Isoprene-like pyrophosphate photoaffinity probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological detection and analysis, and particularly relates to an isoprenyl pyrophosphate photoaffinity probe as well as a preparation method and application thereof. Specifically, on the basis that the structure of isoprenyl pyrophosphate is connected with small photoaffinity side chain modification, a novel small-molecule active probe for detecting the isoprenyl pyrophosphate binding protein is developed; the probe can be used for effectively marking the isoprenoid pyrophosphate binding protein in a cell lysis solution for fluorescent gel imaging analysis and high-flux proteomics analysis, and the isoprenoid interaction protein is identified by combining quantitative proteomics analysis; the probe can also be used for marking living cells and corresponding fluorescence imaging analysis and protein spectrum analysis, so that the probe has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection and analysis, and particularly relates to an isoprenoid pyrophosphate photoaffinity probe and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Isoprenoid pyrophosphates are key intermediates in the biosynthesis of important molecules such as terpenes, steroids, squalene, and coenzyme Q. Isoprenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP), produced by the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) pathway, serve as building blocks for these biosyntheses. IPP reacts with DMAPP to form geranyl pyrophosphate (GPP), which in turn reacts with IPP to form farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP).

[0004] Dysregulation of isoprenoid synthesis or protein prenylation is associated with a variety of diseases, including cancer, inflammatory diseases, infectious diseases, and Alzheimer's disease. Statins are inhibitors of HMG-CoA reductase, an enzyme upstream of isoprenoid synthesis, and are widely used as lipid-lowering drugs due to their inhibition of cholesterol biosynthesis. In addition to their effects on plasma cholesterol levels, statins have been reported to have a variety of effects on various biological processes, such as cell proliferation and apoptosis, angiogenesis, inflammatory responses, and immune regulation. These functions may be related to their inhibition of isoprenoid synthesis, but the specific mechanisms are still unclear. In addition to the extensive research on the functions of isoprenoids in biosynthesis and protein prenylation modification, new functions of isoprenoids are constantly being reported.

[0005] To uncover new biological functions of isoprenoids, an increasing number of isoprenoid probes have been reported. These probes typically incorporate a reporter group at the end of the isoprenyl group of isoprenoid pyrophosphate or isoprenol, allowing for labeling, enrichment, and analysis of prenylated proteins. Early studies used probes containing biotin or fluorophores to analyze prenylated proteins. However, these groups are large and may interfere with the binding between the probe and the prenyltransferase, necessitating structural modification of the prenyltransferase active pocket to accommodate the probe. By introducing smaller reporter groups, such as azide and alkyne, onto the isoprenyl group, combined with click chemistry, better analysis of prenylated proteins is possible. These probes are very useful for analyzing prenylated proteins, but cannot be used to analyze proteins that interact with isoprenoid pyrophosphate. To investigate proteins that interact with isoprenoids or prenylated proteins, several probes with photoreactive groups have been reported. Zhao et al. developed a photoaffinity probe in which a photoreactive benzophenone group is linked to the isoprenyl end of GPP for covalent binding to target proteins, and a biotin group is linked to the pyrophosphate end for protein enrichment. Using this probe, they identified 30 proteins with diverse activities and functions from the Saccharomyces cerevisiae proteome. Distefano et al. reported another photoaffinity probe in which a benzophenone group is linked to the pyrophosphate end of FPP. This probe can be used to analyze FPP-interacting proteins, such as farnesyltransferase and farnesyl diphosphate synthase, but it lacks a group for protein enrichment.

[0006] The inventors previously developed a desthiobiotin-GPP acyl phosphate probe that, upon binding, transfers desthiobiotin to lysine residues near the probe-binding site of the target protein. Combined with quantitative proteomics analysis based on stable isotope labeling in cell culture (SILAC), this probe identified several known and potential isoprenoid pyrophosphate-interacting proteins. While this probe is highly efficient at labeling target proteins, its high reactivity with nucleophiles results in poor stability, hindering its practical application. Summary of the Invention

[0007] In response to the deficiencies of the above-mentioned prior art, the present invention provides an isoprenoid pyrophosphate photoaffinity probe, its preparation method, and application. Specifically, based on the structural modification of isoprenoid pyrophosphate with a smaller photoaffinity side chain, the present invention has developed a novel small molecule active probe for detecting isoprenoid pyrophosphate-binding proteins. This probe can effectively label isoprenoid pyrophosphate-binding proteins in cell lysates for fluorescent gel imaging analysis and high-throughput proteomics analysis, combined with quantitative proteomics analysis to identify isoprenoid-interacting proteins. This type of probe can also be used for labeling living cells, and for corresponding fluorescent imaging analysis and protein spectrum analysis. Based on the above research results, the present invention was completed.

[0008] Specifically, the present invention relates to the following technical solutions:

[0009] The first aspect of the present invention provides an isoprenoid pyrophosphate photoaffinity probe, wherein the photoaffinity probe has the following structure:

[0010]

[0011] The present invention designs the aforementioned photoaffinity probe, which contains a GPP / FPP / GGPP binding group, a photoreactive diazirine group, and a bioorthogonal alkynyl group. The photoreactive group can form a carbene under ultraviolet irradiation, which inserts into the NH, OH, and C-H bonds of amino acids to form a covalent bond. The alkynyl group can undergo click chemistry with azide compounds to link to a fluorescent group or biotin group for use in fluorescence imaging or affinity enrichment. This photoaffinity probe has the ability to covalently capture GPP / FPP / GGPP-binding proteins for subsequent analysis.

[0012] The second aspect of the present invention provides a method for preparing the above-mentioned photoaffinity probe, which comprises the following synthetic route:

[0013]

[0014] The third aspect of the present invention provides a kit, which at least comprises the above-mentioned photoaffinity probe.

[0015] A fourth aspect of the present invention provides use of the above-mentioned photoaffinity probe or kit in research related to isoprenoid pyrophosphate binding proteins.

[0016] Beneficial technical effects of one or more of the above technical solutions:

[0017] The above technical solution is based on the structural connection of GPP / FPP / GGPP with a small photoaffinity side chain modification, developing a new small molecule active probe for detecting GPP / FPP / GGPP-binding proteins. This probe has the following three main advantages: First, the use of a minimized linker group reduces the impact of the modification on GPP / FPP / GGPP-protein binding, better simulating the binding of GPP / FPP / GGPP to proteins; second, the probe contains an alkyne group for bioorthogonal reactions, which can undergo click chemistry with azide compounds, link to the fluorescent group rhodamine for fluorescence gel imaging, or link to a biotin group for affinity enrichment and mass spectrometry detection; third, the probe can be used for labeling and analysis of living cells.

[0018] In summary, the above fluorescent probes can effectively label GPP / FPP / GGPP-binding proteins in cell lysates and living cells for fluorescent gel analysis, high-throughput proteomics analysis, and fluorescence imaging analysis, thus having great practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 : The structures of the probes of the present invention; (a) is a GPP probe; (b) is an FPP probe; (c) is a GGPP probe; and (d) is an FPP control probe.

[0021] Figure 2 The design of the probe of the present invention; (a) indicates that the GPP / FPP / GGPP probes all contain an isoprenoid chain (binding group), a diazirine (photoreactive group) and an alkynyl group (bioorthogonal group); (b) indicates that the alkynyl group and the azide compound undergo a click chemistry reaction to connect a rhodamine fluorescent group or a biotin group; (c) indicates that based on a photocross-linking reaction, the photoreactive group can form a carbene under ultraviolet irradiation conditions and insert between the NH bonds, OH bonds, and CH bonds of amino acids.

[0022] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the GPP photoaffinity probe of the present invention.

[0023] Figure 4 This is the carbon nuclear magnetic resonance spectrum of the GPP photoaffinity probe of the present invention.

[0024] Figure 5 This is the nuclear magnetic resonance phosphorus spectrum of the GPP photoaffinity probe of the present invention.

[0025] Figure 6 This is the secondary mass spectrometry of the GPP photoaffinity probe of the present invention in negative ion mode.

[0026] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the FPP photoaffinity probe of the present invention.

[0027] Figure 8 This is the carbon nuclear magnetic resonance spectrum of the FPP photoaffinity probe of the present invention.

[0028] Figure 9 This is the nuclear magnetic resonance phosphorus spectrum of the FPP photoaffinity probe of the present invention.

[0029] Figure 10 This is the secondary mass spectrometry of the FPP photoaffinity probe of the present invention in negative ion mode.

[0030] Figure 11 This is the hydrogen nuclear magnetic resonance spectrum of the FPP control probe of the present invention.

[0031] Figure 12 This is the carbon NMR spectrum of the FPP control probe of the present invention.

[0032] Figure 13 This is the nuclear magnetic resonance phosphorus spectrum of the FPP control probe of the present invention.

[0033] Figure 14 This is the secondary mass spectrum of the FPP control probe of the present invention in negative ion mode.

[0034] Figure 15 This is the hydrogen nuclear magnetic resonance spectrum of the GGPP photoaffinity probe of the present invention.

[0035] Figure 16 This is the carbon nuclear magnetic resonance spectrum of the GGPP photoaffinity probe of the present invention.

[0036] Figure 17 This is the nuclear magnetic resonance phosphorus spectrum of the GGPP photoaffinity probe of the present invention.

[0037] Figure 18 This is the secondary mass spectrometry of the GGPP photoaffinity probe of the present invention in negative ion mode.

[0038] Figure 19 This is a high-resolution mass spectrum of the GPP / FPP / GGPP probe of the present invention in negative ion mode.

[0039] Figure 20 The effects of different labeling conditions on the labeling effect of the GPP / FPP / GGPP probe of the present invention are shown; (a) Comparison of the labeling effect of a 10 μM FPP photoaffinity probe and a 10 μM FPP control probe under the conditions of UV irradiation and without NAP-5 or protein precipitation before the click chemistry reaction; (b) Effects of the labeling effect of a 10 μM FPP photoaffinity probe under the conditions of adding different types and concentrations of divalent metal salts.

[0040] Figure 21 The figure shows the effect of different labeling conditions on the labeling effect of the probe in the present invention; (a) labeling effect of GPP probe with increasing concentration; (b) labeling effect of FPP probe with increasing concentration; (c) labeling effect of GGPP probe with increasing concentration; (d) competitive effect on GPP probe labeling under the condition of increasing GPP concentration; (e) competitive effect on FPP probe labeling under the condition of increasing FPP concentration; (f) competitive effect on GGPP probe labeling under the condition of increasing GGPP concentration; except for the conditions marked in the figure, the competitor incubation time is 10 minutes, the probe incubation time is 10 minutes, the UV irradiation time is 30 minutes, and the click chemistry reaction time is 2 hours.

[0041] Figure 22 This is a consistency analysis between the forward and reverse SILAC mass spectrometry samples of proteins labeled with the FPP / GGPP probe of the present invention; (a) Fluorescent gel analysis of HEK293T light lysate (L) or heavy lysate (H) labeled with 10 μM FPP probe under 1 mM FPP conditions; (b) Fluorescent gel analysis of HEK293T light lysate (L) or heavy lysate (H) labeled with 10 μM GGPP probe under 1 mM GGPP conditions; (c) Comparison of 10 μM FPP probe forward labeling and reverse labeling; (d) Comparison of 10 μM GGPP probe forward labeling and reverse labeling (forward ratio = light / heavy, reverse ratio = heavy / light, ratios are the average of 3 biological replicates).

[0042] Figure 23 Figure 3. Mass spectrometry sample analysis of proteins labeled with the SILAC-based FPP / GGPP probe of the present invention; (a) Volcano plot showing proteins identified by the 10 μM FPP probe; (b) Volcano plot showing proteins identified by the 10 μM GGPP probe; (c) Correlation analysis of total proteins identified by the FPP and GGPP probes; the data shown are proteins commonly identified in three sets of forward and reverse experiments.

[0043] Figure 24 Comparison of the protein labeling results of the FPP probe and the GGPP probe of the present invention; (a) Venn diagram showing the comparison of proteins identified by the FPP probe and the GGPP probe under the screening conditions of ratio>2 and p<0.05; (b) Venn diagram showing the subcellular localization of 86 proteins jointly identified by the FPP probe and the GGPP probe; (c) Comparison of the subcellular localization and biological processes of proteins identified by the FPP probe and the GGPP probe under the screening conditions of ratio>2 and p<0.05.

[0044] Figure 25Fluorescence imaging of Hela cells labeled with (a) FPP probe and (b) GGPP probe of the present invention at different concentrations for 12 hours; scale bar: 20 μm.

[0045] Figure 26 Fluorescence imaging of Hela cells labeled with 100 μM (a) FPP probe and (b) GGPP probe of the present invention at different times; scale bar is 20 μm.

[0046] Figure 27 This is the subcellular localization analysis of Hela cells labeled with 100 μM FPP probe of the present invention for 2 hours; the scale bar is 10 μm.

[0047] Figure 28 This is the subcellular localization analysis of Hela cells labeled with 100 μM GGPP probe of the present invention for 2 hours; the scale bar is 10 μm. DETAILED DESCRIPTION

[0048] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0049] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are generally in accordance with conventional methods and conditions within the art, and such techniques and conditions are fully explained in the literature.

[0050] The present invention will be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the sales company. Materials and reagents used in the examples are commercially available unless otherwise specified.

[0051] As mentioned above, the currently reported isoprenoid pyrophosphate probes generally have problems such as poor detection sensitivity, poor stability, or lack of groups for protein enrichment, and cannot be used for the analysis of isoprenoid pyrophosphate-interacting proteins in living cells.

[0052] In light of this, the present invention provides a series of novel isoprenoid pyrophosphate photoaffinity probes. These probes introduce minimal photoreactive groups to reduce the effects of modification on probe function. These probes also contain a bioorthogonal alkyne group, enabling the introduction of a fluorescent group or biotin via click chemistry, for fluorescence analysis of labeled proteins and enrichment and proteomic analysis, respectively. These probes can be used for fluorescence gel imaging of labeled proteins and for localization analysis of labeled proteins in living cells. Combined with quantitative proteomic analysis, they can be used to analyze isoprenoid pyrophosphate-interacting proteins and proteins associated with prenylation.

[0053] Specifically, in a typical embodiment of the present invention, an isoprenoid pyrophosphate photoaffinity probe is provided, wherein the photoaffinity probe has the following structure:

[0054]

[0055] The present invention designs the above-mentioned photoaffinity probe, which contains a binding group GPP / FPP / GGPP, a photoreactive group diazirine and a bioorthogonal group alkynyl. Among them, the photoreactive group can form a carbene under ultraviolet irradiation conditions, which is inserted between the NH bonds, OH bonds, and CH bonds of amino acids to form a covalent connection. The alkynyl group can undergo click chemistry reactions with azide compounds to connect fluorescent groups (such as rhodamine) or biotin groups (such as biotin-DADPS-azide, CAS: 1260247-50-4) for fluorescence imaging or affinity enrichment, etc. The present invention has verified through experiments that the above-mentioned photoaffinity probe has the ability to covalently capture GPP / FPP / GGPP binding proteins for subsequent analysis.

[0056] In another embodiment of the present invention, a method for preparing the above-mentioned photoaffinity probe is provided, and the preparation method includes the following synthetic route:

[0057]

[0058] In another embodiment of the present invention, a kit is provided, comprising at least the above-mentioned photoaffinity probe. The kit may also comprise any other buffer, reaction reagent, etc., which are not specifically limited herein.

[0059] In another embodiment of the present invention, there is provided the use of the above-mentioned photoaffinity probe or kit in research related to isoprenoid pyrophosphate binding proteins.

[0060] The application at least includes:

[0061] (a) Fluorescence gel imaging analysis of isoprenoid pyrophosphate binding proteins in cell lysates;

[0062] (b) Protein profiling sample analysis of isoprenoid pyrophosphate binding proteins in cell lysates;

[0063] (c) Immunofluorescence imaging and subcellular localization analysis of isoprenoid pyrophosphate binding protein in cells.

[0064] Wherein, in (a)-(b), the cells may be HEK293T cells;

[0065] In the above-mentioned (c), the cells may be Hela cells.

[0066] The isoprenoid pyrophosphate binding protein may be any one or more of GPP / FPP / GGPP binding proteins.

[0067] In another specific embodiment of the present invention, the specific method in the application (b) includes: adding the photoaffinity probe to a SILAC cell lysate (HEK293T), pre-incubating and UV irradiating, and then performing a click chemistry reaction with biotin-DADPS-azide; specifically, using LC-MS / MS to detect the protein labeled with the probe, using MaxQuant (v2.1.4.0) software to attribute the obtained mass spectrometry data, and using the DAVID website to perform GO analysis.

[0068] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0069] Example 1 Design and Synthesis of Novel Isoprenoid Pyrophosphate Photoaffinity Probes

[0070] 1. Materials and Methods

[0071] 1.1 Experimental Reagents

[0072] Methanol and acetonitrile (Opsun), tetrahydrofuran (THF, Tissin), N,N-dimethylformamide (DMF, Adamas), phosphorus tribromide (PBr3, Adamas), tetrabutylammonium hydroxide (Adamas), cation exchange resin Dowex50WX4100-200(H) (Alfa Aesar), disodium dihydrogen pyrophosphate (Aladdin), geraniol (McLean), farnesol (Bidel Pharmaceuticals), geranylgeraniol (Annage), 1-ethyl-(3-dimethylaminopropyl), carbodiimide hydrochloride (EDC, Bidel Pharmaceuticals), 3-aminoethyl-3-(but-3-ynyl)diaziridine (Bidel Pharmaceuticals), 5-hexyn-1-amine, ammonium bicarbonate (Bidel Pharmaceuticals), sodium chloride (Titan), sodium bicarbonate (Bidel Pharmaceuticals), petroleum ether (Fuyu).

[0073] 1.2 Experimental instruments

[0074] Constant temperature magnetic stirrer (Aika), centrifuge (Eppendorf), mass spectrometer (LTQ-Orbitrap Velos Pro, Thermo Fisher), freeze dryer (Beijing Sihuan Freeze Drying), Nanodrop (Thermo Fisher), Ultimate AQ-C18 preparative column (Yuexu, 5 μm, 10×250 mm), preparative liquid phase system (Aijer), Flash column (Welflash AQ-C18, 20-40 μm, 40 g / 25 g / 4 g), CHEETAH Pro purification system (Aijer), constant temperature metal bath (Hangzhou Aosheng), nuclear magnetic resonance spectrometer (Bruker), pH meter (Sartorius).

[0075] 1.3 Experimental steps

[0076] 1.3.1 Synthesis and purification of isoprenoid pyrophosphate

[0077]

[0078] Synthesis of geranyl pyrophosphate (GPP): Under light-shielding conditions, 1a (1.5 g, 10 mmol, 1 equivalent) was dissolved in 30 mL of anhydrous tetrahydrofuran, cooled to 0°C, and phosphorus tribromide (1.35 g, 5 mmol, 0.5 equivalent) was slowly added; the reaction mixture was stirred at 0°C for 1 hour, quenched by adding pre-cooled saturated sodium bicarbonate solution, and extracted with 30 mL of petroleum ether. The aqueous layer was extracted once with petroleum ether, and the combined organic phases were washed twice with appropriate amounts of saturated sodium bicarbonate and brine, dried over Na2SO4, filtered, and spin-dried. Using disodium pyrophosphate as the raw material, Tetrabutylammonium pyrophosphate (NBu4)3HP2O7 was prepared by cation exchange with 50WX4200-400(H) resin. The crude product 2a and (NBu4)3HP2O7 (8.30 g, 9.2 mmol, 1.1 equivalents) were dissolved in 8 mL of dry N,N-dimethylformamide (DMF). Under nitrogen, the (NBu4)3HP2O7 solution was slowly added to the crude product solution. The reaction was allowed to proceed at room temperature for 24 hours, and then dried. The crude product was dissolved in water and purified by Welflash AQ-C18 column (40 g) with gradient elution conditions: 0-30 min, 0% B; 30-100 min, 0-55% B; 100-120 min, 55-55% B; 120-130 min, 55-70% B; 130-150 min, 70-70% B; 150-160 min, 70-85% B; 160-170 min, 85-85% B; 170-175 min, 85-100% B; 1 The gradient was 100-100% B from 75 to 185 minutes; 100-80% B from 185 to 186 minutes; and 80-80% B from 186 to 196 minutes (this gradient represents the purification gradient for the GPP / FPP / GGPP mixture). The GPP peak (approximately 28% acetonitrile) elutes at a retention time of approximately 100 minutes (55% B). Mobile phase A consisted of 25 mM aqueous ammonium bicarbonate, and mobile phase B consisted of 50% acetonitrile in 25 mM ammonium bicarbonate. The flow rate was 10 mL / min, and the detection wavelength was 214 nm. The GPP-containing eluates were combined and lyophilized.

[0079] Synthesis of Farnesyl Pyrophosphate (FPP): FPP was synthesized using the same method as GPP. PBr (1.35 g, 5 mmol, 0.5 equiv) was reacted with farnesol (1b, 2.22 g, 10 mmol, 1 equiv). The crude product 2b was dissolved in 8 mL of DMF and reacted with a solution of (NBu)3HP2O7 (9.95 g, 11 mmol, 1.5 equiv) in 8 mL of DMF. Purification was performed using the same procedure as for GPP. FPP elutes at a retention time of approximately 130 minutes (70% B) (approximately 35% acetonitrile).

[0080] Synthesis of Geranylgeranyl Pyrophosphate (GGPP): GGPP was synthesized using the same method as GPP. Geraniol 1c (1 g, 3.4 mmol, 1 equiv) was reacted with PBr (0.46 g, 1.7 mmol, 0.5 equiv). Crude 2c was dissolved in 4 mL of DMF and reacted with a solution of (NBu)3HP2O7 (3.17 g, 3.5 mmol, 1.1 equiv) in 4 mL of DMF. Purification was performed using the same procedure as for GPP. GGPP elutes at a retention time of approximately 155 minutes (85% B) (approximately 42% acetonitrile).

[0081] 1.3.2 Synthesis and purification of isoprenoid pyrophosphate probes

[0082]

[0083] Synthesis of the GPP photoaffinity probe (GPP-P): GPP (3a, 97.50 mg, 0.1 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (95.85 mg, 0.5 mmol) were dissolved in 2 mL of H₂O. 3-Aminoethyl-3-(but-3-ynyl)bis(ethyleneimine) (4, 15.15 mg, 0.11 mmol) was dissolved in 2 mL of DMF. The bis(ethyleneimine) solution was slowly added dropwise to the GPP solution. The mixture was allowed to react at room temperature for 4 hours and then dried. The crude product was dissolved in a small amount of water and purified on a preparative liquid chromatography system using an UltimateAQ-C18 column with gradient elution. The GPP probe peak elutes at a retention time of approximately 75 minutes (65% B) (approximately 39% acetonitrile). Mobile phase A consisted of water and mobile phase B consisted of 60% acetonitrile. The flow rate was 2 mL / min, and the detection wavelength was 214 nm. The eluates containing the GPP probe were mixed and lyophilized. The probe concentration was determined by ultraviolet absorption of its aqueous solution at 214 nm. The yield was about 15%. The probe was aliquoted and frozen at -80°C. Its structure and purity were determined by nuclear magnetic resonance and high-resolution mass spectrometry. 1 H NMR (600MHz, MeOD) δ5.43(t,J=6.7Hz,1H),5.11(t,J=7.1Hz,1H),4.50(t,J=6.3Hz,2H),3.27–3.10(m,17Hfrom NBu4 + ),2.82(q,J=8.1Hz,2H),2.12(q,J=7.4Hz,2H),2.03(q,J=8.3Hz,4H),1.74–1.57(m,32H,15H from GPP-P,17H fromNBu4 + ),1.42(m,17H fromNBu4 + ),1.03(t,J=7.4Hz,26H fromNBu4 + ). 13 C NMR(151MHz,D2O)δ141.48,132.67,123.54,119.38(d,J=8.3Hz),83.90,69.35,62.13(d,J= 5.0Hz),38.33,35.88,33.62(d,J=7.7Hz),30.69,27.29,25.23,24.32,16.47,15.19,12.01. 31P NMR(162MHz,D2O)δ-2.05(d,J=21.9Hz),-10.84(d,J=21.9Hz).HRMS(ESI)m / z calcd.for C 17 H 28 N3O6P2 - :432.1459,found:432.1438.

[0084] Synthesis of FPP photoaffinity probe (FPP-P) and FPP control probe (FPP-CP): The synthesis method of the FPP probe and FPP control probe is the same as that of the GPP probe. FPP (3b, 92.5 mg, 0.1 mmol) was reacted with 4 (15.15 mg, 0.11 mmol) to synthesize the FPP probe, and with 6 (10.69 mg, 0.11 mmol) to synthesize the FPP control probe. The crude product was dissolved in a small amount of water and purified on a CHEETAH Pro purification system using a Welflash AQ-C18 column (4 g) with gradient elution. The FPP probe and control probe elute at a retention time of approximately 120 minutes (60% B) (approximately 42% acetonitrile). Mobile phase A was water, and mobile phase B was 70% acetonitrile, with a flow rate of 10 mL / min. The eluates containing the FPP probe and the FPP control probe were combined and lyophilized. The probe concentrations were determined by UV absorption of their aqueous solutions at 214 nm. The yields of the FPP probe and the control probe were 29% and 25%, respectively. The probes were aliquoted and frozen at -80°C. Their structures and purities were confirmed by nuclear magnetic resonance and high-resolution mass spectrometry. FPP photoaffinity probe (5b): 1 HNMR(600MHz,MeOD)δ5.43(t,J=6.0Hz,1H),5.17–5.04(m,2H),4.50(t,J=6.5Hz,2H),3.27–3.20(m,15H fromNBu4 + ),2.83(q,J=8.1Hz,2H),2.19–1.94(m,11H),1.77–1.58(m,31H,16H from FPP-P,15H from NBu4 + ),1.42(m,J=7.4Hz,15H from NBu4 + ),1.02(t,J=7.4Hz,22H fromNBu4 + ). 1313C NMR (151 MHz, D2O) δ 139.85, 134.60, 130.73, 124.83, 124.05 (d, J = 2.8 Hz), 120.57, 83.43, 69.63, 62.16 (d, J = 4.7 Hz), 39.52, 39.30, 36.34, 34.12 (d, J = 7.5 Hz), 31.55, 27.25, 26.34, 26.24, 25.05, 17.03, 15.88, 15.39, 12.62. 31 31P NMR (162 MHz, D2O) δ -2.37, -11.10. HRMS (ESI) m / z calcd. for C 22 H 36 N3O6P2 - : 500.2085, found: 500.2061. FPP control probe (7b): 1 1H NMR (600 MHz, D2O) δ 5.45 (d, J = 6.6 Hz, 1H), 5.19 (t, J = 7.2 Hz, 1H), 5.15 (t, J = 6.9 Hz, 1H), 4.50 (t, J = 6.1 Hz, 2H), 3.34–3.12 (m, 17H from NBu4 + ), 2.92 (q, J = 7.5 Hz, 2H), 2.29–2.24 (m, 3H), 2.22–1.97 (m, 9H), 1.79–1.54 (m, 34H, 16H from FPP-CP, 17H from NBu4 + ), 1.41 (m, J = 7.4 Hz, 17H from NBu4 + ), 1.00 (t, J = 7.3 Hz, 25H from NBu4 + ). 13 13C NMR (151 MHz, D2O) δ 140.55, 135.27, 131.63, 125.15, 124.38 (d, J = 4.5 Hz), 120.84, 85.82, 69.05, 62.49 (d, J = 5.8 Hz), 41.32, 39.52, 39.47, 30.40 (d, J = 8.7 Hz), 26.53, 26.37, 25.69, 25.33, 17.74, 17.33, 16.08, 15.67. 31 31P NMR (162 MHz, D2O) δ -1.30 (d, J = 22.2 Hz), -10.89 (d, J = 22.2 Hz). HRMS (ESI) m / z calcd. for C 21 H 36 NO6P2- :460.2023,found:460.2000.

[0085] Synthesis of GGPP photoaffinity probe (GGPP-P): The synthesis method of the GGPP probe was the same as that of the GPP probe. GGPP (3c, 76.7 mg, 0.1 mmol) was reacted with EDC (95.85 mg, 0.5 mmol) and 4 (15.15 mg, 0.11 mmol). The crude product was dissolved in a small amount of water and purified using a Welflash AQ-C18 column (25 g) on ​​a CHEETAH Pro purification system. Gradient elution was used, and the GGPP probe elutes at a retention time of approximately 140 minutes (50% B) (approximately 50% acetonitrile). Mobile phase A consisted of water and mobile phase B consisted of acetonitrile at a flow rate of 10 mL / min. The eluates containing the GGPP probe were combined and lyophilized. The probe concentration was determined by UV absorption of the aqueous solution at 214 nm. The yield of the GGPP probe was 38%. The probe was stored frozen in aliquots at -80°C. Its structure and purity were confirmed by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry. GGPP probe (5c): 1 H NMR(600MHz,MeOD)δ5.45(t,J=6.1Hz,1H),5.12(m,3H),4.52(t,J=6.3Hz,2H),3.27–3.20(m,13H fromNBu4 + ),2.90-2.81(m,2H),2.25–1.93(m,15H),1.74–1.55(m,37H,19H from GGPP-P,12H from NBu4 + ),1.42(m,J=7.4Hz,13H fromNBu4 + ),1.03(t,J=7.4Hz,19H from NBu4 + ). 13 C NMR (151MHz, D2O) δ 139.68, 134.73, 134.35, 130.62, 124.39, 124.33, 121.13, 121.07, 83.53, 69.81, 62.38 (d, J = 4.9Hz), 42. 87,39.80,39.78,39.72,36.67,34.57(d,J=8.1Hz),32.04,27.47,26.79,26.75,25.48,17.41,16.33,15.82,15.79,13.04. 31P NMR(162MHz,D2O)δ-2.58(d,J=21.8Hz),-11.25(d,J=21.6Hz).HRMS(ESI)m / z calcd.for C 27 H 44 N3O6P2 - :568.2711,found:568.2683.

[0086] 2. Results and Discussion

[0087] We synthesized GPP, FPP, and GGPP photoaffinity probes, respectively. These probes contain a binding group, a photoreactive group (diaziridine), and a bioorthogonal group (alkynyl). The photoreactive group can be activated by ultraviolet light (365 nm) to form a covalent bond with an amino acid residue near the probe binding pocket. The bioorthogonal group (alkynyl) reacts with an azide through a click chemistry reaction to attach a fluorescent group or biotin group for subsequent fluorescence imaging or affinity enrichment. An FPP control probe, lacking a photoreactive group, is used for comparison with the photoaffinity probes to analyze the effect of the photoreactive group on protein labeling.

[0088] We characterized the four synthesized probes using nuclear magnetic resonance and mass spectrometry, assigned their secondary ions, and verified the structures of the probes ( Figure 3-Figure 19 ).

[0089] Example 2: Isoprenoid pyrophosphate photoaffinity probe for labeling and fluorescence gel imaging analysis of proteins in cell lysates

[0090] To verify whether the new isoprenoid pyrophosphate photoaffinity probe can effectively label its binding protein, we first used fluorescence gel imaging experiments to screen conditions, mainly including the effects of UV irradiation, precipitation conditions, metal ions and probe concentration on probe-labeled proteins, and used GPP / FPP / GGPP competition experiments to verify the specificity of the probe labeling its binding protein.

[0091] 1. Materials and Methods

[0092] 1.1 Experimental Reagents

[0093] Methanol (Fuyu), glacial acetic acid (Fuyu), triethanolamine (Tianjin Fuyu), sodium chloride (Titan), chloroform (Iron Tower), sodium bicarbonate (Biden Pharmaceuticals), rhodamine B-PEG3-azide, tris-(2-carboxyethyl)phosphine hydrochloride (Biden Pharmaceuticals), copper sulfate (Sigma), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, bromophenol blue (Aladdin), β-mercaptoethanol (Biden Pharmaceuticals), 4-hydroxyethylpiperazineethanesulfonic acid (Biyuntian), ethylenediaminetetraacetic acid (Solaibo), sodium lauryl sulfate (Genview), Triton X-100 (Biyuntian), magnesium chloride (McLean), tetramethylethylenediamine (Titan), 30% acrylamide (Dingguo), Coomassie Brilliant Blue R-250 (Biden Pharmaceuticals), Tris(hydroxymethyl)aminomethane hydrochloride, phosphatase inhibitor A (Emmer), phosphatase inhibitor B (Emmer), protease inhibitor (Emmer), Bradford kit (Biyuntian), BCA kit (Biyuntian), fetal bovine serum (FBS, ExCell Bio), penicillin-streptomycin (PS, Gibco), high glucose DMEM (Gibco), GPP, FPP and GGPP and their corresponding probes.

[0094] 1.2 Experimental instruments

[0095] Pipette, centrifuge (Eppendorf), constant temperature metal bath (Thermo Fisher), microplate reader, electrophoresis instrument (Bio-Rad), 365 nm UV lamp (ZF-1), vortexer (Aika), horizontal shaker (Dalong), pH meter (Sartorius), NanoDrop one (Thermo Fisher), fluorescence imager (Amersham Imager600, GE), multispectral laser imager (Amersham Typhoon 5, Storafen), incubator (Thermo Fisher).

[0096] 1.3 Experimental steps

[0097] Fluorescent gel imaging of HEK 293T cell lysate labeled with GPP / FPP / GGPP probes

[0098] Take an appropriate amount of HEK293T cells and lyse them on ice for 30 minutes using 1% Triton X-100 lysis buffer (containing 1% Triton X-100, 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 7.4, 150 mM sodium chloride; add protease inhibitors, phosphatase inhibitor A, and phosphatase inhibitor B before use). Vortex the cells for 3-5 seconds every 5-10 minutes. Centrifuge at 10,000 rpm for 30 minutes at 4°C. Collect the supernatant and measure the concentration using the BCA assay. 100 μg of protein was diluted in 0.1% Triton X-100 buffer (containing 0.1% Triton X-100, 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 7.4, 150 mM sodium chloride, with protease inhibitors, phosphatase inhibitor A, and phosphatase inhibitor B added before use). Various concentrations of competitor and probe were added to the target concentration (competitor was added first and incubated for 10 minutes before adding the probe). The final volume was 100 μL and the final protein concentration was 1 μg / μL. After incubation on ice for 10 minutes, the sample was spread on ice and irradiated with 365 nm UV light for 30 minutes to covalently bind the probe to the target protein. The protein was then precipitated with a mixture of chloroform:methanol:water (4:3:1 by volume) and repeated three times to remove excess competitor, probe, and other small molecules. After the protein was air-dried, it was redissolved in 20 μL of 4% SDS solution (containing 50 mM triethanolamine, pH 7.4, 150 mM sodium chloride, and 4% sodium dodecyl sulfate) and diluted to 100 μL with 4-hydroxyethylpiperazineethanesulfonic acid buffer (containing 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 7.4, 150 mM sodium chloride). Freshly prepared click chemistry reagents (final concentrations: 1 mM tris-(2-carboxyethyl)phosphine hydrochloride, 0.1 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, 1 mM copper sulfate, and 0.1 mM rhodamine B-PEG3-azide) were added to the solution and vortexed at room temperature for 2 hours. Subsequently, the protein was precipitated with a mixture of chloroform:methanol:water (4:3:1 by volume) three times. After the protein was air-dried, it was redissolved in 4% SDS buffer and the concentration was determined using the BCA method. Approximately 20 μg of protein was added to Laemmli loading buffer (containing 25 mM Tris, pH = 6.8, 4% sodium dodecyl sulfate, 40% glycerol, 0.02% bromophenol blue, and 20% β-mercaptoethanol was added before use), mixed, and heated at 95°C for 5 minutes. The sample was then loaded onto an SDS-PAGE gel (4% stacking gel, 10% separating gel) and subjected to electrophoresis at a constant voltage of 90 V for approximately 110 minutes. After electrophoresis, the stacking gel was removed and fluorescent gel imaging was performed using an Amersham Imager 600 (excitation light source 520 nm) or an Amersham Typhoon 5 imaging system (excitation light source 532 nm).After imaging is completed, the gel is stained with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue, 10% glacial acetic acid, 50% methanol) for 30 minutes, and the background color is removed with destaining solution (10% glacial acetic acid, 30% methanol). The consistency of the sample loading is determined based on the staining results.

[0099] 2. Results and Discussion

[0100] In order to achieve better probe labeling effects, we first used in-gel fluorescence imaging technology to explore the labeling conditions of cell lysates, including UV irradiation, sedimentation conditions, metal ions, and probe concentrations. The fluorescence gel results showed that the FPP probe had a good labeling effect under UV irradiation. In the absence of UV, the fluorescence of the labeled protein was very weak, similar to the labeling results of the FPP control probe. Considering that these bands may come from the nonspecific reaction of excess probes, we tried to remove the unreacted probes after UV irradiation. Protein precipitation before the click chemistry reaction greatly reduced the fluorescence intensity of the labeled bands ( Figure 20 a) This may be because the hydrophobic probe is embedded in the protein after resolubilization. Although removing excess probe using a NAP-5 column also reduced the fluorescence intensity of the labeled band, the impact was relatively small. To eliminate the potential nonspecific labeling reaction caused by excess probe, a simple protein precipitation procedure was performed in the in-gel fluorescence imaging experiment. During mass spectrometry sample preparation, NAP-5 columns were used to remove excess probe and other small molecules to minimize the impact on labeling.

[0101] Addition of different metal cations during probe incubation had no apparent effect on overall labeling, suggesting that metal cations may not be required for isoprenoid pyrophosphate binding to the target protein ( Figure 20 b). Labeling intensity increases with increasing probe concentration ( Figure 21 ac). Because 10 μM FPP probe labeling was effective, this concentration was chosen for all subsequent experiments. The three XPP probes showed similar labeling patterns, while the GPP-P-labeled protein band was much weaker compared to the FPP and GGPP probes.

[0102] When there is an excess of GPP, FPP, or GGPP during probe treatment, the fluorescence intensity of the labeled protein band decreases with increasing XPP concentration ( Figure 21 df). Similar to the lowest labeling efficiency of the GPP probe, GPP also presents the least competitive effect on probe labeling. Given the high labeling efficiencies of the FPP and GGPP probes, we selected these two probes for SILAC-based proteomics analysis.

[0103] Example 3: Labeling and proteomic analysis of FPP / GGPP binding proteins in cell lysates using isoprenoid pyrophosphate photoaffinity probes

[0104] Under optimal probe labeling conditions, SILAC cell (HEK293T) lysate was labeled with FPP / GGPP photoaffinity probe, and the probe-labeled proteins were detected and analyzed using high-resolution liquid chromatography-mass spectrometry. The competitive effect of FPP / GGPP on probe-labeled proteins verified the specificity of the FPP / GGPP probe for labeling FPP / GGPP-binding proteins.

[0105] 1. Materials and Methods

[0106] 1.1 Experimental Reagents

[0107] Ammonium bicarbonate (Biodex Pharmaceuticals), iodoacetamide (Biodex Pharmaceuticals), methanol (Thermo Fisher), formic acid (Thermo Fisher), acetonitrile (Thermo Fisher), water (Thermo Fisher), urea (Genview), biotin-DADPS-azide (Leyan), 1× PBS buffer (Solebol), 10× PBS buffer (Solebol), high-glucose DMEM lacking L-lysine and L-arginine (Thermo Fisher), dialyzed fetal bovine serum (FBS, Gibco), penicillin-streptomycin (PS, Gibco), 13 C6-arginine hydrochloride (CIL), 13 C6, 15 N2-lysine hydrochloride (CIL), L-arginine monohydrochloride (Sigma), L-lysine monohydrochloride (Sigma), PBS (Thermo Fisher), streptavidin agarose beads (Thermo Fisher), trypsin (Thermo Fisher). The remaining reagents are the same as those in 1.1 of Example 2.

[0108] 1.2 Experimental instruments

[0109] Centrifugal concentrator (Eppendorf), centrifuge (Eppendorf), NAP-5 (GE), UV 365 nm lamp, constant temperature metal bath (Mini H100), rotary shaker (Thermo Fisher), ultrasonic cleaner (Shandong Boco), microplate reader (Bio-Rad), vortexer (IKA), horizontal shaker (Dalong), pH meter (Sartorius), C18Tip (Thermo Fisher), Vanquish Neo liquid chromatography (Thermo Fisher), Orbitrap Exploris 480 mass spectrometer (Thermo Fisher).

[0110] 1.3 Experimental steps

[0111] 1.3.1 Sample preparation for protein profiling of HEK293T cell lysate using SILAC probes

[0112] Preparation of SILAC-specific DMEM medium and cell culture: 10% dialyzed FBS and 1% PS were added to 89% high-glucose DMEM lacking L-lysine and L-arginine, and the medium was divided into two parts: one as light standard (L) medium: supplemented with 0.4M L-arginine monohydrochloride (Sigma-Aldrich) and 0.8M L-lysine monohydrochloride; the other as heavy standard (H) medium: supplemented with 0.4M 13 C6-arginine monohydrochloride (CIL) and 0.8M 13 C6, 15 HEK 293T cells were cultured in parallel for more than five passages in either light-labeled or heavy-labeled SILAC medium to ensure complete incorporation of the isotopically labeled amino acid.

[0113] SILAC-based mass spectrometry sample preparation: In the forward experiment, 0.5 mg of light lysate (L) was labeled with 10 μM FPP probe or 10 μM GGPP probe, and 0.5 mg of heavy lysate (H) was labeled with 10 μM FPP probe in the presence of 1 mM FPP or 0.5 mg of heavy lysate (H) was labeled with 10 μM GGPP probe in the presence of 1 mM GGPP; in the reverse experiment, the labeling conditions were interchanged. After labeling, 50 μg of each sample was removed and a fluorescent gel was prepared according to the method of 1.3 in Example 2. Equal amounts of the corresponding light-labeled and heavy-labeled lysates were mixed and passed through a NAP-5 column to remove excess small molecules. The CuAAC reaction was performed according to the method of 1.3 in Example 2, wherein biotin-DADPS-azide (final concentration 0.01 mM) was used instead of rhodamine-PEG3-azide. The precipitated protein was redissolved in 100 μL of 4-hydroxyethylpiperazineethanesulfonic acid buffer containing 4% sodium dodecyl sulfate (50 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH = 7.4, 150 mM sodium chloride) and diluted to 1 mL with 4-hydroxyethylpiperazineethanesulfonic acid buffer without sodium dodecyl sulfate. 50 μL of streptavidin agarose beads were added, and the mixture was incubated at room temperature for 2 hours. After incubation, transfer the entire mixture to a centrifugal filter column to wash away unbound proteins: wash with 1% SDS in 1× PBS, 1× PBS, 10× PBS, 1× PBS, and 8M urea in 50mM ammonium bicarbonate, 1 mL each, five times. After washing, resuspend the protein-bound streptavidin beads in 500 μL of 8M urea in 50mM ammonium bicarbonate and transfer to a new centrifuge tube. Add tris-(2-carboxyethyl)phosphine hydrochloride to a final concentration of 20mM to reduce disulfide bonds and react at 37°C for 1 hour. Add iodoacetamide to a final concentration of 50mM for alkylation and react at room temperature in the dark for 30 minutes. After the reaction was completed, the cells were washed three times with 500 μL of 25 mM ammonium bicarbonate solution, resuspended in 100 μL of 25 mM ammonium bicarbonate solution (centrifuged at 3000 rpm for 2 minutes), and 1 μL of trypsin (1 μg / μL) was added for enzymatic hydrolysis at 37°C for 16 hours.

[0114] After enzymatic digestion, add 200 μL of 25 mM ammonium bicarbonate solution to each sample and resuspend the streptavidin beads. Centrifuge at 10,000 rpm for 2 minutes and remove the supernatant. Repeat five times. Combine the supernatants, concentrate by centrifugation, desalt using a C18 tip, and store frozen at -80°C until LC-MS / MS analysis. Dissolve the sample in 20 μL of pure water, and inject 4 μL.

[0115] 1.3.2 LC-MS / MS Detection Method and Data Analysis

[0116] Samples were analyzed using an Orbitrap Exploris 480 mass spectrometer equipped with a nanoelectrospray ionization source, coupled to a Vanquish Neo ultra-high performance liquid chromatography system. A self-prepared nano-liquid phase C18 capillary column was used, packed with Reprosil-Pur120C18-AQ (Mike, Germany), with a 3 μm particle size, a 75 μm inner diameter, and an 18 cm column length. Mobile phase A consisted of 98% water containing 0.1% formic acid, and mobile phase B consisted of 98% acetonitrile containing 0.1% formic acid. The flow rate was set at 300 nL / min, and the elution gradient was as follows: 5% B (0-5 min); 26% B (130 min); 36% B (140 min); 72% B (141 min); and 80% B (151-166 min). The spray voltage was 1.9 kV, and the ion transfer tube temperature was 320°C. Full scan mass spectra were acquired with an Orbitrap mass analyzer at 120,000 resolution over the m / z range of 350–1500. The normalized AGC target was 300%, and the maximum injection time was 25 ms. Data dependent acquisition (DDA) mode was used with a 3 s MS scan cycle time and a data acquisition time of 0–151 min. Ions with a charge state ≥2 in the full scan mass spectra were fragmented in an HCD collision cell (normalized collision energy of 30%), and tandem mass spectra were acquired with an Orbitrap mass analyzer at 15,000 resolution. The normalized AGC target for the secondary mass spectra was 50%, and the maximum injection time was automatically selected.

[0117] The acquired MS raw files were analyzed using MaxQuant (v2.1.4.0) software and searched against the Uniprot human proteome database (20,401 total proteins, downloaded on June 28, 2023) to identify and quantify the labeled proteins. For SILAC analysis, Lys8 and Arg6 were selected, with cysteine ​​carbamidomethylation as fixed modifications, methionine oxidation and N-terminal acetylation as variable modifications, and enzyme specificity set to trypsin. GO enrichment analysis was performed on the identified proteins using the DAVID database.

[0118] 2. Results and Discussion

[0119] To identify isoprenoid pyrophosphate-interacting proteins, we combined competitive probe labeling experiments with a SILAC-based quantitative proteomics approach. In the forward experiment, light lysate (L) was labeled with either an FPP probe or a GGPP probe, and heavy lysate (H) was labeled with an FPP probe in the presence of FPP or with a GGPP probe in the presence of GGPP. In the reverse experiment, labeling conditions were interchanged. After UV irradiation, equal amounts of light and heavy lysates were mixed and passed through a NAP-5 column to remove excess probe and other small molecules. Probe-labeled proteins were biotinylated via click chemistry, subsequently enriched with streptavidin beads, digested with trypsin, and analyzed by LC-MS / MS.

[0120] Fluorescence gel and correlation analysis of forward and reverse experiments showed that the ratios of forward and reverse experiments were relatively consistent ( Figure 22 ab). In three forward and three reverse experiments, with ratio>2 and p<0.05 as screening conditions, the FPP probe identified a total of 126 proteins, and the GGPP probe identified a total of 132 proteins ( Figure 23 ab). For most proteins, the ratio of GGPP probe is higher than that of FPP probe ( Figure 23 c), this may be because the longer fatty chain of GGPP has a higher affinity for proteins.

[0121] FPP synthase (FDPS) catalyzes the synthesis of GPP from IPP and DMAPP, and subsequently FPP from GPP and IPP. Both probes identified FPP synthase, but it did not meet the screening criteria, likely due to the probe's longer fatty chain than the FDPS substrate. The average ratio of FDPS was higher in the FPP probe set compared to the GGPP probe set. CAAX prenyl protease 1 (ZMPSTE24), which removes three amino acids from the C-terminus of prenylated proteins, showed a ratio >1.5 in both the FPP and GGPP probe mass spectrometry data, with p < 0.05. Prevalerylcysteine ​​oxidase 1 (PCYOX1), which oxidatively cleaves the thioether bond of free valerylcysteine, showed a ratio >2 in both probe mass spectrometry data but was not identified in the GGPP probe set due to lack of quantitative data in two of six replicates. These results suggest that this method has promising applications in analyzing isoprenoid pyrophosphate-interacting proteins and proteins involved in prenylation.

[0122] Among the 86 proteins identified by both FPP and GGPP probes, 48 ​​proteins were membrane proteins and 28 proteins shuttled between the cytoplasm and the nucleus ( Figure 24GO analysis of 172 proteins identified using FPP or GGPP probes showed that proteins located in the endoplasmic reticulum, mitochondria, and Golgi apparatus were highly enriched ( Figure 24 c). The GGPP probe has a certain preference for endoplasmic reticulum proteins, while the FPP probe has a certain preference for mitochondrial proteins. Of the identified proteins, 57 are involved in cellular transport, particularly protein transport, of which 18 are annotated as small G protein binding proteins. Because prenylation of the terminal CAAX motif of small G proteins is crucial for their transport, localization, and function, studying proteins that interact with prenylated proteins is important for understanding the underlying mechanisms. Furthermore, 15 proteins are associated with lipid metabolism, particularly lipid biosynthesis, indicating that this method can be used to study the roles of FPP and GGPP in biosynthesis and to explore enzymes that utilize isoprenoid pyrophosphate in these pathways. Combined with these results, this analytical method shows great potential for investigating new cellular functions of prenylation-related proteins and isoprenoid pyrophosphate.

[0123] Example 4 Fluorescent labeling of Hela cells with photoaffinity probes

[0124] 1.1 Experimental Reagents

[0125] Cell slides BS-14-RC ​​(14 mm, White Shark), glass slides (Sita), DAPI (Biyuntian), paraformaldehyde (Annaiji), anti-fluorescence quenching solution (Biyuntian), BSA (Solar), Tween-20 (Biyuntian), Organelle Localization IFAntibody Sampler Kit (CST), fluorescent secondary antibody (Alexa Fluor 488 nm, ABclonal). Other reagents were the same as in 1.1 of Example 2.

[0126] 1.2 Experimental instruments

[0127] Pipette (Thermo Fisher), clean bench (Shandong Boco), incubator (Thermo Fisher), pH meter (Sartorius), horizontal shaker (Dalong), inverted fluorescence microscope (OLYMPUS), rare cell analyzer (Dragonfly 200).

[0128] 1.3 Experimental steps

[0129] Fluorescent labeling of Hela cells with photoaffinity probes and immunofluorescence imaging analysis

[0130] In the time gradient and concentration gradient experiments of probe labeling, Hela cells were plated in a 24-well plate with a cell slide at a cell density of approximately 50-60%. Different concentrations of FPP probe or GGPP probe were added and incubated at 37°C for the specified time. After incubation, the culture medium was replaced with 1×PBS, and the cells were placed on ice and irradiated with UV (365nm) for 10 minutes. The cells were fixed with 200μL 4% paraformaldehyde for 15 minutes. After fixation, the cells were washed three times with 1×PBS for 3-5 minutes each time, and 0.1% triton X-100 buffer (50mM 4-hydroxyethylpiperazineethanesulfonic acid, pH=7.4, 150mM sodium chloride, 0.1% triton) was added. X-100) was infiltrated for 10 minutes, and a click chemistry reaction was performed for 2 hours according to the method in 1.3 of Example 2, wherein the final concentration of rhodamine B-PEG3-azide was 10 μM. After the reaction, the reaction solution was removed, and the cells were washed 3 times with 1× PBS for 3-5 minutes each time, 200 μL DAPI staining solution (10 μg / mL) was added and stained in the dark for 5 minutes, and the cells were washed 3 times with 1× PBS. The slide was taken out and placed with the cell side down on a slide with anti-fluorescence quenching solution, and the slide was fixed with transparent nail polish. After drying, fluorescence imaging was performed, and the image was taken using an inverted microscope with the objective lens set to 40 times, the probe exposure time was 50 ms, and the DAPI exposure time was 1 ms.

[0131] For probe-labeled immunofluorescence imaging, HeLa cells were plated and incubated with 100 μM FPP or GGPP probe for 2 hours. The cells were treated as described above and permeabilized. The cells were blocked with 5% BSA in PBST buffer for 2 hours at room temperature. The blocking buffer was removed and the cells were incubated overnight at 4°C with antibodies against AIF, EEA1, LAMP1, PDI, or RCAS1 (prepared in 5% BSA in PBST buffer). After incubation, the cells were washed three times with PBST and incubated with the corresponding rabbit secondary antibody (488 nm) for 2 hours at room temperature. Images were acquired using a laser confocal microscope using a 63× oil-immersion objective, an exposure time of 50 ms, and a laser output power of 30%. Images were analyzed using Fiji software.

[0132] 2. Results and Discussion

[0133] Since more than 50% (99 out of 172) of the identified proteins were clearly localized in the endoplasmic reticulum, mitochondria, and Golgi apparatus, we used FPP probes and GGPP probes to study the subcellular localization of FPP and GGPP.

[0134] Fluorescence imaging showed that the fluorescence of labeled cells increased with the increase of probe concentration. At a probe concentration of 20 μM, both FPP probe and GGPP probe were partially labeled ( Figure 25ab). Fluorescence also increases with the increase of probe incubation time ( Figure 26 ab).

[0135] To investigate the localization of FPP and GGPP, cells were fluorescently labeled with 100 μM FPP or GGPP probe for 2 hours and then incubated with antibodies against different organelle marker proteins. Laser confocal microscopy analysis showed that FPP and GGPP probes were mainly colocalized with the endoplasmic reticulum and mitochondria, and partially colocalized with the Golgi apparatus, lysosomes, and early endosomes ( Figure 27 and Figure 28 ), which is consistent with the LC-MS / MS data. In the FPP and GGPP probe labeling experiments, the intense, concentrated fluorescence signal observed near the nuclear membrane did not completely colocalize with the labeled proteins. This phenomenon may be related to the role of FPP and GGPP in mediating nucleocytoplasmic protein trafficking, but the precise mechanism requires further investigation. The results of the cell imaging experiments also suggest that this type of probe can be used in the future for the omics analysis of isoprenoid pyrophosphate-interacting proteins in living cells.

[0136] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An isoprenoid pyrophosphate photoaffinity probe, characterized in that The photoaffinity probe has the following structure:

2. The method for preparing the photoaffinity probe according to claim 1, characterized in that: The preparation method includes the following synthetic route:

3. A kit, characterized in that The kit at least comprises the photoaffinity probe according to claim 1.

4. The kit according to claim 3, wherein The kit further comprises a buffer solution and reaction reagents.

5. Use of the photoaffinity probe according to claim 1 or the kit according to claim 3 in research related to isoprenoid pyrophosphate binding proteins.

6. The use according to claim 5, characterized in that The application at least includes: (a) Fluorescence gel imaging analysis of isoprenoid pyrophosphate binding proteins in cell lysates; (b) Protein profiling sample analysis of isoprenoid pyrophosphate binding proteins in cell lysates; (c) Immunofluorescence imaging and subcellular localization analysis of isoprenoid pyrophosphate binding protein in cells.

7. The use according to claim 6, characterized in that In (a)-(b), the cells are HEK293T cells.

8. The use according to claim 6, characterized in that In the above-mentioned (c), the cells are Hela cells.

9. The use according to claim 6, characterized in that The isoprenoid pyrophosphate binding protein is any one or more of GPP / FPP / GGPP binding proteins.

10. The use according to claim 6, characterized in that The specific method of application (b) includes: adding the photoaffinity probe to SILAC cell lysate (HEK293T) for pre-incubation and UV irradiation, and then performing a click chemistry reaction with biotin-DADPS-azide; further, using LC-MS / MS to detect the protein labeled with the probe, attributing the obtained mass spectrometry data, and performing GO analysis.

Images