Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

1. Analysis of IP- or PI-binding proteins by affinity chromatography and Western blotting

1. Cell growth, lysis and affinity chromatography
   1. Grow T. brucei cells to mid-log phase and monitor cell viability and density. A total of 5.0 x 10⁷ cells is sufficient for one binding assay.
      1. For bloodstream forms, grow cells in HMI-9 media supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂. Keep cell density between 8.0 x 10⁵ to 1.6 x 10⁶ cells/mL.
         NOTE: Density higher than 1.8 x 10⁶ cells/mL may affect cell viability. The doubling-time of T. brucei 427 strain grown in vitro is between 5.5 and 6.5 h.
      2. For procyclic forms, grow cells in SDM-79 medium supplemented with 10% FBS at 27 °C and keep cell density between 1.0 x 10⁷ and 3.0 x 10⁷ cells/mL.
      3. For purified proteins (e.g., recombinant proteins), take 0.5 to 1 µg of protein and dilute in 450 µL of binding buffer (25 mM HEPES, 150 mM NaCl, 0.2% 4-nonyl phenyl-polyethylene glycol, pH 7.4). Keep 5% of the diluted protein (input) for Western blot analysis. Proceed to step 1.1.7.
   2. Centrifuge cells at 1,600 x g for 10 min at room temperature (RT). Discard the supernatant.
      NOTE: See step 2.1.2 for additional information on centrifugation of large culture volumes.
   3. Gently resuspend the pellet in 10 mL of phosphate buffered saline pH 7.4 supplemented with 6 mM glucose (PBS-G) and pre-heated at 37 °C to wash the cells. Then, centrifuge the cells at 1,600 x g for 5 min at RT. Repeat the procedure twice.
   4. Resuspend the pellet in 1 mL of PBS-G. Then, transfer the volume to a 1.5 mL tube and centrifuge at 1,600 x g for 5 min. Discard the supernatant.
      NOTE: Cell pellets can be flash frozen in liquid nitrogen and stored at -80 °C or liquid nitrogen.
   5. Resuspend the pellet in 0.5 mL of lysis buffer (25 mM HEPES, 150 mM NaCl, 1% t-octylphenoxypolyethoxyethanol, pH 7.4) supplemented with 1.5x protease inhibitor cocktail and 1x phosphatase inhibitor cocktail (Table of Materials) pre-chilled in ice to lyse the cells. Incubate lysate for 10 min rotating at 50 rpm at 4 °C.
      NOTE: This is a critical step because proteins can degrade if not handled as indicated. Check integrity of protein lysate by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/PAGE) if necessary.
      CAUTION: T-octylphenoxypolyethoxyethanol is toxic and can cause skin and eye irritation. Use gloves, eyeshield and faceshield protection.
   6. Centrifuge the lysate at 14,000 x g for 10 min at 4 °C. Collect the supernatant into a new 1.5 mL tube for binding assays. Keep 5% of total lysate (input) for Western blotting analysis. The supernatant contains parasite proteins extracted with lysis buffer.
   7. Collect 50 µL of IPs or PIs conjugated to agarose beads (i.e., 50 µL of slurry) or 50 µL of agarose beads, and centrifuge for 1 min at 1,000 x g. Discard the supernatant and resuspend in 50 µL of binding buffer to equilibrate the beads. Use non-conjugated beads as a control. Use IP/PI-beads with different phosphate configuration including non-phosphorylated forms to control for unspecific interactions.
   8. Add 50 µL of IP- or PI-beads to the cell lysate or purified proteins (each 1 mL of beads contains 10 nmol of conjugated IPs or PIs). Keep the volume of IP- or PI-beads within 10% of the total lysate and if necessary, adjust the binding reaction volume with binding buffer.
      1. For competition assays, add to the binding reaction various concentrations of non-conjugated IPs or PIs (e.g., 1-, 10-, 100-fold molar excess compared to IP- or PI-beads).
   9. Incubate the reaction for 1 h, or overnight, at 4 °C and rotating at 50 rpm.
      NOTE: Binding reactions with purified proteins can be done at RT depending on the stability of the protein. If using IPs or PIs conjugated to biotin only proceed to step 1.1.9.1, otherwise proceed to step 1.1.10.
      1. Add 50 µL of streptavidin-conjugated to magnetic beads to the binding reaction and incubate for 1 h at 4 °C rotating at 50 rpm.
   10. Centrifuge the mix for 1 min at 1,000 x g at 4 °C. Remove the supernatant (flow-through) and keep the pellet. Keep 5% of the supernatant for Western blot analysis.
       NOTE: If using magnetic beads, remove supernatants and perform subsequent washes using a magnetic stand (centrifugations are not necessary).
   11. Add 1 mL of washing buffer (25 mM HEPES, 300 mM NaCl, 0.2% 4-nonyl phenyl-polyethylene glycol, pH 7.4) and resuspend the resin by tapping or swirling the tube (do not use a pipette because beads can attach to pipette tips). Centrifuge the reaction for 1 min at 1,000 x g at 4 °C and discard the supernatant. Repeat the procedure for a total of five washes.
       CAUTION: 4-nonyl phenyl-polyethylene glycol is toxic and can cause skin and eye irritation. Use gloves, eyeshield and faceshield protection.
   12. Add 50 µL of 2x Laemmli buffer supplemented with 710 mM 2-mercaptoethanol to the beads and mix by tapping or vortex to elute the proteins. Heat at 95 °C for 5 min, then centrifuge for 10,000 x g for 1 min and collect the supernatant (contain eluted proteins). Alternatively, elute proteins with 8 M urea/100 mM glycine pH 2.9 to avoid using SDS. Freeze the eluate at -80 °C, otherwise proceed to Western blot analysis.
       CAUTION: 2-mercaptoethanol is toxic and may cause skin, eye and respiratory irritations. Use gloves and work in the chemical hood.
2. Western blotting analysis
   1. Mix 15 µL of input (from step 1.1.6) or flow-through (from step 1.1.10) samples with 5 µL of 4x Laemmli buffer. Heat input and flow-through samples for 5 min at 95 °C. For samples eluted in 8 M urea/100 mM glycine pH 2.9, mix 15 µL of eluate with 5 µL of 4x Laemmli buffer, and heat for 5 min at 95 °C.
      NOTE: This step is not necessary for samples eluted in 2x Laemmli buffer.
   2. Load wells of 4-20% SDS/PAGE gel with 2.5 µL of input, 2.5 µL flow-through, and 20 µL of eluted samples, and load protein ladder according to the manufacturer’s recommendation.
      NOTE: Choose gel% according to the molecular weight of the protein of interest.
   3. Run SDS/PAGE at 150 V for 30-45 min in running buffer, or until the blue dye of the Laemmli buffer is at the end of the gel.
      NOTE: Time of run may vary according to laboratory equipment.
   4. Remove the gel from the glass (or plastic) plates, and soak in transfer buffer for 15 min.
   5. Transfer the proteins to polyvinylidene difluoride (PVDF) membrane or nitrocellulose membrane. Soak membranes and 3 mm filter paper in transfer buffer. Assemble a sandwich with three sheets of filter paper, nitrocellulose or PVDF membrane, gel, and an additional three sheets of filter paper. Make sure no air bubbles are trapped in the sandwich. Use a roller to remove bubbles if necessary. Set the membrane on the cathode and the gel on the anode side of the cassette.
      NOTE: Check the membrane manufacturer’s instructions for information on membrane activation or blot preparation.
   6. Place the cassette containing the sandwich in the transfer tank with transfer buffer. Place the tank in an ice bucket or at 4 °C (e.g., in the cold room). Transfer proteins at 100 V for 1 h (current varies between 200-400 mA). Alternatively, transfer overnight at a constant current of 15 mA at 4 °C.
   7. Remove the membrane from the cassette. Incubate the membrane in 6% non-fat dry milk diluted in PBS with 0.05% of polysorbate 20 (PBS-T), or compatible blocking solution, for 1 h at RT to block the membrane.
      NOTE: Before blocking the membrane, the quality of the transfer can be checked using Ponceau S stain. Incubate the membrane for 1 min in 15 mL of Ponceau S, rinse in water and visualize bands.
   8. Remove the blocking solution and incubate the membrane for 1 h at RT with 50 rpm rotation in primary antibodies diluted in 6% non-fat dry milk diluted in PBS-T. Alternatively, incubate membrane overnight at 4 °C with 50 rpm rotation.
      NOTE: The time of incubation may vary according to the quality of the antibodies; however, most antibodies will work with incubations of 1-3 h at RT. Follow the manufacturer’s recommendation for antibodies concentration or dilution.
   9. Wash the blot by incubating the membrane in PBS-T for 5 min with 50 rpm rotation at RT. Repeat the procedure 3-5 times. More washes may be needed depending on the quality of antibodies.
   10. Incubate the membrane in horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT in 6% non-fat dry milk diluted in PBS-T with 50 rpm rotation.
       NOTE: Follow the manufacturer’s recommendation for antibodies concentration or dilution.
   11. Wash the blot as indicated in step 1.2.9.
   12. Add the chemiluminescent substrate to cover the membrane. Remove the excess of substrate and incubate for 5 min at RT in the dark.
       NOTE: Check the manufacturer’s instructions for recommendations on the chemiluminescence reagents.
   13. Capture the chemiluminescent signal using a camera-based imager. Alternatively, use an X-ray film to capture the chemiluminescent signal.

2. Analysis of IP/PI-binding proteins by affinity chromatography and mass spectrometry

1. Cell growth, lysis and affinity chromatography
   1. Grow T. brucei cells to mid-log phase and monitor cell viability and density.
      NOTE: A total of 1.0 x 10¹⁰ cells is enough for two binding assays. Using fewer cells than indicated here may affect the detection of low abundance proteins by mass spectrometry.
      1. For T. brucei bloodstream forms, grow cells at mid-log phase (8.0 x 10⁵-1.6 x 10⁶ cells/mL) in HMI-9 media supplemented with 10% FBS at 37 °C and with 5% CO₂. For 427 strain, 5 L of culture will yield 0.5-1.0 x 10¹⁰ cells. Monitor the cell growth to avoid density higher than 1.8 x 10⁶ cells/mL which may affect cell viability. Keep the cell culture volume to 1/10 of the flask volume; otherwise, the growth rate of the cells will be affected due to poor aeration.
         NOTE: The 427 strain has a doubling time of 5.5-6.5 h.
      2. For procyclic forms, grow cells in SDM-79 medium supplemented with 10% FBS at 27 °C and keep cell density between 1.0 x 10⁷ and 3.0 x 10⁷ cells/mL. 500 mL of culture will yield 0.5-1.5 x 10¹⁰ cells.
   2. Centrifuge the cells at 1,600 x g for 15 min at RT. Discard the supernatant and resuspend the pellet in 200 mL of PBS-G pre-heated at 37 °C. Use round bottom centrifugation tubes (Table of Materials) because T. brucei bloodstream form pellets are easily disturbed when using fixed-angle centrifuge rotors. Centrifuge the cells again at 1,600 x g for 5 min at RT.
   3. Remove the supernatant and resuspend the pellet in 10 mL of PBS-G. Centrifuge the cells at 1,600 x g for 5 min at RT. Repeat the procedure and after the final wash, discard the supernatant.
      NOTE: Pellets may be flash frozen in liquid nitrogen and stored at -80 °C or liquid nitrogen.
   4. Resuspend the cell pellet in 5 mL of lysis buffer pre-chilled in ice and supplemented with 1.5x protease inhibitor cocktail and 1x phosphatase inhibitor cocktail (Table of Materials). Incubate the lysate for 10 min at 4 °C rotating at 50 rpm.
   5. Centrifuge the lysate at 10,000 x g for 10 min at 4 °C. Collect the supernatant (solubilized proteins) and dilute it in 20 mL of binding buffer.
   6. Collect 400 µL of IPs or PIs conjugated to agarose beads (i.e., 400 µL of slurry) or 400 µL of control beads, and centrifuge for 1 min at 1,000 x g at RT. Discard the supernatant and resuspend in 400 µL of binding buffer to equilibrate the beads. Use agarose beads as a negative control to determine specific enrichment of protein-metabolite interactions compared to unspecific interactions (e.g., proteins that bind nonspecifically to the beads). Use IPs or PIs with different phosphate configurations including non-phosphorylated forms to control for unspecific interactions due to phosphate charges or binding to biotin.
   7. Add 400 µL of IP/PI-beads or control beads to 10 mL of lysate and incubated for 1 h, or overnight, at 4 °C rotating at 50 rpm. If using IPs or PIs conjugated with biotin only (without beads) proceed to step 2.1.7.1, otherwise proceed to step 2.1.8.
      1. Add 100 µL of streptavidin-conjugated to magnetic beads and incubate for 1 h at 4 °C rotating at 50 rpm.
   8. Centrifuge the binding reaction for 1 min at 1,000 x g at 4 °C. Remove the supernatant (flow-through) and keep the pellet. Keep 5% of the supernatant for Western blot analysis.
   9. Add 5 mL of washing buffer to the pellet, gently mix by swirling the tube, and then centrifuge for 1 min at 1,000 x g at 4 °C. Discard the supernatant. Repeat the wash five times. If using magnetic beads, collect supernatants and perform washes using a magnetic stand (centrifugations are not necessary).
   10. Add 50 µL of 2x Laemmli buffer (or 8 M urea/100 mM glycine pH 2.9, to avoid using SDS) to the beads and mix by tapping or vortex (avoid pipetting because beads can attach to pipette tips), and then heat at 95 °C for 5 min. Centrifuge for 10,000 x g for 1 min and collect the supernatant (eluted proteins). Repeat the procedure twice to collect a total of three fractions.
   11. Freeze the eluate at -80 °C, otherwise separate proteins in SDS/PAGE or keep in solution for trypsinization and mass spectrometry analysis.
2. Trypsin digestion of proteins for mass spectrometry
   NOTE: Two variations of this procedure are shown for section 2.2.1 (in gel) or section 2.2.2 (in solution) digestion of proteins. Protein low binding tubes are recommended to prevent sample losses. Consult with an analytical chemist at the proteomics facility the suitability of the protocol for samples and mass spectrometer instruments available.
   1. In-gel trypsinization of proteins
      1. After protein separation in SDS/PAGE, briefly rinse the gel in high-purity water. Transfer the gel onto a clean glass plate. Excise protein bands with a clean blade and avoid cutting extra gel outside bands. Cut the gel pieces into small pieces (i.e., approximately 1 mm square) and transfer them into a 1 mL tube. Use a pipette tip if necessary, but make sure that the pipette tip is rinsed in ethanol before use.
         NOTE: Use gloves to avoid gel contamination. Gel pieces can be stored at -20 °C.
      2. Add 100 μL of high-purity or high-performance liquid chromatography grade water to tubes to rinse gel pieces. Discard the water.
         1. For Coomassie or ruthenium-based fluorescent stained gel pieces, incubate the gel pieces in destaining solution (25 mM NH₄HCO₃ in 50% acetonitrile) for 1 h, then discard the solution. Repeat the procedure until staining is not visible.
            NOTE: Prepare solutions containing NH₄HCO₃ by dilution from a stock solution at 100 mM NH₄HCO₃, pH 7.8.
            CAUTION: Acetonitrile is a volatile solvent, flammable and toxic. NH₄HCO₃ can cause skin or eye irritation. Use gloves and work under a chemical hood.
         2. For silver stained gel pieces, incubate gel pieces in 50 μL of destaining solution for silver stain (15 mM K₃[Fe(CN)₆], 50 mM Na₂S₂O₃) in water for 30 min. Discard the solution and wash gel pieces with 200 μL of water. Repeat wash five times or until gel yellow color is not visible.
            CAUTION: K₃[Fe(CN)₆] may cause skin or eye irritation. Use gloves.
      3. Dehydrate the gel pieces using 200 μL of acetonitrile for 10 min at RT. Then, discard the solution.
         NOTE: Dehydrated gel pieces are smaller in volume, opaque, and tacky. If several pieces of gel are combined in one tube, repeat the procedure for efficient dehydration of gel pieces.
      4. Add 50 µL (or enough volume to cover the gel pieces) of reducing solution (10 mM dithiothreitol [DTT] in 100 mM NH₄HCO₃) and incubate at 56 °C for 1 h. Afterwards, cool the tubes to RT and discard the excess of reducing solution.
      5. Add 50 µL (or enough volume to cover the gel pieces) of alkylation solution (50 mM iodoacetamide in 100 mM NH₄HCO₃) and incubate for 30 min at RT in the dark. Afterwards, discard the excess of alkylation solution.
      6. Dehydrate the gel pieces with 200 μL of acetonitrile for 10 min at RT. Remove the acetonitrile and hydrate the gel pieces with 100 mM NH₄HCO₃ for 10 min at RT.
      7. Dehydrate the gel pieces again with 200 μL of acetonitrile for 10 min at RT and discard the excess of solution.
      8. Add 15 µL of mass spectrometry grade trypsin diluted in 50 mM NH₄HCO₃ buffer, or enough volume to cover hydrated gel pieces and incubate for 4 h, or overnight, at 37 °C. Keep total trypsin amounts between 100 to 500 ng (or 20 ng trypsin/µg of protein).
      9. Cool the sample to RT, and centrifuge for 1 min at 2,000 x g in a microcentrifuge. Add 10-20 μL of 5% formic acid diluted in water and incubate for 10 min at RT.
         CAUTION: Formic acid is flammable, corrosive and toxic. Use gloves and work under a chemical hood.
      10. Centrifuge as indicated in step 2.2.1.9, then collect the supernatant (contain extracted peptides) to a different tube. Add 20 μL of 5% formic acid diluted in 50-60% acetonitrile to the tube and incubate for 10 min at RT. Collect extracted peptide fractions in the same tube.
      11. Dry the sample in a vacuum concentrator and reconstitute in 10 μL of 0.5% acetic acid and 2% acetonitrile for mass spectrometry analysis. Centrifuge and collect the solution at the bottom of the tube; store solution at -20 °C or -80 °C.
   2. In solution trypsinization of proteins
      1. Precipitate proteins to reduce sample volume, desalting, and buffer exchange. Add six volumes of chilled (-20 °C) acetone to the sample, e.g., 600 μL of acetone to 100 μL of sample. Vortex and incubate at -20 °C for 15 min to 1 h. The solution will turn cloudy or form a precipitate.
         CAUTION: Acetone is toxic and flammable. Use gloves and work under a chemical hood.
      2. Centrifuge samples at 4 °C for 30 min. Decant the acetone and air-dry the pellet for 15 min.
      3. Add 10 µL of 6-8 M urea or 1% SDS in 50 mM NH₄HCO₃ to dissolve the pellet and vortex to mix. For larger amounts of proteins (> 10 µg), use up to 20 µL of dissolution buffer.
      4. Add 5 µL of reducing solution and vortex. Spin the volume down using a microcentrifuge. If samples are diluted in urea, then incubate for 1 h at RT. If samples are diluted in SDS, then incubated for 1 h at 56 °C.
      5. Spin the volume down using a microcentrifuge. Add 3 µL of alkylation solution and vortex. Then, spin the volume down and incubate in the dark for 30 min at RT.
      6. Add 3 μL of reducing solution to neutralize the reaction. Slowly dilute the sample to 1 M urea or 0.05% SDS using 50 mM NH₄HCO₃.
         NOTE: Trypsin digestion buffer must have detergent or denaturant. Concentration limits for denaturants are: 0.05% SDS; 0.1% octyl B-D-glucopyranoside; 0.1% 4-nonylphenyl-polyethylene glycol; 0.1% t-octylphenoxypolyethoxyethanol; 0.1% polysorbate 20; 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; < 1 M urea or thiourea.
      7. Add 5 µL of mass spectrometry grade trypsin diluted in 50 mM NH₄HCO₃ buffer and incubate for 4 h, or overnight, at 37 °C. Keep total trypsin amounts between 100 and 500 ng (or 20 ng trypsin/µg of protein).
      8. Cool the sample to RT, and spin the volume down using a microcentrifuge. Add 5% acetic acid (or 5% formic acid in 50% acetonitrile) to quench the reaction.
      9. Dry the samples in a vacuum concentrator as indicated in step 2.2.1.11. Store samples at -80 °C. Desalt and concentrate peptides using a reversed phase column such as C18 zip-tip and then analyze by mass spectrometry.