Protocols for rapid identification of small-molecule metabolite ligands interacting with proteins

Summary

Metabolite-protein interactions have not been systematically studied due to a lack of effective techniques. Here, we present a protocol for identifying small-molecule metabolite ligands interacting with proteins. We describe steps for mixing the sample with antibodies for immunoprecipitation and applying organic solvent to extract small-molecule metabolites. We then detail procedures for using high-resolution mass spectrometry to quantify small-molecule metabolites bound to proteins. We validate protocol feasibility using the example of an arachidonic acid-Menin protein interaction.

For complete details on the use and execution of this protocol, please refer to He et al.¹

Graphical abstract

Highlights

• •Instructions for preparing endogenous, exogenous, and purified proteins
• •Steps for identifying metabolite ligands interacting with proteins
• •Guidance on recognizing and validating potential protein-metabolite interacting complex

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Metabolite-protein interactions have not been systematically studied due to a lack of effective techniques. Here, we present a protocol for identifying small-molecule metabolite ligands interacting with proteins. We describe steps for mixing the sample with antibodies for immunoprecipitation and applying organic solvent to extract small-molecule metabolites. We then detail procedures for using high-resolution mass spectrometry to quantify small-molecule metabolites bound to proteins. We validate protocol feasibility using the example of an arachidonic acid-Menin protein interaction.

Before you begin

The protocol below describes the specific steps for the identification of interactions between arachidonic acid and subunit proteins of the compass complex, including Menin, WDR5, or WDR82. This protocol provides a detailed description for the detection of interactions between endogenous, exogenous, or purified proteins with arachidonic acid.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit monoclonal antibody anti-WDR5 (IP: 1:200)	Cell Signaling Technology	Cat# 13105; RRID: AB_2620133
Rabbit monoclonal antibody anti-WDR82 (IP: 1:200)	Cell Signaling Technology	Cat# 99715; RRID: AB_2800319
Rabbit monoclonal antibody anti-Menin (IP: 1:200)	Abcam	Cat# ab92443; RRID: AB_10564144
Rabbit polyclonal antibody anti-IgG (IP: 1:200)	Cell Signaling Technology	Cat# 2729; RRID: AB_1031062
Bacterial and virus strains
DH5α-competent cells	Beyotime	Cat# D1031S
Rosetta competent cells	Beyotime	Cat# D1065S
Chemicals, peptides, and recombinant proteins
Anti-Flag affinity gel	Bimake	Cat# B23101
IPTG	Biomed	Cat# SH401-01
NP-40	Sangon Biotech	Cat# A600385
5 M NaCl	Beyotime	Cat# ST347
1 M Tris-HCl pH 6.8	Beyotime	Cat# ST768
0.5 M EDTA	Beyotime	Cat# C0196
100 mM MgCl2	Beyotime	Cat# R0058
3 M KCl	Beyotime	Cat# ST345
0.5 M DTT	Beyotime	Cat# ST041
100× Protease Inhibitor Cocktail	GlpBio	Cat# GK10019
TritonX-100	Beyotime	Cat# P0096
Arachidonic acid	GlpBio	Cat# GC31725
Protein A/G agarose	MCE	Cat# HY-K0230
Bradford Protein Quantification Kit	GlpBio	Cat# GK10027
Methanol	Sangon Biotech	Cat# A601617
Acetonitrile	Sangon Biotech	Cat# A610870
Formic acid	Sangon Biotech	Cat# A503066
Acetonitrile	Sangon Biotech	Cat# A610870
RE BamHI	NEB	Cat# R0136V
RE XhoI	NEB	Cat# R0146V
RE KpnI-HF	NEB	Cat# R3142V
RE HindIII	NEB	Cat# R0104V
RE EcoRI	NEB	Cat# R0101V
Trypsin	Gibco	Cat# R001100
DMEM medium, high glucose (DMEM)	Gibco	Cat# 11960077
Fetal bovine serum (FBS)	Gibco	Cat# 10099-141
Lipofectamine 3000	Thermo Fisher Scientific	Cat# L3000001
Opti-MEM medium	Thermo Fisher Scientific	Cat# 31985070
Critical commercial assays
USER cloning system	NEB	Cat# M5505L
His-tag protein purification Kit	QUALITYARD	Cat# QYP1062
Gel Extraction Kit	TIANGEN	Cat# DP208
Plasmid Extraction Kit	TIANGEN	Cat# DP105
PfuTurbo Cx Hotstart DNA Polymerase Kit	Agilent	Cat# 600410
PrimeScript RT Reagent Kit	Takara	Cat# RR037A
Experimental models: Cell lines
Human: 293T	ATCC	Cat# CRL-3216
Recombinant DNA used in this paper
Homo sapiens menin 1 (MEN1), transcript variant 3, mRNA	GeneCopoeia	Cat# L1372
Homo sapiens WD repeat domain 5 (WDR5), transcript variant 10, mRNA	GeneCopoeia	Cat# L4393
Homo sapiens WD repeat domain 82 (WDR82), mRNA	GeneCopoeia	Cat# H1270
pET-28a-Menin WT	This paper	N/A
Others
T25 cell culture flask	Corning	Cat# 430639
60 mm dish	Corning	Cat# 430166
24-well plate	Corning	Cat# 3524
1.5 mL tubes	Axygen	Cat# MCT-150-C
cell strainers	BD Falcon	Cat# 352235
0.22 mm filter	Sartorius	Cat# 17597-K
1 mL injection syringes	BD Pharmingen	Cat# 300841
EP tube	Eppendorf	Cat# 3810X
20G and 25G needles	Kunming Huangbao Trading Co.	Cat# ZSZT-6

Materials and equipment

aIP buffer	Final concentration	Amount
NP-40	N/A	100 μL
5 M NaCl	15 mM	300 μL
1 M Tris-HCl pH 6.8	20 mM	200 μL
0.5 M EDTA	10 mM	200 μL
100 mM MgCl2	1.5 mM	150 μL
3 M KCl	15 mM	50 μL
0.5 M DTT	5 mM	100 μL
100 × Protease Inhibitor Cocktail	1×	100 μL
Autoclaved Milli-Q H2O	N/A	8,800 μL
Total	N/A	10 mL

Note: ① Protease Inhibitor Cocktail should be added right before buffer use. Store at 4°C. If needed, the protease inhibitor can be stored at 4°C for up to 2 h. ② aIP buffer can be stored at 4°C for up to 3 days.

PCR reaction reagent	Final concentration	Volume
CDS expressing vector	1 μg	2 μL
Turbo cx	N/A	1 μL
10 × pfu Turbo cx buffer	1 ×	5 μL
DMSO	N/A	3 μL
dNTP (10 mM)	200 μM	1 μL
Forward primer (10 μM)	0.5 μM	0.5 μL
Reverse primer (10 μM)	0.5 μM	0.5 μL
Autoclaved Milli-Q H2O	N/A	37 μL
Total	N/A	50 μL

Always prepare fresh before each assay. Store at 4°C for up to 1 h.

Thermocycler settings included lid temperature at 105°C and volume of 50 mL

PCR reaction system	Temperature	Time	Number of cycles
Initial Denaturation	95°C	3 min	1
Denaturation	95°C	3 min	30–35
Annealing	54°C	30 s
Extension	68°C	6 min
Final extension	72°C	10 min	1
Hold	4°C	Forever	N/A

Always prepare fresh before each assay. Store at 4°C for up to 1 h.

Digest reaction reagent	Volume
PCR DNA/Plasmid	4 μL
CutSmart Buffer 10 ×	1 μL
Restriction endonucleases-1	0.5 μL
Restriction endonucleases-2	0.5 μL
Autoclaved Milli-Q H2O	4 μL
Total	10 μL

Always prepare fresh before each assay. Store at 4°C for up to 1 h.

Ligation reaction reagent	Volume
PCR DNA	6 μL
Plasmid DNA	2 μL
CutSmart Buffer 10 ×	1 μL
USER enzyme	1 μL
Total	10 μL

Always prepare fresh before each assay. Store at 4°C for up to 1 h.

Step-by-step method details

Timing: 2–4 days

Timing: 1–2 days (for step 1)

Timing: 3–4 h (for step 2)

Timing: 1–2 days (for step 3)

Interactions between endogenous proteins and arachidonic acid

Below we provide a detailed step-by-step protocol for metabolite–protein immunocomplex preparation, metabolite extraction, and quantitation of arachidonic acid interacting with proteins.

• 1.
  Preparation of immunocomplexes.

  • a.
    Wash the 293 cells twice with 1.0 mL pre-cooled phosphate buffer twice.
  • b.
    Prepare the adjusted immunoprecipitation (aIP) buffer following the instruction described in the materials and equipment section.

    • i.
      Pre-cool the aIP buffer at 4°C for least 2 h in advance.
    • ii.
      Add pre-cooled aIP buffer (1 mL/10⁷ cells) to cultured cells.
  • c.
    Use a pre-cooled cell scraper to scrape the cells off the culture dish or bottle and transfer the suspension to a clean 1.5 mL microcentrifuge tube.
  • d.
    Place it on a low-speed shaker and gently shake at 4°C for 15 min.

    Note: Insert the microcentrifuge tube into ice and place it on a horizontal shaker).

  • e.
    Centrifuge at 4°C and 12,000× g for 15 min, and immediately transfer the supernatant to a new pre-cooled centrifuge tube.
  • f.
    Extract 50 μL into a new microcentrifuge tube as 5% input lysate.
  • g.
    Wash the protein A/G agarose beads twice with 1.0 mL aIP buffer and prepare a 50% protein A/G agarose bead working solution using aIP Buffer.

    CRITICAL: Remove the tip of the tips to avoid damaging the agarose beads during operations.

  • h.
    Add 100 μL of the protein A/G agarose beads solution to the 1 mL sample.
  • i.
    Shake horizontally at 4°C for 10 min to remove non-specifically bound proteins.²
  • j.
    Centrifuge at 4°C and 12,000× g for 15 min, transfer the supernatant to a new centrifuge tube, and remove the protein A/G agarose beads.
  • k.
    Prepare a protein standard curve (using the Bradford method) and determine the protein concentration.
  • l.
    Dilute the total protein by least 1:10 times or more before measurement to reduce the impact of descaling agents in the cell lysate.

    Note: Can be stored at −20°C for one month.

  • m.
    Add a certain volume of antibody to 500 μL of total protein.

    Note: The antibody usage varies depending on the amount of total protein lysate. The specific usage amounts of antibodies are usually recommend by the manufacturer. For our tests, 1.0 μg Menin, 2.5 μg WDR5, 2.5 μg WDR82, and 2.0 μg IgG antibodies are applied for 1.0 mg total protein lysate.

  • n.
    Slowly shake the antigen antibody mixture at 4°C for 12–16 h or at 25 for 2°C for 4 h.
  • o.
    Add 100 μL protein A/G agarose beads to capture the antigen–antibody complexes, and slowly shake the antigen–antibody mixture for 12–16 h at 4°C.
  • p.
    Centrifuge at 12,000× g for 5 s, collect the agarose beads, and wash it thrice with 800 μL pre-cooled aIP buffer.

    CRITICAL: Three times of wash with pre-cooled aIP buffer is necessary to remove non-specific bindings in IP assays.

• 2.
  Metabolites extraction.

  • a.
    Add 800 μL of cold methanol/acetonitrile (1:1, v/v) to the agarose beads to extract the metabolites.
  • b.
    Collect the mixture into a new centrifuge tube. centrifuge at 14,000× g for 5 min at 4°C to collect the supernatant.
  • c.
    Dry the supernatant in a vacuum freeze dryer (BILON-FD80CE). Subsequently, perform the following operations:

    • i.
      For the vacuum drying, place the sample in the pre-freezing chamber of the freeze dryer and cool it down to −50°C.
    • ii.
      Start the vacuum pump to remove the air from the freezer and create a vacuum environment.
    • iii.
      Maintain the temperature of the freezer at −40°C–50°C, gradually sublime the moisture in the sample, and collect it into the condenser bottle through the condenser.
    • iv.
      After vacuum drying, remove the sample rack from the vacuum drying chamber and thaw it at 25°C.
    • v.
      After thawing is complete, the sample can be taken out and further operations can be carried out.
    • vi.
      For LC-MS analysis, the samples were re-dissolved in 100 μL acetonitrile/water (1:1, v/v) solvent.
• 3.
  Quantitation of arachidonic acid using LC-MS.

  • a.
    Sonicate the homogenate on ice for 30 min. Centrifuge the mixture for 10 min at 14,000× g, 4°C.
  • b.
    Use 500 μL of supernatant to extract arachidonic acid through the HLB elution system. Subsequently, perform the following operations:

    • i.
      Preactivate the HLB elution system with 200 μL methanol.
    • ii.
      Equilibrate the HLB elution system with 200 μL water.
    • iii.
      Add 1.0 mL of methanol to activate well plate for 2 times.
    • iv.
      Add 1.0 mL of pure water to the equilibrium orifice plate for 2 times.
    • v.
      Add 1.0 mL of Washing Solution A for 2 times.
    • vi.
      Add 1.0 mL of Washing Solution B for 2 times, and discard the filtrate.
    • vii.
      Add 1.0 mL of pure methanol to elute the sample, wash it twice, collect the eluent, dry it with nitrogen, and freeze it at −80°C.
  • c.
    Wash the loaded system with 200 μL water and 200 μL 10% aqueous methanol. Elute with 50 μL acetonitrile.
  • d.
    Performed analyses with an ultra-high performance liquid chromatography (UHPLC, I-Class LC, Waters) system coupled to a QTRAP system (AB Sciex 5500).
  • e.
    The mobile phase contains A: 0.1% formic acid (FA) in water and B: 0.1% formic acid in acetonitrile (ACN). Subsequently, perform the following operations:

    • i.
      Place the samples in the automatic sampler at 4°C.
    • ii.
      Keep the column temperatures constant at 45°C.
    • iii.
      The flow rate is 400 μL/min, and inject a 4 μL aliquot of each sample.
    • iv.
      The gradient is first 30% B for 0–1 min, 30% B linearly increased to 80% for 1–7 min, and then increased to 90% for 7–9 min and kept for 9–11 min.
  • f.
    Use the QC samples for testing and evaluation of the stability and repeatability of this system. In the ESI negative mode, the conditions settings as as follows: source temperature 450°C, ion Source Gas1 (Gas1): 55, Ion Source Gas2 (Gas2): 60, Curtain gas (CUR): 30, ionSapary Voltage Floating (ISVF) 4,500 V; the MRM mode is adopted to detect ion pairs (Table 1).
  • g.
    Quantitate the arachidonic acid abundance based on the isotope internal standard method.

    • i.
      Calculate the absolute abundance of arachidonic acid using the peak area ratio of arachidonic acid to internal standard response abundance and the concentration of internal standard.
    • ii.
      The specific quantitative steps are as follows: first, obtain the standard curve equation based on the mass spectrometry signals of different concentrations of standard samples.
    • iii.
      Then, using the standard curve (Figure 1), the software automatically calculates the concentration of different metabolites in each sample.
    • iv.
      Calculate the content information (μmol/g or μmol/L) based on the initial mass or volume of the sample.

Table 1.

The MRM ion pair parameters

Metabolite name	Arachidonic acid
Mass information	303.2/259.2
Retention Time (min)	7.96
Linear	Y = 9.794E-4X + 0.0080
R value	0.9993
Linear range (ng/mL)	0.25–1000
Limit of detection (LOD, ng/mL)	0.05
Limit of quantitation (LOQ, ng/mL)	0.25
Upper limit of quantification (ULOQ, ng/mL)	1000

Figure 1.

The standard curve equation based on the mass spectrometry signals of different concentrations of standard arachidonic acid

Interactions between exogenous proteins with arachidonic acid

Timing: 2–3 weeks

Timing: 3–4 days (for step 4)

Timing: 3–4 days (for step 5)

Timing: 2–3 days (for step 6)

Timing: 3–4 h (for step 7)

Timing: 1–2 days (for step 8)

Below we provide a detailed step-by-step protocol regarding Menin (WDR5, or WDR82) coding sequence (CDS)-expressing construct preparation, transfection, metabolite–protein immunocomplex preparation, metabolites extraction, and quantification of arachidonic acid interacting with proteins.

• 4.
  Construct preparation.

  • a.
    Purchase expressing vectors containing full-length CDS of Menin (WDR5, or WDR82) from GeneCopoeia.
  • b.
    Conduct polymerase chain reaction (PCR) using a PfuTurbo Cx Hotstart DNA Polymerase kit described in the materials and equipment section. The PCR reaction conditions are listed in the materials and equipment section.

    Note: Here, we use Menin (-WDR5, or -WDR82) full-length CDS expressing plasmid as a template, design primers with a USER specific sequence at both ends, and use high fidelity enzymes to amplify Menin, WDR5, or WDR82 full-length CDS into the pCMV-3 × FLAG vector.

  • c.
    Separate PCR products by agarose gel electrophoresis and then isolate the required gene fragments using a gel extraction kit.

    Note: In our assay, add 20 μL ddH₂O to elute and dissolve Menin (WDR5, or WDR82) CDS fragments.

  • d.
    Use two restriction endonucleases (REs) to cleave the pCMV-3 × FLAG vector or CDS fragments following the instruction described in the materials and equipment section.
  • e.
    Incubate at 37°C for 3 h. REs for Menin: BamHI and XhoI; REs for WDR5: BamHI and KpnI; REs for WDR82: HindIII and EcoRI.
  • f.
    Ligate the CDS fragments of Menin, WDR5, or WDR82 to the pCMV-3 × FLAG linear vector following the instruction described in the materials and equipment section. Ligation reaction conditions: 37°C for 30 min, followed by 25°C for 1 h.
  • g.
    Bacterial transformation.

    • i.
      Mix 1–5 μL of DNA (usually 10 pg to 100 ng) into 20–50 μL of DH5α competent cells in a microcentrifuge or falcon tube.
    • ii.
      Gently mix by flicking the bottom of the tube with your finger a few times.
    • iii.
      Incubate the competent cell/DNA mixture on ice for 20–30 min.³
    • iv.
      Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube in a 42°C water bath for 30–60 s.

      Note: 45 s is usually ideal, but this varies depending on the competent cells you are using.

    • v.
      Add 250–1,000 μL LB media (without antibiotics) to the bacterial culture and grow in a 37°C shaking incubator for 45 min.

      CRITICAL: Make sure the LB media without antibiotics was used in this step.

    • vi.
      Plate some or all of the transformants onto a 10 cm LB agar plate containing kanamycin. Incubate the plates at 37°C for 12–16 h.
    • vii.
      Select bacterial clones, amplify bacterial, extract plasmids, and perform Sanger-sequence and identification, and obtain recombinant plasmids pCMV-3 × FLAG-Menin (-WDR5, or -WDR82).
• 5.
  Transfection in 293 cells.

  • a.
    Wash 293 cells twice with 5.0 mL PBS, add appropriate amount of trypsin, and place them at 37°C for 2–3 min.

    Note: When all cells are detached, add FBS-containing medium to terminate digestion.

  • b.
    Centrifuge at 1,000× g for 5 min, discard the supernatant, add an appropriate amount of culture medium to resuspend the cells, and seed the corresponding number of cells according to the surface area of different culture vessels.
  • c.
    Cultivate the cells for 12–16 h at 37°C in a humidified atmosphere with 5% CO₂ in conventional conditions.
  • d.
    When the cell confluence reaches 70%–90%, perform transfection of plasmids.

    • i.
      Dilute the culture using Lipofectamine 3000 reagent with Opti-MEM medium to obtain solution A.
    • ii.
      Dilute plasmids with Opti-MEM medium to obtain solution B.
    • iii.
      Mix solution A with solution B and incubate at 25°C for 10–15 min.
  • e.
    Add the transfectants to the medium and incubate the cells at 37°C for 48 h.
• 6.
  Preparation of immunocomplexes.

  • a.
    After 48 h of transfection, wash the cells twice with 5.0 mL PBS and scrape the cells off.
  • b.
    Centrifuge at 1,200× g for 10 min, discard the supernatant, add 1 mL of aIP buffer, mix the cells, and lyse on ice for 15 min.
  • c.
    Centrifuge at 12,000× g for 10 min at 4°C, transfer the supernatant to a new microcentrifuge tube, and discard the precipitate.
  • d.
    Extract 50 μL into a new microcentrifuge tube as 5% input lysate.
  • e.
    Add the 100 μL washed anti-FLAG agarose beads to the lysis buffer and rotate at 4°C for at least 4 h.
  • f.
    Centrifuge at 1,500× g, 4°C for 3 min, discard the supernatant, and wash the agarose beads 3 times with 1.0 mL aIP buffer.
• 7.Metabolite extraction.

The experimental steps are same as described in the section of 2.

• 8.Quantitation of arachidonic acid using LC-MS.

The experimental steps are same as described in the section of 3.

Interactions between purified proteins with arachidonic acid

Timing: 2–3 weeks

Timing: 3–4 days (for step 9)

Timing: 2–3 days (for step 10)

Timing: 2–3 days (for step 11)

Timing: 3–4 h (for step 12)

Timing: 1–2 days (for step 13)

Below we provide a detailed step-by-step protocol regarding Menin protein purification, in-vitro binding, and quantification of arachidonic acid interacting with Menin protein.

• 9.
  Construct preparation.

  • a.
    Subclone DNA for full-length Menin into a pET28a vector using gene-specific primers.

    Note: In our assay, design primers based on the sequence of the Menin gene to amplify it.

  • b.
    Conduct PCR using the information described in the materials and equipment section. The PCR reaction conditions are listed in the materials and equipment section.
  • c.
    Run a 1% agarose gel to verify the size and purity the PCR products.
  • d.
    Perform enzyme digestion of the target gene, Menin, and pET-28a plasmid following the instruction described in the materials and equipment section.
  • e.
    The enzyme digestion products are cut and recovered using a gel extraction kit.
  • f.
    Ligate both the PCR and linearized plasmid following the instruction described in the materials and equipment section.
  • g.
    Incubate the ligation reaction at 20°C–25°C for 10–15 min.

    Note: Use the insert and vector DNA at a 3:1 molar ratio.

  • h.
    Transform DH5α competent cells and select the transformants on LB agar plates containing 100 mg/mL ampicillin.

    • i.
      Incubate the plates at 37°C for 12–15 h.
    • ii.
      Inoculate a single colony in LB media supplemented with 100 mg/mL ampicillin and extract and purify the plasmid DNA using a Plasmid Extraction Kit.
    • iii.
      Sequence the DNA to confirm that the construct sequence is correct before proceeding.
• 10.
  Induced expression of Menin and identification by SDS-PAGE.

  • a.
    Transform the pET-28a Menin recombinant vector into Rosetta competent cells.
  • b.
    Cultivate the transformed bacterial Rosetta cells in LB medium containing kanamycin and chloramphenicol until the OD600 is approximately 0.6.
  • c.
    Add 0.1 M IPTG (final concentration is 0.1 mM) and incubate at 16°C for 12 h.

    CRITICAL: The optimal conditions for IPTG-induced protein expression need to consider factors including IPTG concentration, cell culture temperature, cell culture time, and cell strain.

  • d.
    Expand the bacterial suspension to a 1,200 mL bacterial suspension.
  • e.
    Centrifuge, discard the supernatant, and resuspend the bacterial solution in 18 mL phosphate buffered saline (PBS).
  • f.
    Add 1 mg of DNase to the bacterial suspension, and crush the suspension twice using a high-pressure homogenizer at a pressure of 16,000 psi.
  • g.
    Add 2 mL of 10% Triton X-100 to the mixture and mix at 4°C for 2–3 h.
  • h.
    Centrifuge the sample at 6,000× g for 30 min at 4°C and obtain the supernatant.
  • i.
    Add 1 mL of nickel column to the supernatant and mix on the Rotator at 4°C for 2 h.
  • j.
    Add the nickel column suspension to the gravity column, allow it to settle by gravity through the column.
  • k.
    Wash the protein with 2 mL eluent.
  • l.
    Centrifuge and concentrate the protein to 250 μL using Millipore ultrafiltration tubes.
  • m.
    Take 2 μL of the protein solution, add 4 μL of 5 × protein loading buffer, boil in water for 5 min, and use this as the loading sample for identification.
• 11.
  In-vitro binding assay.

  • a.
    Add 0.1 mg purified Menin protein and 5 μM arachidonic acid to the aIP buffer to make a final 1 mL volume.
  • b.
    Incubate for 6 h at 37°C.
  • c.
    Add anti-IgG or anti-Menin antibodies for the IP assay.⁴
  • d.
    The subsequent steps for the preparation of immunocomplexes are same as those described before.
• 12.Metabolite extraction.

The experimental steps are same as described in the section of 2.

• 13.Quantitation of arachidonic acid using LC-MS method.

The experimental steps are same as described in the section of 3.

Expected outcomes

This protocol describes three approaches for the confirmation of direct bindings between arachidonic acid and endogenous proteins including Menin, WDR5, and WDR82 (Figure 2), arachidonic acid and exogenous proteins including pCMV-3 × FLAG-Menin, -WDR5 and -WDR82 (Figure 3), arachidonic acid and purified Menin protein (Figure 4).

Figure 2.

Identification of interactions between endogenous proteins and arachidonic acid

293 cell lysates were immunoprecipitated using antibodies against Menin, WDR5 and WDR82. Metabolites in the immunocomplex were extracted using methanol. Quantitative abundance of arachidonic acid was measured using a LC-MS-based metabolites detection method.

Figure 3.

Identification of interactions between exogenous proteins with arachidonic acid

Expression constructs of six regulatory subunits of the Compass complex (pCMV Flag-Menin, -WDR5, -WDR82) were transfected into 293T cells for 72 h. Cell lysates were immunoprecipitated using anti-FLAG agarose beads. Metabolites in the immunocomplex were extracted using methanol. Quantification of arachidonic acid was measured using a LC-MS-based metabolites detection method.

Figure 4.

Identification of interactions between purified protein with arachidonic acid

Incubation of 0.1 mg purified 6 × His-Menin WT protein with 5 μM AA was conducted at 37°C for 6 h. Immunoprecipitation was performed using anti-Menin antibody. Metabolites in the immunocomplex were extracted using methanol. Quantitative abundance of arachidonic acid was measured using a LC-MS-based metabolites detection method.

Limitations

Only metabolites that have been characterized can be detected using this protocol, and the protocol cannot be used for the detection of unknown metabolites.

Troubleshooting

Problem 1

No PCR fragment amplified or incorrect size of the PCR product (step 1 of the Constructs preparation).

Potential solution

Optimize the primer annealing temperature by running a gradient PCR and optimize the primer extension time.

Problem 2

Few or no transformants (constructs preparation).

Potential solution

Check the efficiency of the competent cells by transforming a control circular plasmid. Also check the temperature of the water bath and time for heat shock.

Problem 3

Less purified protein obtained.

Potential solution

Optimize the conditions for inducing protein expression. For example, induce expression for 12–16 h at 16°C, 20°C, 24°C, 28°C, 32°C, and 36°C, respectively.

Problem 4

Few or no metabolites detected in the immunocomplexes of purified proteins.

Potential solution

Prolong the incubation time to a maximum of 16 h at 37°C in the in-vitro binding assay.

Problem 5

How to compare the differences in metabolite levels bound to the equivalent amount of exogenous protein?

Potential solution

Due to the different expression efficiency of each exogenous overexpressing vector in cells, one can adjust the plasmid transfection amount by quantifying the band grayscale values during western blotting to ensure that the amount of each protein precipitated by Tag-beads in the IP experiment remains equivalent.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Qingli Bie (xiaobie890101@163.com).

Technical contact

Technical questions on executing this protocol should be directed to Baoyu He (hebaoyu99@sina.com).

Materials availability

This study did not generate any new unique reagents. Further information requests about materials and reagents should be directed to the lead contact, Dr. Qingli Bie (xiaobie890101@163.com).

Data and code availability

This study did not generate any unique data sets or code.

Acknowledgments

The authors would like to thank our funding sources, including the National Natural Science Foundation of China (no. 82472959, 82173371, 82273447, 82273069, and 82372679), the Tai Shan Young Scholar Foundation of Shandong Province (no. tsqn201909192 and tsqn202312383), and the Research Fund for Lin He’s Academician Workstation of New Medicine and Clinical Translation in Jining Medical University, Shandong, China (no. YHL2022F2D04).

Author contributions

B.H. and R.Z. performed the experiments. B.Z. and Q.B. wrote the manuscript.

Declaration of interests

The authors declare no competing interests.