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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Abstract

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

Introduction

Inositol phosphates (IPs) and phosphoinositides (PIs) play a central role in eukaryote biology through the regulation of cellular processes such as the control of gene expression1,2,3, vesicle trafficking4, signal transduction5,6, metabolism7,8,9, and development8,10. The regulatory function of these metabolites results from their ability to interact with proteins and thus regulate protein function. Upon binding by proteins, IPs and PIs may alter protein conformation11, catalytic activity12, or interactions13 and hence affect cellular function. IPs and PIs are distributed in multiple subcellular compartments, such as nucleus2,3,14,15, endoplasmic reticulum16,17, plasma membrane1 and cytosol18, either associated with proteins3,19 or with RNAs20.

The cleavage of the membrane-associated PI(4,5)P2 by phospholipase C results in the release of Ins(1,4,5)P3, which can be phosphorylated or dephosphorylated by IP kinases and phosphatases, respectively. IPs are soluble molecules that can bind to proteins and exert regulatory functions. For example, Ins(1,4,5)P3 in metazoan can act as a second messenger by binding to IP3 receptors, which induces receptor conformational changes and thus release of Ca2+ from intracellular stores11. Ins(1,3,4,5)P4 binds to the histone deacetylase complex and regulates protein complex assembly and activity13. Other examples of IPs regulatory function include the control of chromatin organization21, RNA transport22,23, RNA editing24, and transcription1,2,3. In contrast, PIs are often associated with the recruitment of proteins to the plasma membrane or organelle membranes25. However, an emerging property of PIs is the ability to associate with proteins in a non-membranous environment3,15,19,26. This is the case of the nuclear receptor steroidogenic factor, which transcriptional control function is regulated by PI(3,4,5)P319, and poly-A polymerase which enzymatic activity is regulated by nuclear PI(4,5)P226. A regulatory role for IPs and PIs has been shown in many organisms including yeast22,27, mammalian cells19,23, Drosophila10 and worms28. Of significance is the role of these metabolites in trypanosomes, which diverged early from the eukaryotic lineage. These metabolites play an essential role in Trypanosoma brucei transcriptional control1,3, development8, organelle biogenesis and protein traffic29,30,31,32, and are also involved in controlling development and infection in the pathogens T. cruzi33,34,35, Toxoplasma36 and Plasmodium5,37. Hence, understanding the role of IPs and PIs in trypanosomes may help to elucidate new biological function for these molecules and to identify novel drug targets.

The specificity of protein and IP or PI binding depends on protein interacting domains and the phosphorylation state of the inositol13,38, although interactions with the lipid part of PIs also occurs19. The variety of IPs and PIs and their modifying kinases and phosphatases provides a flexible cellular mechanism for controlling protein function which is influenced by metabolite availability and abundance, the phosphorylation state of the inositol, and protein affinity of interaction1,3,13,38. Although some protein domains are well-characterized39,40,41, e.g., pleckstrin homology domain42 and SPX (SYG1/Pho81/XPR1) domains43,44,45, some proteins interact with IPs or PIs by mechanisms that remain unknown. For example, the repressor-activator protein 1 (RAP1) of T. brucei lacks canonical PI-binding domains but interacts with PI(3,4,5)P3 and control transcription of genes involved in antigenic variation3. Affinity chromatography and mass spectrometry analysis of IP or PI interacting proteins from trypanosome, yeast, or mammalian cells identified several proteins without known IP- or PI-binding domains8,46,47. The data suggest additional uncharacterized protein domains that bind to these metabolites. Hence, the identification of proteins that interact with IPs or PIs may reveal novel mechanisms of protein-metabolite interaction and new cellular regulatory functions for these small molecules.

The method described here employs affinity chromatography coupled to Western blotting or mass spectrometry to identify proteins that bind to IPs or PIs. It uses biotinylated IPs or PIs that are either cross-linked to streptavidin conjugated to agarose beads or alternatively, captured via streptavidin-conjugated magnetic beads (Figure 1). The method provides a simple workflow that is sensitive, non-radioactive, liposome-free and is suitable for detecting the binding of proteins from cell lysates or purified proteins3 (Figure 2). The method can be used in label-free8,46 or coupled to amino acid-labelled quantitative mass spectrometry47 to identify IP or PI-binding proteins from complex biological samples. Hence, this method is an alternative to the few methods available to study the interaction of IPs or PIs with cellular proteins and will help in understanding the regulatory function of these metabolites in trypanosomes and perhaps other eukaryotes.

Protocol

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.

Results

Analysis of RAP1 and PI(3,4,5)P3 interaction by affinity chromatography and Western blotting
This example illustrates the application of this method to analyze the binding of PIs by RAP1 from T. brucei lysate or by recombinant T. brucei RAP1 protein. Lysates of T. brucei bloodstream forms that express hemagglutinin (HA)-tagged RAP1 were used in binding assays. RAP1 is a protein involved in transcriptional control of variant surface glycoprotein (VSG) genes

Discussion

The identification of proteins that bind to IPs or PIs is critical to understand the cellular function of these metabolites. Affinity chromatography coupled to Western blot or mass spectrometry offers an opportunity to identify IP or PI interacting proteins and hence gain insights on their regulatory function. IPs or PIs chemically tagged [e.g., Ins(1,4,5)P3 chemically linked to biotin] and crosslinked to agarose beads via streptavidin or captured by streptavidin magnetic beads allows the isolation of interacting protein...

Disclosures

The author has nothing to disclose.

Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2019-04658); NSERC Discovery Launch Supplement for Early Career Researchers (DGECR-2019-00081) and by McGill University.  

Materials

NameCompanyCatalog NumberComments
AcetoneSigma-Aldrich650501Ketone
AcetonitrileSigma-Aldrich271004Solvent 
Ammonium bicarbonateSigma-AldrichA6141Inorganic salt
Centrifuge Avanti J6-MIBeckman CoulterAvanti J6-MICentrifuge for large volumes (e.g., 1L)
Centrifuge botlesSigma-AldrichB1408Bottles for centrifugation of 1L of culture
Control BeadsEchelonP-B000-1mlAffinity chromatography reagent - control
D-(+)-GlucoseSigma-AldrichG8270Sugar, Added in PBS to keep cells viable
Dithiothreitol (DTT) Bio-Rad1610610Reducing agent
Dynabeads M-270 StreptavidinThermoFisher Scientific65305Streptavidin beads for binding to biotin ligands
EDTA-free Protease Inhibitor CocktailRoche11836170001Protease inhibitors
Electrophoresis running bufferBio-Rad161073225 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3
Falcon 15 mL Conical Centrifuge TubesCorning Life Sciences430052To centrifuge 10 mL cultures
Formic acidSigma-Aldrich106526Acid
GlycineSigma-AldrichG7126Amino acid
HMI-9 cell culture mediumThermoFisher ScientificME110145P1Cell culture medium for T. brucei bloodstream forms
Imperial Protein StainThermoFisher Scientific24615Coomassie staining for protein detection in SDS/PAGE
Ins(1,4,5)P3 BeadsEchelonQ-B0145-1mlAffinity chromatography reagent 
Instant Nonfat Dry MilkThomas ScientificC837M64Blocking reagent for Western blotting
IodoacetamideSigma-AldrichI6125Alkylating reagent for cysteine proteins or peptides
Lab RotatorThomas Scientific1159Z92For binding assays
LoBind Microcentrifuge TubesThermoFisher Scientific13-698-793Low protein binding tubes for mass spectrometry
Nonidet P-40 (Igepal CA-630)Sigma-Aldrich21-3277Detergent
PBS, pH 7.4ThermoFisher Scientific10010031Physiological buffer
Peroxidase substrate for chemiluminescenceThermoFisher Scientific32106Substrate for Western bloting detection of proteins
PhosSTOP Phosphatase Inhibitor Cocktail TabletsRoche4906845001Phosphatase inhibitors
PI(3)P PIP BeadsEchelonP-B003a-1mlAffinity chromatography reagent 
PI(3,4)P2 PIP BeadsEchelonP-B034a-1mlAffinity chromatography reagent 
PI(3,4,5)P3 diC8EchelonP-3908-1mgAffinity chromatography reagent 
PI(3,4,5)P3 PIP BeadsEchelonP-B345a-1mlAffinity chromatography reagent 
PI(3,5)P2 PIP BeadsEchelonP-B035a-1mlAffinity chromatography reagent 
PI(4)P PIP BeadsEchelonP-B004a-1mlAffinity chromatography reagent 
PI(4,5)P2 diC8EchelonP-4508-1mgAffinity chromatography reagent 
PI(4,5)P2 PIP BeadsEchelonP-B045a-1mlAffinity chromatography reagent 
PI(5)P PIP BeadsEchelonP-B005a-1mlAffinity chromatography reagent 
Ponceau S solutionSigma-AldrichP7170Protein staining (0.1% [w/v] in 5% acetic acid)
Potassium hexacyanoferrate(III)Sigma-Aldrich702587Potassium salt 
PtdIns PIP BeadsEchelonP-B001-1mlAffinity chromatography reagent 
PVDF MembraneBio-Rad1620177For Western blotting 
Refrigerated centrifugeEppendorf5910 RMicrocentrifuge for small volumes (e.g., 1.5 mL)
Sodium dodecyl sulfateSigma-Aldrich862010Detergent
Sodium thiosulfateSigma-Aldrich72049Chemical 
SpeedVac Vacuum ConcentratorsThermoFisher ScientificSPD120-115Sample concentration (e.g., for mass spectrometry)
T175 flasks for cell culture ThermoFisher Scientific159910To grow 50 mL T. brucei culture
Trypsin, Mass Spectrometry GradePromegaV5280Trypsin for protein digestion
UreaSigma-AldrichU5128Denaturing reagent
VortexFisher Scientific02-215-418For mixing reactions
Western blotting transfer bufferBio-Rad161073425 mM Tris, 192 mM glycine, pH 8.3 with 20% methanol
Whatman 3 mm paperSigma-AldrichWHA3030861Paper for Wester transfer
2-mercaptoethanol (14.2 M)Bio-Rad1610710Reducing agent
2x Laemmli Sample BufferBio-Rad161-0737Protein loading buffer
4–20% Mini-PROTEAN TGX Precast Protein GelsBio-Rad4561094Gel for protein electrophoresis
4x Laemmli Sample BufferBio-Rad161-0747Protein loading buffer

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