A method for enriching protein palmitoyltransferase substrates
By integrating APEX2 proximity labeling technology with Acyl-RAC technology in a tandem enrichment strategy, the specificity problem of identifying protein palmitoylation modification enzyme substrates was solved, achieving efficient identification of specific protein palmitoylation enzyme substrates, reducing false positives, and promoting the progress of palmitoylation modification research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DALIAN INSTITUTE OF CHEMICAL PHYSICS CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2024-11-26
- Publication Date
- 2026-05-26
AI Technical Summary
Existing detection methods have poor specificity in identifying protein palmitoylation-modified enzyme substrates, resulting in false positives and making it difficult to accurately identify the substrate proteins of ZDHHC enzymes.
A tandem enrichment strategy integrating proximity labeling and palmitoylation modification enrichment techniques was adopted. The APEX2 proximity labeling technique was used to label the interacting proteins of specific palmitoylation transferases, and the biotinylated proteins were enriched using the biotin-streptavidin system. The palmitoylated modified proteins were enriched by combining Acyl-RAC technology, and finally, the specific protein palmitoylation substrates were identified by proteomics techniques.
This improved the identification specificity of cellular protein palmitoylase substrates, reduced false positives, and promoted the exploration of the catalytic kinetics of palmitoylation-modified enzymes.
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Figure CN122084902A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biochemical analysis technology, specifically a method for enriching protein palmitoylation catalytic enzyme substrates by integrating a tandem enrichment strategy of proximity labeling technology and palmitoylation modification enrichment technology. Background Technology
[0002] Palmitoylation is a dynamic and reversible post-translational modification of proteins catalyzed by palmityltransferase (ZDHHC). Palmitoylation affects the localization and stability of proteins and has a wide range of effects on cell signal transduction, immune response, tumor development, transcription and metabolism.
[0003] In mammalian cells, there are 23 palmitoyltransferases. Some ZDHHC enzymes undergo palmitoylation modification through mutual catalysis before they can exert their catalytic function. From a proteomics perspective, more than a quarter of human proteins undergo palmitoylation modification, but only a few hundred ZDHHC enzymes have clearly defined substrate pairings. Clarifying the correspondence between ZDHHC enzymes and their substrates helps to reveal the molecular mechanisms of disease development, thus providing new ideas and targets for disease treatment. Generally, changes in protein palmitoylation modification profiles can be detected by altering the expression level of ZDHHC enzymes, or by detecting interacting proteins of ZDHHC enzymes through methods such as immunoprecipitation. These two approaches are used to discover ZDHHC enzyme substrates. Palmitoylation modification may affect protein stability; therefore, increasing or decreasing the level of ZDHHC enzymes will directly or indirectly affect the abundance of substrate proteins, leading to changes in the protein palmitoylation modification profile and resulting in false positives or false negatives. Palmitoylation proteomics typically enriches palmitoylated proteins using acyl-biotin exchange (ABE) or acyl-resin-assisted capture (Acyl-RAC). Both methods are based on thioester bonds, and modifications linked by thioester bonds, such as ubiquitin modification, can also be enriched by these methods, naturally leading to irreversible false positives. Furthermore, not all interacting proteins of ZDHHC enzymes are their substrates; some ZDHHC enzymes bind to many proteins without directly catalyzing palmitoylation. Therefore, directly detecting ZDHHC enzyme interacting proteins via immunoprecipitation is prone to false positives.
[0004] To address the shortcomings of existing detection methods in terms of poor specificity, the inventors of this application propose to provide a method for identifying protein palmitoylation catalytic enzyme substrates using a tandem enrichment strategy that integrates proximity labeling technology and palmitoylation modification enrichment technology. This application integrates APEX2-based proximity labeling technology with palmitoylation modification substrate enrichment technology represented by Acyl-RAC, tandemly enriching specific protein palmitoylation enzyme substrates. Specific protein palmitoylation enzyme substrates are then identified using proteomics technology, improving the specificity of large-scale identification of protein palmitoylation enzyme substrates at the cellular level, accelerating the construction of palmitoylation enzyme-substrate interaction networks, initially promoting the exploration of the kinetic process catalyzed by protein palmitoylation modification enzymes at the cellular level, and advancing the research progress of palmitoylation modification. Summary of the Invention
[0005] To address the aforementioned technical problems, the present invention aims to provide a method for enriching protein palmitoyltransferase substrates. This method integrates a tandem enrichment strategy combining proximity labeling technology and palmitoylation modification enrichment technology to enrich and identify protein palmitoylation catalytic enzyme substrates. This method improves the specificity of large-scale identification of protein palmitoyltransferase substrates at the cellular level and can be used to explore the kinetic processes catalyzed by protein palmitoylation modification enzymes.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for enriching protein palmitoyltransferase substrates, wherein the method integrates APEX2 proximity labeling technology with acyl-RAC tandem enrichment of specific protein palmitoyltransferase substrates, and identifies specific protein palmitoyltransferase substrates at the cellular level through proteomics technology.
[0007] It includes the following steps:
[0008] (1) Biotin labeling of specific palmitoyltransferase-interacting proteins in cells was performed using APEX2 proximity labeling technology;
[0009] (2) Enrich APEX2-near biotinylated proteins using the biotin-streptavidin system; (3) Elute APEX2-near biotinylated proteins enriched by streptavidin.
[0010] (4) Based on step (3), palmitoylated proteins are enriched using Acyl-RAC technology;
[0011] (5) Use proteomics techniques to identify substrates of specific palmitoyltransferases enriched in tandem.
[0012] Further, in step (1), biotin is labeled with the interacting protein of a specific palmitoyltransferase in the cell using the APEX2 proximity labeling system, which specifically includes the following steps:
[0013] ① Construct a cell line that stably expresses the APEX2 fusion protein with a specific palmitoyltransferase;
[0014] ②Culture cells expressing the APEX2 system to a density of 60-80%;
[0015] ③ Discard the culture medium and wash the cells with PBS;
[0016] ④ Add 3-7 mL of preheated culture medium containing 0.1-1 mM biotinylate (CAS: 41994-02-9) at 37℃ to a 10 cm culture dish, preferably 5 mL of 0.5 mM, and incubate at 37℃ for 15-60 minutes; 30 minutes is preferred.
[0017] ⑤ Discard the culture medium and wash the cells with PBS;
[0018] ⑥ Add 2-5 mL of DPBS (Dubor's phosphate buffer) containing 0.1-5 mM H2O2 to a 10 cm culture dish, preferably 3 mL of 1 mM solution, and label on a shaker for 30 seconds to 3 minutes, preferably 1 minute.
[0019] ⑦ Add 2-5 mL of quenching solution to terminate the reaction, wash on a shaker for 30 seconds to 3 minutes, preferably 1 minute, and repeat three times;
[0020] ⑧ Wash three times with an appropriate amount of PBS, quench the cells with liquid nitrogen, and store at -80℃.
[0021] Furthermore, step (2) involves enriching APEX2-labeled biotinylated proteins using the biotin-streptavidin system, specifically including the following steps:
[0022] ① Add 200-500 μL of Lysis Buffer containing protease inhibitors, phosphatase inhibitors, and ML211 (CAS: 2205032-89-7) at a final concentration of 10 μM to the quenched cells in a 10 cm culture dish. Scrape the cells, preferably 300 μL, and sonicate for 5-6 seconds.
[0023] ② Centrifuge to extract the supernatant containing protein and determine the protein concentration; centrifuge at 12000-16000 rpm for 10-20 minutes, preferably at 14000 rpm for 15 minutes;
[0024] ③ Add 3-10 μL of cleaned biotin-capturing enrichment carrier to each 1 mg of protein, and enrich overnight at 4°C. Biotin-capturing enrichment carriers include, but are not limited to, pre-coupled streptavidin agarose beads, pre-coupled streptavidin magnetic beads, pre-coupled avidin agarose beads, and pre-coupled avidin magnetic beads.
[0025] Further, in step (3), the APEX2-adjacent biotinylated proteins enriched by streptavidin are eluted, which specifically includes the following steps:
[0026] ①Wash the enrichment vector containing biotinylated proteins with Lysis Buffer at room temperature or 4°C, for a total of 3 washes, each for 10 minutes;
[0027] ② Use Lysis Buffer containing 1-10 mM desulfurized biotin (CAS: 533-48-2), preferably 2.5 mM, and mix at room temperature or 4°C for 10-45 minutes by rotation to competitively elute and enrich carrier-bound biotinylated proteins; or use Lysis Buffer containing 6 M guanidine hydrochloride (CAS: 50-01-1) or 8 M urea (CAS: 57-13-6), heat at 98°C for 5-15 minutes to denature and elute carrier-bound biotinylated proteins.
[0028] Step (4) involves enriching palmitoylated proteins among the eluted biotinylated proteins using Acyl-RAC technology, specifically including the following steps:
[0029] ① Add TCEP (CAS: 115-96-8) to the eluent at a final concentration of 1-50 mM, preferably 10 mM, and mix by rotating at 25°C for 10-60 minutes, preferably 30 minutes;
[0030] ② Add freshly prepared maleimide (CAS: 541-59-3) to a final concentration of 5-50 mM or freshly prepared iodoacetamide (CAS: 144-48-9) to a final concentration of 10-100 mM. Maleimide is preferably 25 mM and iodoacetamide is preferably 50 mM. Mix at 25°C in the dark by rotating for 60-180 minutes, preferably 120 minutes.
[0031] ③The protein was precipitated using the MeOH / CHCl3 / H2O system, the methanol was evaporated, and the protein precipitate was dried.
[0032] ④ Use an appropriate amount of Lysis Buffer containing 4% SDS to reconstitute the protein, and promote dissolution by sonication or heating at 37°C;
[0033] ⑤ Add 1-2 μL of a cleaned enrichment carrier capable of capturing free sulfhydryl groups per 1 mg of protein, preferably 1.25 μL, to a final concentration of 200-1200 mM hydroxylamine (CAS: 7803-49-8), preferably 600 mM. Mix by rotation at room temperature for 60-180 minutes to enrich palmitoylated modified proteins, preferably 120-150 minutes. The enrichment carrier capable of capturing free sulfhydryl groups includes, but is not limited to, activated thiol agarose beads, thiopropyl agarose beads, activated thiol magnetic beads, and thiopropyl magnetic beads.
[0034] ⑥ Wash three times at room temperature with Lysis Buffer containing 1% SDS, 10 minutes each time;
[0035] ⑦ Wash the sample three times at room temperature with a 100mM NH4HCO3 aqueous solution containing 8M urea for 10 minutes each time. After enzymatic desalting, the sample can be used for proteomics detection.
[0036] The precautions for operating this invention include:
[0037] ① The quenching solution described in steps (1)-⑦ consists of: 5mM Trolox (CAS: 53188-07-1), 10mM sodium ascorbate (CAS: 134-03-2), 10mM NaN3, and the remainder is PBS.
[0038] ② The Lysis Buffer designed in steps (2), (3), and (4) consists of: 50mM Tris-HCl, 150mM NaCl, 4mM EDTA, 0.5% NP-40 (CAS: 9016-45-9), with the remainder being water. The pH is between 7.0 and 8.0, with 7.4 being the most suitable.
[0039] ③ The step of precipitating protein in the MeOH / CHCl3 / H2O system described in step (4) is to add methanol to the protein reaction system and vortex for 20-40 seconds, then add chloroform and vortex for 20-40 seconds, then add water and vortex for 20-40 seconds, centrifuge at 0-4℃ and 14000-20000 rpm and discard the supernatant, then add methanol again and vortex for 30 seconds, centrifuge at 0-4℃ and 14000-20000 rpm and discard the supernatant.
[0040] ④ The volume ratio of the MeOH / CHCl3 / H2O system described in step (4) is 350~500:100~500:200~500, preferably 400:150:400. The sample volume should be included in the water volume. All of these ratios can be adjusted appropriately. Proteins can be precipitated.
[0041] Advantages and beneficial effects of the present invention:
[0042] This invention integrates APEX2-based proximity labeling technology with palmitoylation-modified substrate enrichment technology represented by Acyl-RAC, to tandemly enrich specific protein palmitoyase substrates. Because APEX2 proximity labeling technology only labels proteins within a 20nm range of specific protein palmitoyases, it reduces false positives in the identification of interacting proteins by immunoprecipitation technology. Furthermore, the tandem palmitoylation-modified enrichment technology reduces false positives for proteins that bind to specific ZDHHC enzymes but have not undergone palmitoylation modification, thereby improving the specificity of identifying specific protein palmitoyase substrates at the cellular level. Attached Figure Description
[0043] Figure 1 The present invention provides the operational procedure for enriching protein palmitoyltransferase substrates.
[0044] Figure 2 For HeLa cells stably transfected with APEX2-2×FLAG and APEX2-2×FLAG-ZDHHC20 in Example 1, with or without hydrogen peroxide labeling, the extracted proteins were then used to capture ZDHHC20 substrates using a tandem enrichment strategy that combines APEX2 neighbor labeling with Acyl-RAC palmitoylation modification. After gel electrophoresis, the proteins were stained with Coomassie Brilliant Blue.
[0045] Figure 3 In Example 2, the ZDHHC9 substrates enriched by tandem APEX2 neighbor markers and Acyl-RAC palmitoylation modification were identified based on DIA proteomics. Differential proteins were analyzed using the Significance Analysis of Microarrays (SAM) algorithm, and the blue markers are potential ZDHHC9 substrates. Detailed Implementation
[0046] This invention discloses a method for identifying protein palmitoylated enzyme substrates using a tandem enrichment strategy integrating proximity labeling technology and palmitoylation modification enrichment technology. The invention is further illustrated with specific embodiments and accompanying drawings to enable those skilled in the art to more clearly understand the technical solution, but this is not intended to limit the invention. Those skilled in the art can refer to the content of this patent and appropriately modify the technical parameters to achieve the desired results. These parameters include the construction method of the APEX2 (The Engineered Ascorbate Peroxidase) and ZDHHC (Zinc fingerDHHC domain-containing protein) fusion protein particle, the type of APEX2 and ZDHHC fusion protein linker, the amount of protein used for enrichment, the conventional enrichment method for palmitoylated modified proteins, the protein precipitation method, the protein drying method, the protein reconstitution method, and the protein enzymatic digestion method. Modifications to these technical parameters that have little impact on the results are all considered to be within the scope of protection of this invention.
[0047] Example 1: Comparison of ZDHHC20 substrates enriched by different enrichment methods
[0048] For example, the method in ZL2016111333727 clones the ZDHHC20 sequence into an expression vector containing APEX2-2×FLAG to construct an APEX2-2×FLAG-ZDHHC20 fusion expression vector. Similarly, the method in ZL2017113145708 constructs HeLa cell lines stably overexpressing APEX2-2×FLAG and APEX2-2×FLAG-ZDHHC20, respectively.
[0049] Collect one 10cm dish each of HeLa cells stably transfected with APEX2-2×FLAG and APEX2-2×FLAG-ZDHHC20. Add 500μL of Lysis Buffer (50mM Tris-HCl, 150mM NaCl, 4mM EDTA, 0.5% NP-40, pH=7.4) containing 1× protease inhibitor (Selleck, B14002) and 1× phosphatase inhibitor (Selleck, B15002), respectively. Scrape the collected cells into 1.5mL EP tubes, sonicate at 20% energy for 5-6 seconds, mix by rotation at 4℃ for 2 hours, centrifuge at 14000rpm for 15 minutes, and retain the supernatant. Take 20μL of the supernatant, add 10μL of 3*SDS Loading Buffer, heat at 98℃ for 10 minutes to denature proteins, and prepare the IP-WCL (FLAG immunoprecipitation group whole cell lysate sample) group sample. Figure 2Left lane 1 / 2); 20 μL of washed Anti-Flag Affinity Gel (Selleck, B23101) was added to each of the remaining supernatant samples, and the mixture was rotated and incubated overnight at 4°C. The samples were then washed three times with Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH = 7.4), 10 minutes each time. 60 μL of 1*SDS Loading Buffer was added, and the proteins were denatured at 98°C for 10 minutes to prepare the FLAG-beads pull-down (FLAG immunoprecipitation) group samples. Figure 2 Left lane 3 / 4).
[0050] Three 10cm dishes containing stable APEX2-2×FLAG-ZDHHC20 cells were cultured to 80% confluence. The culture medium was discarded, and the cells were washed once with PBS. 5 mL of preheated RPMI-1640 medium (gibco, C11875500BT) containing 0.5 mM biotinylate was added, and the cells were incubated at 37°C for 30 minutes. After discarding the culture medium, the cells were washed again with PBS. Two dishes were labeled with 3 mL of DPBS (Dubor's phosphate-buffered saline) containing 1 mM H2O2 for 1 minute on a shaker. The unlabeled dish was the one without H2O2. 3 mL of quenching solution (5 mM Trolox, 10 mM sodium ascorbate, 10 mM NaN3, the remainder PBS) was added to each dish to terminate the reaction. The cells were washed for 1 minute on a shaker, and this process was repeated three times. Finally, 3 mL of PBS was added to each dish, and the cells were washed for 1 minute on a shaker, and this process was repeated three times. The cells were then quenched with liquid nitrogen. Three cell trays were each added with 500 μL of Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH = 7.4) containing ML211 (Taoshu Biotechnology, T8974) to a final concentration of 10 μM, 1× protease inhibitor (Selleck, B14002), and 1× phosphatase inhibitor (Selleck, B15002). The lysate was scraped and collected into 1.5 mL EP tubes, sonicated at 20% energy for 5-6 seconds, mixed by rotation at 4°C for 2 hours, and centrifuged at 14000 rpm for 15 minutes, retaining the supernatant. 20 μL of supernatant was taken from both the unlabeled group and one of the labeled groups, and 10 μL of 3*SDS Loading Buffer was added to each. The cells were then incubated at 98°C for 10 minutes to denature the proteins, preparing two WCL (whole cell lysate) samples for both the unlabeled and labeled groups. Figure 2Lanes 1 and 2 on the right side); 20 μL of washed Streptavidin-Sepharose Beads (BioVision, 6565-5) were added to each of the three remaining supernatant samples (one unlabeled group and two labeled groups). After mixing at 4℃ overnight, the mixture was washed three times with Lysis Buffer for 10 minutes each time. 60 μL of 1*SDS Loading Buffer was added to the unlabeled group and one of the labeled groups, and the protein was denatured at 98℃ for 10 minutes. Two sets of Streptavidin-beads pull-down (streptavidin immunoprecipitation) samples were prepared, one unlabeled and one labeled. Figure 2 Right lanes 3 and 4).
[0051] Add 280 μL of Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH 7.4) containing 6 M guanidine hydrochloride (GuHCl) without inhibitors to the remaining labeled sample group. Denature the protein at 98 °C for 10 min, then centrifuge at 14,000 rpm for 5 min at room temperature and retain all supernatant. Add 20 μL of 200 mM TCEP to the supernatant and incubate at 25 °C, 800 rpm for 30 min with a constant temperature shaker. Add freshly prepared 1 M maleimide to a final concentration of 25 mM and incubate at 25 °C, 800 rpm for 120 min with a constant temperature shaker. Add 600 μL of ice-cold methanol and vortex at speed up to level 10 for 30 seconds. Then add 225 μL of ice-cold chloroform and vortex at speed up to level 30 for 30 seconds. Finally, add 300 μL of ice-cold water and vortex at speed up to level 30 for 5 minutes. Centrifuge at 15,000 rpm for 5 minutes at 4°C. Three layers will form with a protein membrane in the middle. Discard the supernatant and add 1 mL of ice-cold methanol. Vortex at speed up to level 30 for 5 minutes. Centrifuge at 15,000 rpm for 5 minutes at 4°C. The protein will precipitate at the bottom. Discard most of the supernatant, leaving approximately 100 μL. Freeze-dry under vacuum for 10 minutes. Add 95 μL of inhibitor-free Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH = 7.4) containing 4% (v / v) sodium dodecyl sulfate (SDS). Incubate at 37°C and 1200 rpm for 15 minutes using a constant temperature shaker to reconstitute the protein. Add 140 μL of inhibitor-free Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH 7.4), 50 μL of washed HighCapacity Acyl-Rac Capture Beads (NANOCS, AR-S3-1), and 15 μL of 50 wt% hydroxylamine aqueous solution (i.e., final concentration 600 mM). Mix by rotation at room temperature for 150 minutes. Wash three times at room temperature for 10 minutes each time with inhibitor-free Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH 7.4) containing 1% SDS. Add 60 μL of 1*SDS Loading Buffer containing 10 mM TCEP and denature the protein at 98 °C for 10 minutes to prepare the tandem enrichment group sample. Figure 2 Right lane 5).
[0052] Samples from the IP-WCL group, FLAG-beads pull-down group, WCL group, Streptavidin-beadspull-down group, and tandem enrichment group were loaded into PAGE gels at concentrations of 3 μL, 30 μL, 30 μL, and 30 μL, respectively. After gel running, samples were stained with Coomassie Brilliant Blue (Beyotime, P0017A) kit. Figure 2 As shown, the Streptavidin-beads pulldown group had lower levels of non-specific binding proteins compared to the FLAG-beads pulldown group, while the tandem enrichment group showed further improved specificity.
[0053] Example 2: Identification of ZDHHC9 substrates enriched by tandem APEX2 proximity markers and Acyl-RAC palmitoylation modification based on DIA proteomics
[0054] For example, the method in ZL2016111333727 clones the ZDHHC9 sequence into an expression vector containing APEX2-2×FLAG to construct an APEX2-2×FLAG-ZDHHC9 fusion expression vector. Similarly, the method in ZL2017113145708 constructs HeLa cell lines stably overexpressing APEX2-2×FLAG and APEX2-2×FLAG-ZDHHC9, respectively.
[0055] Twenty 10 cm dishes each of HeLa cells stably transfected with APEX2-2×FLAG and APEX2-2×FLAG-ZDHHC9 were cultured to 80% confluence. After discarding the culture medium, the cells were washed with PBS. 5 mL of preheated RPMI-1640 medium (gibco, C11875500BT) containing 0.5 mM biotinylate was added, and the cells were incubated at 37°C for 30 minutes. The culture medium was discarded, and the cells were washed once with PBS. 3 mL of DPBS containing 1 mM H2O2 was added, and the cells were labeled by shaking for 1 minute. The reaction was terminated by adding 3 mL of quenching buffer (5 mM Trolox, 10 mM sodium ascorbate, 10 mM NaN3, the remainder PBS). The cells were washed by shaking for 1 minute, and this process was repeated three times. Finally, the cells were quenched with liquid nitrogen. Add 300 μL of Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH = 7.4) containing ML211 (Taoshu Biotechnology, T8974), 1× protease inhibitor (Selleck, B14002), and 1× phosphatase inhibitor (Selleck, B15002) to each cell tray. Scrape and collect the cells into 15 mL centrifuge tubes, sonicate at 20% energy for 5-6 seconds, mix by rotation at 4°C for 2 hours, and centrifuge at 14000 rpm for 15 minutes to collect the supernatant. Protein concentration was determined using a BCA protein quantification kit (Tiangen, PA115-02). BSA (bovine serum albumin) was added at concentrations of 2000 / 1500 / 1000 / 750 / 500 / 250 / 125 / 25 / 0 μg / mL to obtain BCA quantification standard curves. Based on the established standard curves, the protein amounts in the APEX2-2×FLAG group and the APEX2-2×FLAG-ZDHHC9 group were approximately 40.21 mg and 39.87 mg, respectively.Based on the calculation of approximately 5 μL of Streptavidin-Sepharose Beads (Bio Vision, 6565-5) per mg of protein, add 200 μL of washed Streptavidin-Sepharose Beads to the supernatant, mix by rotation at 4°C overnight, discard the solution, and wash Streptavidin-Sepharose Beads three times with 5 mL Lysis Buffer for 10 minutes each time. Add 280 μL of Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH = 7.4) containing 6 M guanidine hydrochloride (GuHCl) without inhibitors to the washed Streptavidin-Sepharose Beads, incubate at 98°C for 10 minutes, then centrifuge at 14,000 rpm for 5 minutes at room temperature, and retain all supernatant. Add 20 μL of 200 mM TCEP to the supernatant and treat with a constant temperature shaker at 25°C and 800 rpm for 30 minutes. Add freshly prepared 1M maleimide to a final concentration of 25mM, and incubate at 25°C and 800rpm for 120 minutes using a constant-temperature shaker. Add 600μL of ice-cold methanol and vortex for 30 seconds at maximum speed, followed by 225μL of ice-cold chloroform and vortex for 30 seconds at maximum speed. Finally, add 300μL of ice-cold water and vortex for 30 seconds at maximum speed. Centrifuge at 15000rpm for 5 minutes at 4°C. Three layers will form, with a protein membrane in the middle. Discard the supernatant, add 1mL of ice-cold methanol, vortex for 30 seconds at maximum speed, and centrifuge at 15000rpm for 5 minutes at 4°C. The protein will precipitate at the bottom. Discard most of the supernatant, leaving approximately 100μL, and freeze-dry under vacuum for 10 minutes. Add 95 μL of inhibitor-free Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH = 7.4) containing 4% SDS, and treat with a constant temperature shaker at 37°C and 1200 rpm for 15 minutes to reconstitute the protein.Add 140 μL of inhibitor-free Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH=7.4), 50 μL of washed High Capacity Acyl-Rac Capture Beads (NANOCS, AR-S3-1), and 15 μL of 50% (wt) hydroxylamine aqueous solution (i.e., final concentration 600 mM). Mix by rotation at room temperature for 150 minutes. Remove the liquid and wash the High Capacity Acyl-Rac Capture Beads three times at room temperature for 10 minutes each time with 1 mL of inhibitor-free Lysis Buffer (50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40, pH=7.4) containing 1 mL of 1% SDS. Wash three times at room temperature for 10 minutes each time with 1 mL of PBS containing 8 M urea and 100 mM ammonium bicarbonate. Wash three times with 1 mL of PBS containing 100 mM ammonium bicarbonate at room temperature for 10 minutes each time. Resuspend High Capacity Acyl-Rac Capture Beads in 200 μL of PBS containing 100 mM ammonium bicarbonate, add 0.5 μg trypsin (Merck, 4370285), and incubate at 37°C and 1600 rpm for 18 hours using a constant temperature shaker. Centrifuge at 14000 rpm for 5 minutes, retain the supernatant to obtain the enzymatically digested peptides, and add 2.5 μL of formic acid to the supernatant to adjust the pH to <3. Desalt using a desalting column (Waters OASIS HLB Extraction Cartridge, WAT094225), and freeze-dry under vacuum for later use. HeLa cells stably transfected with APEX2-2×FLAG and APEX2-2×FLAG-ZDHHC9 were designated as the HAC group and HD9C group, respectively. Each sample was biologically replicated three times.
[0056] Both the HAC and HD9C groups used the DIA method to collect proteomics data. The DIA proteomics data were searched using Spectronaut software, and differentially expressed proteins were analyzed using Perseus software.
[0057] Two sets of proteomics samples were reconstituted with 0.1% formic acid aqueous solution and injected at 500 ng. Separation was performed on an EASY-nLC1200 liquid chromatography system, and the samples were compared with Orbitrap Exploris. TMIdentification was performed using a 480 MHz mass spectrometer. Peptides were separated using a C18 analytical column (150 μm × 150 mm, 2 μm). Mobile phase A was 0.1% (v / v) formic acid, and mobile phase B was 80% (v / v) acetonitrile / 0.1% (v / v) formic acid. Gradient elution conditions were as follows: 3% to 15% B, 0.5 min; 15% to 50% B, 15 min; 50% to 90% B, 1.5 min; 90% B, 3 min. The flow rate was 1000 nL / min. Orbitrap Exploris TM The DIA mode parameters for the 480 mass spectrometer were set as follows: ion transport capillary temperature 320℃; spray voltage 2.5 kV; MS / MS resolution 30000 at a mass-to-charge ratio of 500; automatic gain control (AGC) target 3 × 10^6; ion implantation time 54 ms; mass range 500-900; 50 windows of 8 m / z each within the mass range; high-energy collision dissociation (HCD) collision energy 32%. All data were acquired in positive ion mode in profilometry, and peptide matching was disabled. Raw DIA files were analyzed using Spectronaut v19 software in a library-free workflow (direct DIA), with default settings used unless otherwise specified. Identification searches were performed in the UniProt Human Proteome Database (UniProt Reference Proteome, proteome ID UP000005640, 20,387 entries, downloaded August 22, 2022). The search parameters for Spectronautv19 were set as follows: Trypsin / P was used as the digestive enzyme, peptide length ranged from 7 to 52 amino acids, the maximum number of missed cuts was set to 2, carbamoyl methylation on cysteine was used as a fixed modification, and acetylation at the N-terminus of the protein and oxidation of methionine were used as variable modifications, with a maximum number of modifications set to 5. The false discovery rate (FDR) at the precursor, peptide, and protein levels was set to 0.01. The protein level results from the Spectronaut search were exported, and proteins with 2 or more missing values in the HD9C group were filtered out. After median standardization for each group, a log2 transformation was performed, and missing values were filled with random decimals (the mean was shifted down by 1.8 standard deviations, and the standard deviation was 0.3 times the original dataset standard deviation). The Significance Analysis of Microarrays (SAM) algorithm was used to analyze differentially expressed proteins, with an FDR of 0.05 and an S0 of 0.1. Figure 3 The blue portion represents the differentially expressed protein, which is the substrate for palmitoylation modification that ZDHHC9 may catalyze.
Claims
1. A method for enriching protein palmitoyltransferase substrates, characterized in that, The method integrates APEX2's proximity labeling technology with Acyl-RAC (Acyl-Rac ...
2. The method as described in claim 1, characterized in that: It includes the following steps: (1) Biotin labeling of specific palmitoyltransferase-interacting proteins in cells was performed using APEX2 proximity labeling technology; (2) Enrichment of APEX2-near-labeled biotinylated proteins using the biotin-streptavidin system; (3) Elution of APEX2-adjacent biotinylated proteins enriched by streptavidin; (4) Based on step (3), palmitoylated proteins are enriched using Acyl-RAC technology; (5) Use proteomics techniques to identify substrates of specific palmitoyltransferases enriched in tandem.
3. The method as described in claim 2, characterized in that: Step (1) involves biotin labeling of specific palmitoyltransferase-interacting proteins in cells using APEX2 proximity labeling technology, which includes the following steps: (1) Construct a cell line that stably expresses the APEX2 fusion protein with a specific palmitoyltransferase; (2) Culture the cells constructed in step (1) to a density of 60-80%, discard the culture medium and wash with PBS; (3) Add 3-7 mL of preheated culture medium containing 0.1-1 mM biotinylate (CAS: 41994-02-9) at 37℃ to a 10 cm culture dish, preferably 5 mL of 0.5 mM, and incubate at 37℃ for 15-60 minutes, preferably 30 minutes; discard the culture medium and wash the cells with PBS. (4) Add 2-5 mL of DPBS (Dubor's phosphate buffer) containing 0.1-5 mM H2O2 to a 10 cm culture dish, preferably 3 mL of 1 mM, and label it on a shaker for 30 seconds to 3 minutes, preferably 1 minute; (5) Add 2-5 mL of quenching solution to a 10 cm culture dish to stop the reaction, preferably 3 mL. Wash on a shaker for 30 seconds to 3 minutes, preferably 1 minute, and repeat three times. Then wash with PBS 3-5 times and quench the cells with liquid nitrogen. The quenching solution consists of: 5 mM quinoline dimethacrylate (Trolox, CAS: 53188-07-1), 10 mM sodium ascorbate (CAS: 134-03-2), 10 mM sodium azide (NaN3), and the remainder is PBS.
4. The method as described in claim 2, characterized in that: Step (2) involves enriching APEX2-near-labeled biotinylated proteins using the biotin-streptavidin system, which includes the following steps: (1) Add 200-500 μL of Lysis Buffer containing protease inhibitor, phosphatase inhibitor, and ML211 (CAS: 2205032-89-7) at a final concentration of 10 μM to the cells quenched in a 10 cm culture dish. Scrape the cells, preferably in 300 μL, and sonicate for 5-6 seconds. Centrifuge at 12000-16000 rpm for 10-20 minutes and take the supernatant to determine the protein concentration. Centrifugation at 14000 rpm for 15 minutes is preferred. (2) Add 3-10 μL of cleaned biotin-capturing enrichment carrier per 1 mg of protein, preferably 5 μL, and enrich overnight at 4°C to obtain the enrichment carrier that binds biotinylated protein. Enrichment carriers capable of capturing biotin include, but are not limited to, pre-conjugated streptavidin agarose beads, pre-conjugated streptavidin magnetic beads, pre-conjugated avidin agarose beads, and pre-conjugated avidin magnetic beads.
5. The method as described in claim 2, characterized in that: In step (3), the APEX2-adjacent biotinylated proteins enriched by streptavidin are eluted, which includes the following steps: (1) Wash the enrichment carriers containing biotinylated proteins with Lysis Buffer at room temperature or 4°C for a total of 3 washes, each lasting 10 minutes. (2) Elution and enrichment of carrier-bound biotinylated proteins were carried out by competitive elution or denaturation elution. (2-1) The competitive elution method is as follows: use 10-20 μL of Lysis Buffer containing 1-10 mM desulfurized biotin (CAS: 533-48-2) for every 10 μL of enrichment carrier, preferably 14 μL of 2.5 mM, and mix by rotation at room temperature or 4°C for 10-45 minutes. (2-2) The denaturation elution method is as follows: use 10-20 μL of Lysis Buffer containing 6M guanidine hydrochloride (CAS: 50-01-1) or 8M urea (CAS: 57-13-6) for every 10 μL of enrichment carrier, preferably 14 μL, and heat at 98℃ for 5-15 minutes.
6. The method as described in claim 2, characterized in that: Step (4) involves enriching palmitoylated proteins among the eluted biotinylated proteins using Acyl-RAC technology, which includes the following steps: (1) Centrifuge the eluent and take the supernatant. Add tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP, CAS: 51805-45-9) to the supernatant with a final concentration of 1-50mM, preferably 10mM. Mix at 25°C for 10-60 minutes, preferably 30 minutes. (2) Add freshly prepared maleimide (CAS: 541-59-3) with a final concentration of 5-50 mM or freshly prepared iodoacetamide (CAS: 144-48-9) with a final concentration of 10-100 mM. Maleimide is preferably 25 mM and iodoacetamide is preferably 50 mM. Mix at 25°C in the dark for 60-180 minutes, preferably 120 minutes. (3) The protein was precipitated using the MeOH / CHCl3 / H2O system, the methanol was evaporated, and the protein precipitate was dried. (4) Reconstitute the protein using Lysis Buffer containing 4% SDS; (5) Add 1-2 μL of a cleaned enrichment carrier that can capture free sulfhydryl groups per 1 mg of protein, preferably 1.25 μL, and hydroxylamine (CAS: 7803-49-8) at a final concentration of 200-1200 mM, preferably 600 mM. Mix at room temperature for 60-180 minutes to enrich palmitoylated modified proteins, preferably 120-150 minutes. Enrichment carriers capable of capturing free thiol groups include, but are not limited to, activated thiol agarose beads, thiopropyl agarose beads, activated thiol magnetic beads, and thiopropyl magnetic beads. (6) Wash with Lysis Buffer containing 1% SDS 3-5 times at room temperature, 8-10 minutes each time; (7) Wash the sample three times at room temperature with a 100mM NH4HCO3 aqueous solution containing 8M urea for 10 minutes each time. After enzymatic desalting, the sample was subjected to proteomics detection by liquid chromatography-mass spectrometry.
7. The method as described in claim 4, 5, or 6, characterized in that: The Lysis Buffer consists of: 50 mM Tris-HCl, 150 mM NaCl, 4 mM EDTA, 0.5% NP-40 (CAS: 9016-45-9), and the remainder is water. The pH is between 7.0 and 8.0, with 7.4 being preferred.
8. The detection method as described in claim 6, characterized in that: The steps for precipitating proteins using the MeOH / CHCl3 / H2O system are as follows: Add methanol to the protein reaction system and vortex for 20-40 seconds, then add chloroform and vortex for 20-40 seconds, then add water and vortex for 20-40 seconds, centrifuge at 0-4℃ and 14000-20000 rpm and discard the supernatant, then add methanol again and vortex for 30 seconds, centrifuge at 0-4℃ and 14000-20000 rpm and discard the supernatant.
9. The detection method as described in claim 6 or 8, characterized in that: The volume ratio of the MeOH / CHCl3 / H2O system is 350-500:100-500:200-500, with 400:150:400 being preferable.