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

Igor Cestari

doi:10.3791/59865

JoVE Journal > Biochemistry

Bioquímica

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

Published: July 26, 2019

doi:

10.3791/59865

Igor Cestari¹

¹Institute of Parasitology,McGill University

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

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 10⁶cells/mL.
    NOTE: Density higher than 1.8 x 10⁶cells/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.
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

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 10⁷and 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.
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. NH₄HCO₃can 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 NH₄HCO₃for 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 NH₄HCO₃buffer, 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 NH₄HCO₃to 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 NH₄HCO₃buffer 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.

Representative 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) genes³^,⁴⁸, which encode for surface proteins involved in parasite immune evasion by antigenic variation⁴⁹. RAP1 interacts within a telomeric protein complex with the phosphatidylinositol 5-phosphatase (PIP5Pase) enzyme³, which also functions in the control of VSG gene transcription¹^,³. RAP1 has an N-terminal breast cancer 1 carboxyl-terminal (BRCT) domain which is followed by a myeloblastosis (myb) DNA-binding domain and a C-terminal myb-like domain³^,⁴⁸. However, it lacks canonical PI binding domains. Binding assays were performed with PIs that are non-phosphorylated or are phosphorylated at different positions of the inositol ring, and with non-conjugated agarose beads. Western analysis shows that RAP1 binds preferentially to PI(3,4,5)P3-beads (Figure 3A), but it also binds to a lesser extent to PI(4,5)P2-beads. However, it did not bind to any other PIs or agarose beads. Because RAP1 is part of a multiprotein complex³, its interaction with some PIs may not be direct and thus results from RAP1-HA interaction with other cellular proteins that bind to PIs.

Hence, to test whether RAP1 binds directly to PIs, a C-terminally tagged 6x-his recombinant RAP1 (rRAP1) protein was expressed and purified to homogeneity from E. coli³. The protein was used in binding assays with PI(3,4,5)P3-beads in the presence of competing concentrations of PI(3,4,5)P3 or PI(4,5)P2. Western blotting shows that increasing concentrations of PI(3,4,5)P3, but not PI(4,5)P2 inhibits the interaction of rRAP1 with PI(3,4,5)P3 (Figure 3B). Moreover, the addition of T. brucei purified PIP5Pase enzyme to the reaction restored PI(3,4,5)P3-binding by rRAP1, which is due to PIP5Pase dephosphorylation of free PI(3,4,5)P3³ and thus indicates that the phosphorylation pattern of this metabolite is essential for rRAP1 binding. Hence, rRAP1 interacts with PI(3,4,5)P3 as it does RAP1-HA from T. brucei lysates. Moreover, the data show that binding of RAP1-HA from lysate to PI(4,5)P2 likely results from RAP1 interaction with other proteins in the complex (e.g., PIP5Pase)³. The data illustrate the complementarity of binding assays with cell lysates and recombinant proteins. It also shows the utility of competitive binding assays to determine the specificity of interactions between proteins and PIs.

Identification of Ins(1,4,5)P3 binding proteins by affinity chromatography and mass spectrometry
In this example, affinity chromatography followed by mass spectrometry was used to identify T. brucei proteins that bind to Ins(1,4,5)P3; therefore, the experiment surveys potential Ins(1,4,5)P3 binding proteins from T. brucei bloodstream forms. T. brucei lysate was incubated with Ins(1,4,5)P3 conjugated to agarose beads or with non-conjugated beads (used as a control), and bound proteins were eluted with Laemmli sample buffer. SDS/PAGE analysis shows enrichment in proteins eluted from Ins(1,4,5)P3-beads compared to proteins eluted from the control agarose beads (Figure 4A). Mass spectrometry analysis of eluted proteins identified over 250 proteins, of which 84 were enriched with Ins(1,4,5)P3 beads compared to control beads (Figure 4B, fold change [FC] ≥ 2, p < 0.05). The enrichment of proteins bound to Ins(1,4,5)P3 compared to control beads correlates with the protein signal detected by SDS/PAGE. The data include proteins that were validated to bind Ins(1,4,5)P3 and proteins which mechanisms of binding to Ins(1,4,5)P3 are unknown⁸. Moreover, the Ins(1,4,5)P3 binding proteome differed greatly from that of Ins(1,3,4,5)P4 and other PIs⁸, which suggests that some of these proteins recognize the specific phosphate configuration of Ins(1,4,5)P3. Hence, biotin-tagged Ins(1,4,5)P3 can be used for affinity chromatography coupled to mass spectrometry to identify proteins that bind to Ins(1,4,5)P3. The approach can be explored to identify proteins that bind to other IPs or PIs³^,⁸^,⁴⁶^,⁴⁷.

Figure 1: Affinity reagents for binding assays. (A) PI(3,4,5)P3 (top) and Ins(1,4,5)P3 (bottom) conjugated to biotin at sn1 position of the inositol. In PI(3,4,5)P3, the biotin is conjugated to the lipid chain at sn1 position of the inositol, whereas in Ins(1,4,5)P3 the biotin is conjugated to the phosphate at position sn1. (B) Ins(1,4,5)P3 is conjugated with biotin and captured via binding to streptavidin conjugated to beads (e.g., agarose or magnetic beads). Variations of these reagents using custom synthesized linkers that substitute the biotin are also possible⁴⁶. Please click here to view a larger version of this figure.

Figure 2: The workflow of protocols describes the steps for the analysis of IP or PI affinity interaction with proteins of T. brucei and detection by (A) Western blotting or (B) mass spectrometry. AC-WB, affinity chromatography and Western blotting; AC-MS, affinity chromatography and mass spectrometry. Please click here to view a larger version of this figure.

Figure 3: Binding of T. brucei RAP1 to phosphoinositides. (A) Lysates of T. brucei (5.0 x 10⁷ parasites) that express HA-tagged RAP1 were incubated for 2 h at 4 °C with 50 µL of PIs (each 1 mL of agarose beads containing 10 nmol of conjugated PIs) or agarose (Ag) beads. The binding reaction was washed and eluted with 2 x Laemmli sample buffer and heated at 95 °C for 5 min. Proteins were separated in 4-20% SDS/PAGE, transferred to a PVDF membrane, and probed with monoclonal antibodies anti-HA (1:5,000, diluted in 6% PBS-milk) followed by anti-mouse IgG-HRP (1:5,000, diluted in 6% PBS-milk), and detected by chemiluminescence. (B) One µg of rRAP1 was incubated for 1 h at RT with 50 µL of PI(3,4,5)P3-agarose beads in the presence or absence of 5 to 50 µM dioctanoylglycerol (diC8) PI(3,4,5)P3, 20 to 50 µM diC8 PI(4,5)P2, or 50 µM diC8 PI(3,4,5)P3 and 250 ng PIP5Pase purified from T. brucei bloodstream forms³. Binding was analyzed by Western blotting with mouse anti-His HRP monoclonal antibodies (1:2,000, diluted in 6% PBS-milk) and developed by chemiluminescence. This figure has been modified from Cestari et al.³. Please click here to view a larger version of this figure.

Figure 4: Affinity chromatography and mass spectrometry analysis of T. brucei proteins that bind to Ins(1,4,5)P3. (A) 10% SDS/PAGE analysis of T. brucei proteins that bind to Ins(1,4,5)P3-beads or agarose beads. Lysates of 5.0 x 10⁹parasites were incubated at 4 °C for 2 h with 400 µL of Ins(1,4,5)P3 conjugated to agarose beads or with agarose beads without Ins(1,4,5)P3 [1 mL of beads contain 10 nmol of conjugated Ins(1,4,5)P3]. The binding reaction was washed, eluted in 2 x Laemmli sample buffer and boiled for 5 min at 95 °C. Proteins were separated in 10% SDS/PAGE and stained with Coomassie staining (Table of Materials). Arrowheads show proteins that are enriched in Ins(1,4,5)P3-beads compared to agarose beads; circles indicate proteins present in both Ins(1,4,5)P3-beads and agarose beads, and the bracket indicates proteins that are present in both but are enriched in Ins(1,4,5)P3-beads compared to agarose beads. (B) Dot-plot shows proteins identified by mass spectrometry that are enriched in Ins(1,4,5)P3-beads compared to agarose beads. Enrichment defined by FC > 2 and p-value <0.05. Four biological replicates were used for agarose-beads AC-MS, and three biological replicates for IP3-beads AC-MS. Fold-change of proteins identified in IP3-beads vs agarose-beads was calculated using peptide spectra intensity using MSstat⁵⁰. Detailed results and list of peptides are available⁸. Mass spectrometry raw data is also available with identifier PXD005907 through the ProteomeXchange Consortium via the PRIDE partner repository. This figure has been modified from Cestari et al.⁸. Please click here to view a larger version of this figure.

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 proteins which can then be identified by mass spectrometry or Western blot. The protocols described here have been used to identify proteins from T. brucei³^,⁸ and mammalian cells⁴⁷ that bind to these metabolites. Variations of the approach that uses customized tags (other than biotin) have also been used in yeast⁴⁶. An important consideration to this approach is the use of controls to discriminate specific from non-specific interactions. Non-conjugated beads are an essential control, but additional controls may include non-phosphorylated PIs or inositol conjugated to beads³. IPs or PIs with different phosphate combinations³^,⁸ may also be used because protein binding to these metabolites may involve domains that discriminate the phosphate configuration of the inositol³⁸^,³⁹^,⁴⁰^,⁴¹. Additionally, sample complexity may affect the sensitivity of the approach, and hence decreasing sample complexity by sample fractionation may help the detection of low abundant proteins in the cell. There are well-established protocols for cell fractionation and isolation of mitochondrion⁵¹, nucleus⁵², glycosome⁵³, and flagellum⁵⁴^,⁵⁵ from trypanosomes. Note that buffers and reagents used in subcellular fractionations may need to be adjusted for compatibility with buffers and reagents used in this protocol. Notably, the identification of proteins that bind to IPs or PIs by affinity chromatography as indicated here depends on protein and metabolite affinity of interaction, and thus proteins which have a weak affinity for IPs or PIs may not be readily detected.

The analysis of IP or PI interaction with proteins from cell lysate may also result in the identification of proteins that do not bind directly to these metabolites, but which interaction results from protein association in complex with other proteins that bind to IPs or PIs. This feature is exemplified in Figure 3A, in which RAP1-HA from T. brucei lysate appears to bind to PI(3,4,5)P3-beads and PI(4,5)P2-beads. However, binding assays with His-tagged rRAP1 show that this protein binds to PI(3,4,5)P3 and not PI(4,5)P2³. This is illustrated in Figure 3B, in which competitive assays show that free PI(3,4,5)P3 but not free PI(4,5)P2 competes for rRAP1-his interaction with PI(3,4,5)P3-beads. The RAP1 seeming interaction with PI(4,5)P2-beads is due to RAP1 association within a complex with proteins that bind PI(4,5)P2 (e.g., PIP5Pase)³. Hence, binding assays from cell lysates may identify proteins that bind directly or indirectly to IPs or PIs. Notably, indirect interactions are distinct from unspecific interactions since the former may have a biological function (in the context of the protein complex) that affects or is affected by metabolite binding. For example, Ins(1,4,5,6)P4 binds to the multi-subunit co-repressor deacetylase complex and controls the complex assembly and activity¹³. Hence, it is essential to validate IP/PI interactions with proteins. The validation of interaction may involve competition assays with an excess of IPs or PIs (as in Figure 3B)³^,⁸, mutations of potential protein domains⁵⁶, or the use of purified proteins to determine direct interactions (Figure 3A,B)³.

Other methods for studying protein and IP or PI interactions include the binding of proteins to radiolabeled IPs or PIs, the use IPs or PIs bound to hydrophobic membranes as matrices for protein capture, or the binding of proteins to PIs incorporated into liposomes⁴²^,⁵⁷^,⁵⁸. Importantly, if protein interaction with PIs requires membrane structures³⁸, liposome-based assays may be used as a complementary approach. Limitations of these approaches include low throughput, low sensitivity, the unknown chemical orientation of IPs or PIs association to matrices⁵⁸, or the use of radioactive materials⁴². The method described here is sensitive, liposome-free, non-radioactive, and IP/PI-beads are commercially available, and thus they do not require customized chemical synthesis. Furthermore, the position of the biotin linked to IPs or PIs is well-defined, and it can also be modified⁴⁶^,⁵⁸, which allows precise analysis of protein and metabolite interaction. The method described here can also be combined with quantitative mass spectrometry approaches such as stable isotope labelling of amino acids in cell culture (SILAC)⁴⁷, which can be used to identify dynamic interactions under different cellular treatments or conditions. Affinity chromatography coupled to Western blot or mass spectrometry has helped to identify numerous IP- or PI-binding proteins from T. brucei, mammalian cells, and yeast³^,⁸^,⁴⁶^,⁴⁷, including proteins that do not have characterized IP- or PI-binding domains, and it has also helped the identification of novel binding domains, e.g., PI(3,4,5)P3 binding domain⁴⁷.

Overall, the protocols described here can be used to survey potential IP or PI interacting proteins from T. brucei, and to study the molecular interaction of proteins with these metabolites. The protocol can be easily adapted to identify IP- or PI-binding proteins from other unicellular parasites or from other organisms such as mammalian cells⁴⁷ and yeast⁴⁶, and it will help to further understand the biological function of IPs and PIs in eukaryotes.

Declarações

The authors have 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

Acetone	Sigma-Aldrich	650501	Ketone
Acetonitrile	Sigma-Aldrich	271004	Solvent
Ammonium bicarbonate	Sigma-Aldrich	A6141	Inorganic salt
Centrifuge Avanti J6-MI	Beckman Coulter	Avanti J6-MI	Centrifuge for large volumes (e.g., 1L)
Centrifuge botles	Sigma-Aldrich	B1408	Bottles for centrifugation of 1L of culture
Control Beads	Echelon	P-B000-1ml	Affinity chromatography reagent – control
D-(+)-Glucose	Sigma-Aldrich	G8270	Sugar, Added in PBS to keep cells viable
Dithiothreitol (DTT)	Bio-Rad	1610610	Reducing agent
Dynabeads M-270 Streptavidin	ThermoFisher Scientific	65305	Streptavidin beads for binding to biotin ligands
EDTA-free Protease Inhibitor Cocktail	Roche	11836170001	Protease inhibitors
Electrophoresis running buffer	Bio-Rad	1610732	25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3
Falcon 15 mL Conical Centrifuge Tubes	Corning Life Sciences	430052	To centrifuge 10 mL cultures
Formic acid	Sigma-Aldrich	106526	Acid
Glycine	Sigma-Aldrich	G7126	Amino acid
HMI-9 cell culture medium	ThermoFisher Scientific	ME110145P1	Cell culture medium for T. brucei bloodstream forms
Imperial Protein Stain	ThermoFisher Scientific	24615	Coomassie staining for protein detection in SDS/PAGE
Ins(1,4,5)P3 Beads	Echelon	Q-B0145-1ml	Affinity chromatography reagent
Instant Nonfat Dry Milk	Thomas Scientific	C837M64	Blocking reagent for Western blotting
Iodoacetamide	Sigma-Aldrich	I6125	Alkylating reagent for cysteine proteins or peptides
Lab Rotator	Thomas Scientific	1159Z92	For binding assays
LoBind Microcentrifuge Tubes	ThermoFisher Scientific	13-698-793	Low protein binding tubes for mass spectrometry
Nonidet P-40 (Igepal CA-630)	Sigma-Aldrich	21-3277	Detergent
PBS, pH 7.4	ThermoFisher Scientific	10010031	Physiological buffer
Peroxidase substrate for chemiluminescence	ThermoFisher Scientific	32106	Substrate for Western bloting detection of proteins
PhosSTOP Phosphatase Inhibitor Cocktail Tablets	Roche	4906845001	Phosphatase inhibitors
PI(3)P PIP Beads	Echelon	P-B003a-1ml	Affinity chromatography reagent
PI(3,4)P2 PIP Beads	Echelon	P-B034a-1ml	Affinity chromatography reagent
PI(3,4,5)P3 diC8	Echelon	P-3908-1mg	Affinity chromatography reagent
PI(3,4,5)P3 PIP Beads	Echelon	P-B345a-1ml	Affinity chromatography reagent
PI(3,5)P2 PIP Beads	Echelon	P-B035a-1ml	Affinity chromatography reagent
PI(4)P PIP Beads	Echelon	P-B004a-1ml	Affinity chromatography reagent
PI(4,5)P2 diC8	Echelon	P-4508-1mg	Affinity chromatography reagent
PI(4,5)P2 PIP Beads	Echelon	P-B045a-1ml	Affinity chromatography reagent
PI(5)P PIP Beads	Echelon	P-B005a-1ml	Affinity chromatography reagent
Ponceau S solution	Sigma-Aldrich	P7170	Protein staining (0.1% [w/v] in 5% acetic acid)
Potassium hexacyanoferrate(III)	Sigma-Aldrich	702587	Potassium salt
PtdIns PIP Beads	Echelon	P-B001-1ml	Affinity chromatography reagent
PVDF Membrane	Bio-Rad	1620177	For Western blotting
Refrigerated centrifuge	Eppendorf	5910 R	Microcentrifuge for small volumes (e.g., 1.5 mL)
Sodium dodecyl sulfate	Sigma-Aldrich	862010	Detergent
Sodium thiosulfate	Sigma-Aldrich	72049	Chemical
SpeedVac Vacuum Concentrators	ThermoFisher Scientific	SPD120-115	Sample concentration (e.g., for mass spectrometry)
T175 flasks for cell culture	ThermoFisher Scientific	159910	To grow 50 mL T. brucei culture
Trypsin, Mass Spectrometry Grade	Promega	V5280	Trypsin for protein digestion
Urea	Sigma-Aldrich	U5128	Denaturing reagent
Vortex	Fisher Scientific	02-215-418	For mixing reactions
Western blotting transfer buffer	Bio-Rad	1610734	25 mM Tris, 192 mM glycine, pH 8.3 with 20% methanol
Whatman 3 mm paper	Sigma-Aldrich	WHA3030861	Paper for Wester transfer
2-mercaptoethanol (14.2 M)	Bio-Rad	1610710	Reducing agent
2x Laemmli Sample Buffer	Bio-Rad	161-0737	Protein loading buffer
4–20% Mini-PROTEAN TGX Precast Protein Gels	Bio-Rad	4561094	Gel for protein electrophoresis
4x Laemmli Sample Buffer	Bio-Rad	161-0747	Protein loading buffer

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Cestari, I. Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry. J. Vis. Exp. (149), e59865, doi:10.3791/59865 (2019).