and a total of 26 MRMs were set. HR Events, corresponding to CE values ​​of 5, 7, 9…55 eV, were collected. The chromatographic peak of m / z876.93→824.97 was extracted and the peak area was integrated using SCIEX OS software. The CE and its corresponding peak area data matrix were imported into GraphPadPrism 9.0, and a fitting curve of CE and peak area was established, which was the fragmentation curve, and the optimal collision energy (OCE) was calculated.

[0052] Example 2: Experimental results

[0053] 2.1 Online screening of Cyt C ligands based on post-column protein injection

[0054] Cytochrome C (Cyt C) is a monohemoglobin globular protein in the mitochondria of all aerobic organisms, and plays a vital role in maintaining and ending life processes through respiration and apoptotic activities. Many studies have revealed that small molecules from Chinese herbal medicines have a strong binding affinity with Cyt C, causing it to be reduced. Many natural products such as flavonoids, saponins and oligosaccharides have been shown to be ligands of Cyt C. In order to verify the applicability of the present invention for online capture of potential ligands, 12 verified Cyt C ligands (including Cat, EC, EGCG, GCG, Tec, Hes, Re, Rb2, Rb3, SSA and SSC) and 2 negative control compounds (BMA and BHA) were used in the model sample solution of the present invention. As Figure 1 As shown in A, all compounds in the single mixed standard injection are present in the EIC, while all ion species belonging to Cyt C are absent. Accordingly, when the small molecule is absent, the ion source continues to receive a constant amount of Cyt C, so each of its quasi-molecular ions (i.e., [M+nH] n+ , n = 8, 9, 10, etc.) have relatively stable chromatograms, while those quasi-molecular ions belonging to small molecules do not exist ( Figure 1 B) After adding Cyt C after the LC column, the EIC graph obtained is ( Figure 1 C) shows the appearance of all relevant small molecule quasi-molecular ion peaks. By comparison Figure 1 C and Figure 1 The response intensity of the 12 known ligands for the small molecule quasi-molecular ion peaks in A was significantly reduced, but this reduction was not observed at the corresponding retention times of BMA and BHA. Figure 1 C and Figure 1 In the EIC diagrams belonging to Cyt C between B, only a few negative signals exist Figure 1 C, indicating that these Cyt C quasi-molecular ions have been significantly reduced. Specifically, these negative signals have the same retention time as 12 known ligands. Therefore, it can be inferred that binding occurs between the known ligands and Cyt C, and other small molecules except BMA and BHA can bind to Cyt C.

[0055] In addition to these indirect evidences, clear clues that the ligand-protein complex corresponds to the MS / MS mass spectra at the same retention time in the three chromatograms can be found. 1 In the mass spectrum, the small molecule quasi-molecular ion peaks dominate, while Figure 1 Each MS extracted from B 1 There is a group of quasi-molecular ion peaks with different charge states (n=8, 9, 10, etc.) distributed in the mass spectrum. Figure 1 MS extracted from C 1In the mass spectrum, in addition to the Figure 1 A and Figure 1 In addition to the mass spectrometry signal in B, some additional ions with a larger mass than the multi-charged quasi-molecular ion peak of Cyt C were observed for all 12 ligands. However, no additional ions were observed for the two negative control compounds. Taking SSA (11.15min) as an example, the binding form and mass spectrometry characteristics of small molecule ligands and Cyt C are explained. [M+H] + m / z781.4655( Figure 2 A), so the binding of SSA to Cyt C can lead to an increase in the mass of the complex by about 780Da. MS of free Cyt C 1 Spectrum Figure 2 As shown in B, its m / z value is between 589.50 and 1766.90, the charge number z is +7 to +21, and its molecular weight after deconvolution is 12358.4078Da. Figure 2 C can be seen that at exactly the same retention time, its MS 1 More ion types appeared in the spectrum than in the SSA and CytC control. After removing the ions associated with SSA and Cyt C, some signals remained and were characterized by calculating the elemental composition as a complex of multiply charged protonated Cyt C and several neutral SSA molecules. Figure 2 As shown in C, there are several signals in the right region of m / z 727.9752, which are annotated as [Cyt C+SSA+17H] 17+ , [Cyt C+2×SSA+17H] 17+ and [Cyt C+3×SSA+17H] 17+ Thus, 12 positive peaks with significantly different responses were successfully captured at the m / z value of the ligand-protein complex, and their retention time was the same as that of the ligand. 15+ ) as an example, the [Cyt C+ligand+15H] 15+ XIC Figure 2 As shown in Figure D, at the corresponding retention time of each compound, the complexes all have obvious signals, but the responses of different complexes are different. The above results all indicate that after the protein is injected after the column, each known ligand binds to Cyt C and forms a protein-ligand complex.

[0056] 2.2 Comparison of the binding ability of Cyt C with various ligands based on online ER-MS

[0057] The formation of complexes between proteins and small molecule ligands mainly depends on hydrogen bonding, electrostatic forces, van der Waals forces, π-π interaction forces, etc. Due to the differences in the structures of the compounds, there are differences in their interaction forces with proteins, which ultimately manifests as differences in the binding forces between the compounds and proteins. Collision-induced dissociation (CID) can achieve covalent or non-covalent bond cleavage, and the binding strength can be positively correlated with the optimal collision energy (OCE) obtained by energy-resolved mass spectrometry (ER-MS). In addition, the gas-phase ligand-protein complex ions are extremely close in the physiological state, so online energy-resolved mass spectrometry (online ER-MS) is used to evaluate the binding strength between each ligand and Cyt C, and MRM is used. HR to obtain the responses of the transitions of interest at increasing CE levels. Regardless of charge state, all MS 1 The signals, especially the abundant signals, belonging to the complex composed of Cyt C and a single ligand molecule, will enter the collision cell individually to generate MS under fixed CE. 2 Spectra. As expected, signals corresponding to the ligand and Cyt C were generated from each complex by CID. The 15+ ) as an example, in MS 2 A group of major fragment ions ( Figure 3 A), for example, m / z 883.76 ([Cyt C+14H] 14+ ) and 824.90([CytC+15H] 15+ ) corresponds to Cyt C, and m / z 781.47, 763.47, 745.46, 455.36, 437.35, and 419.33 belong to SSA. HR The program measured the ion pair m / z 876.94→824.97 to obtain a Gaussian-shaped mass spectral fragmentation curve in response to ligand fragmentation. Figure 3 As shown in B, according to the regression equation Y = 526621 × exp (-0.5 × ((X-24.00) / 7.617) 2 ), the OCE generated when Cyt C and SSA bind and dissociate is calculated to be 24.00 eV. When focusing on all the protonated molecular signals of the SSA-Cyt C complex ( Figure 2 C), by online ER-MS analysis of [Cyt C+SSA+nH] n+ →[Cyt C+nH] n+ The experiments of ion pairs (n=13-19) obtained different OCE values ​​and observed this phenomenon in different charge states ( Figure 4). Exactly the same phenomenon was observed for other ligands. In addition, after considering all protonated molecular signals of each ligand, a large amount of OCE information was obtained ( Figure 4 ), which poses a great challenge to comparing binding strength levels between different ligands. A careful comparison of OCE values ​​for different charge states revealed that OCE has a linear positive correlation with the mass of the precursor ion, but less correlation with the product ion. Therefore, by converting the experimental OCE (E OCE ) and the precursor ion mass normalized to N OCE , can have the opportunity to reflect the binding strength of the ligand. The conversion formula is as follows:

[0058] N OCE =E OCE ×1000÷M Prec Formula 1

[0059] N of the 12 known ligands OCE The values ​​are shown in Table 2 and Figure 5 As shown. According to N OCE The 12 compounds can be divided into three grades (Grade I-III) based on the values ​​of β-hydroxyl groups. Grade I includes Rb2, Rb3, Re, SSA, and SSC, which show stronger binding ability. Grade III contains Daiz, Cat, and EC, which have weaker binding ability to Cyt C. Other compounds including Hes, Tec, EGCG, and GCG belong to Grade II, which show moderate binding ability. It is worth noting that all compounds in Grade I are saponins, and their greater binding potential is attributed to the formation of more hydrogen bonds between saponin sugar residues and Cyt C functional groups (such as free carboxyl and amino groups). Among Grade II compounds, Hes and Tec are flavonoid glycosides, while EGCG and GCG are composed of flavonoids and polyphenols. Their ability to form hydrogen bonds with Cyt C is weaker than that of saponins, but due to the presence of multiple benzene rings and p-π conjugated systems in their structures, they can interact with aromatic amino acid residues (such as phenylalanine, tryptophan, and tyrosine) in Cyt C under π-π stacking interactions and improve the binding strength. Daiz, Cate, and EC showed the weakest binding ability with Cyt C due to the limited number of hydroxyl groups or benzene rings in their structures that can form hydrogen bonds or π-π interactions.

[0060]

[0061]

[0062] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for online screening of protein ligands and evaluating their binding ability, characterized in that: The method comprises the following steps: Step 1: Screening potential protein ligands in complex systems by post-column protein injection combined with liquid chromatography-high resolution mass spectrometry (LC-HRMS); Step 2: Use high-resolution multiple reaction monitoring (MRM) HR ) Collect the mass spectrometry fragmentation curve of protein-ligand complex precursor ions → free protein ions, fit its optimal collision energy (OCE) parameters, and evaluate the binding ability between different ligands and proteins based on OCE.

2. The method according to claim 1, characterized in that: In step 1, a post-column protein injection system is first constructed, and the chromatographic column and the front end of the ion source are connected with a PEEK pipeline using a tee, and the remaining position is connected to an external injection pump using a PEEK pipeline.

3. The method according to claim 1, characterized in that: In step one, during sample analysis, the potential protein ligand is separated through a chromatographic column of a liquid phase system, while a syringe pump continuously injects protein. The liquid phase flow path and the syringe pump flow path merge at a tee and then flow into a mass spectrometry ion source through a PEEK pipeline for subsequent mass spectrometry acquisition.

4. The method according to claim 1, characterized in that: In step 1, the mass spectrometry was acquired in ESI positive ionization mode and data dependent acquisition (DDA) mode was used to acquire MS 1 and MS 2 data.

5. The method according to claim 4, characterized in that: During the mass spectrometry acquisition process, the parameters of the ion source were set as follows: positive ion mode acquisition; nebulizer, heater, curtain gas or collision gas were all N2; curtain gas (CUR): 30 psi; nebulizer gas (GS1): 55 psi; heater gas (GS2): 55 psi; ion spray needle voltage: 5500 V; heater gas temperature: 550 ° C; collision activated dissociation (CAD) gas: 9 psi; declustering voltage (DP): 50 V, MS 1 and MS 2 The scanning ranges were m / z 100–2000 and m / z 50–2000, respectively; collision energy (CE): 30 eV; Collision energy spread (CES): ±15 eV, and SCIEXOS 3.1 software was used for data acquisition and analysis.

6. The method according to claim 1, characterized in that: In step 1, by comparing the TIC graphs of the protein injected after column injection alone, the potential protein ligand injected after column injection alone, and the potential protein ligand injected after column injection, the position of the inverted peak in the TIC graph of the potential protein ligand injected after column injection of the protein is the position of the potential protein ligand, and the MS at the inverted peak is 1 Multiply charged signals of the protein-ligand complex can be observed in the spectrum.

7. The method according to claim 1, characterized in that: In step 2, high-resolution multiple reaction monitoring (MRM) HR ) mode, a set of progressive CE values ​​was set for each group of ion pairs, the range of the CE values ​​was 5-55 eV, the step was set to 2 eV, the peak area was used as the ordinate, and the progressive collision energy was used as the abscissa. Gaussian curve fitting was further performed using GraphPad Prism to obtain the optimal collision energy (OCE) value of the target ion pair.

8. The method according to claim 1, characterized in that: The potential protein ligand is a small molecule sample. Preferably, the small molecule sample includes traditional Chinese medicine, natural medicine, single reference solution, mixed reference solution, etc.

9. Use of the method according to any one of claims 1 to 8 in rapid screening of protein ligands and rapid preliminary determination of protein ligand binding ability.

10. The use according to claim 9, characterized in that: The protein ligand is a small molecule sample. Preferably, the small molecule sample includes traditional Chinese medicine, natural medicine, single reference solution, mixed reference solution, etc.

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