1.Preparing APEX2 fusion constructs
1.1.Clone the cDNA or gene of the POI in-frame with the APEX2 cDNA in a Dictyostelium expression vector of choice. If possible, two APEX2-POI constructs should be made, one with APEX2 fused in N-terminus and one fused at the C-terminus to test both conformations.
1.2.Ideally, an APEX2 labeling control protein that localizes to the same subcellar compartment as the POI should also be prepared, which will help distinguish between specific interactions and bystander proteins.
1.3.An epitope tag or GFP can also be added to the APEX2 constructs to facilitate their analysis by western blot and imaging.
2.Validating the APEX2 constructs
2.1.Culture and transform cells according to standard protocols .
2.2.Verify the expression and integrity of APEX2 fusion proteins by western blot of whole cell lysates using a POI- or tag-specific antibody and standard SDS-PAGE and western blot protocols.
2.3.Verify the functionality of the APEX2-fused POI using appropriate activity assay and/or phenotypic rescue of null strains. This will vary with the POI under study.
2.4.Verify the localization of the APEX2-fused POI and APEX2 control by confocal immunofluorescence or fluorescence imaging if using GFP.
2.5.Verify APEX2 functionality in a small-scale labeling assay after cell permeabilization, described in Sections 3 and 4 below.
3.1.Transfer the developed cells (1×108 for small scale assay; 1×109 for large scale assay) to 50 mL conical tubes, pellet by centrifugation at 500 xg for 3 minutes at 4oC and wash twice with 40 mL cold 12 mM Na/K phosphate buffer, pelleting the cells by centrifugation at 500 xg for 3 minutes at 4oC after each wash.
3.2.Resuspend the cells to 2×107 cells/mL in ice-cold 12 mM Na/K phosphate buffer (5 mL for small scale assay; 50 mL for large scale assay). Transfer cells to an ice-cold 125 mL Erlenmeyer flask.
3.3.Add 1 volume of ice-cold 0.2 mM digitonin to the cells (0.1 mM final). Incubate at 4oC or on ice with gentle shaking on orbital shaker at ~100 rpm for 5 minutes.
3.4.Transfer the cells to cold 50 mL conical tubes and pellet by centrifugation at 500 xg for 3 minutes at 4oC.
3.5.Wash the cells twice with 40 mL ice-cold 12 mM Na/K phosphate buffer with gentle up and down pipetting, pelleting the cells by centrifugation at 500 xg for 3 minutes at 4oC after each wash.
3.6.Resuspend the cells to 1×108 cells/mL in 12 mM Na/K phosphate buffer (1 mL small scale assay; 10 mL large scale assay) at room temperature.
4.Small scale APEX2 labeling
4.1.Perform the cell permeabilization steps (Section 3 above) using APEX2-POI-expressing cells, APEX2-control-expressing cells, and wild-type cells as reference control. For each strain or condition to be tested, resuspend the 1×108 developed, permeabilized cells in 1 mL room temperature 12 mM Na/K phosphate buffer in a 50 mL conical tube.
4.2.Add 2 µL of 500 mM BP (1 mM final) and incubate for 30 minutes at 22oC with shaking on orbital shaker at ~140 rpm.
4.3.Start the labeling reaction by adding 20 µL of 100 mM H2O2 (2 mM final). Mix by swirling or pipetting up and down and incubate 30 seconds.
4.4.Stop the reaction by adding 1 mL ice-cold quenching solution and mix well by pipetting up and down. Transfer to 5 mL tubes.
4.5.Pellet the cells by centrifugation at 500 xg for 3 minutes at 4oC and quickly wash twice with 1 mL ice-cold quenching solution, pelleting the cells by centrifugation at 500 xg for 3 minutes at 4oC after each wash.
4.6.Resuspend the cells in 0.5 mL RIPA lysis buffer, vortex and incubate on ice for 2 minutes.
4.7.Analyze the samples by western blot as described in Section 7 below. If the labeling is satisfactory, proceed with the large-scale labeling for subsequent MS/MS analysis.
5.Large-scale APEX2 labeling
5.1.For each cell strain, resuspend 1×109 developed cells in 10 mL room temperature 12 mM Na/K phosphate buffer in a 50 mL conical tube.
5.2.Add 20 µL of 500 mM BP (1 mM final) and incubate for 30 minutes at 22oC with shaking at ~140 rpm.
5.3.Start the labeling reaction and stimulate with cAMP at the same time by simultaneously adding 200 µL of 50 µM cAMP solution (1 µM final) and 200 µL of 100 mM H2O2 (2 mM final). Mix and incubate 30 seconds.
5.4.Stop the reaction by adding 40 mL ice-cold quenching solution and mix well by pipetting up and down.
5.5.Pellet the cells by centrifugation at 500 xg for 3 minutes at 4oC and quickly wash the cells twice with 40 mL ice-cold quenching solution, pelleting the cells by centrifugation at 500 xg for 3 minutes at 4oC after each wash.
5.6.Resuspend the cells in 5 mL RIPA lysis buffer, vortex and incubate at 4oC or on ice with shaking on orbital shaker at ~140 rpm for 4 hours.
5.7.Vortex and transfer to polycarbonate tubes and clarify the cell lysates by centrifugation at 15,000 xg for 10 minutes at 4oC in a high-speed centrifuge.
5.8.Transfer the clarified lysates to 15 mL conical tubes and keep on ice.
5.9.Take 10 µL aliquots for total biotin-labeled protein analysis by western blot as described in Section 7 below, mix with 2 µL 6X protein sample buffer in 1.7 mL tube and store at −20oC until needed.
6.Purifying biotin-labeled proteins
6.1.Quantify the lysates’ protein content using a reducing agent compatible assay (e.g. PierceTM 660 nm Protein Assay Reagent) according to the manufacturer’s protocol.
6.2.Add 60 µL of streptavidin-agarose beads (6% slurry) to the lysates for every 360 µg of protein in lysates, and incubate at 4oC with agitation (on rotator or rocker) for at least 1 hour, and up to 16 hours.
6.3.Pellet the beads by centrifugation at 1,000 xg for 1 minute at 4oC and transfer the supernatants to new 1.7 mL tubes. These are the “flowthrough” samples containing unbound proteins and are used to evaluate purification efficiency. Take 10 µL aliquots and mix with 2 µL 6X protein sample buffer for analysis by western blot and store at −20oC until needed.
6.4.Always keeping buffers and samples on ice, wash the beads as follows: twice with 10 mL RIPA lysis buffer, once with 10 mL 1 M KCl, once with 10 mL 0.1 M Na2CO3, once with 10 mL 2 M Urea in Tris-HCl pH 8.0, and twice with 10 mL RIPA lysis buffer, pelleting the beads by centrifugation at 1,000 xg for 1 minute at 4oC between washes.
6.5.Elute the purified proteins by adding 300 µL of 3X biotin elution protein sample buffer to the beads, mix and incubate at 95oC for 10 minutes. Vortex, cool on ice, and centrifuge briefly using a mini centrifuge to bring down condensation.
6.6.Mix and then pellet the beads by centrifugation at 1,000 xg for 1 minute at 4oC, transfer eluates to new 1.7 mL tubes and keep on ice or store at −20oC until needed.
7.Analyzing biotin labeling by western blot
7.1.Thaw the clarified lysates, flowthroughs, and eluate samples containing sample buffer to room temperature, then centrifuge briefly in a mini centrifuge before incubating at 95oC for 10 minutes.
7.2.Centrifuge the heated samples in a mini centrifuge, and then vortex to ensure sample homogeneity before loading and separating on 8% SDS-PAGE gel.
7.3.Transfer the proteins to a nitrocellulose membrane and incubate in blocking buffer at 4oC for 16 hours with rocking on platform rocker.
7.4.Wash the membrane once with 20 mL TBST and incubate with 0.3 µg/mL streptavidin-HRP in blocking buffer at room temperature for 1 hour with rocking on platform rocker.
7.5.Wash the membrane in 20 mL TBST for 30 minutes with rocking on platform rocker, followed by 4 similar washes of 5 minutes each.
7.6.Develop using chemiluminescence and reveal by autoradiography or using a digital imaging system.
8.Preparing samples for LC-MS/MS
8.1.Load the entirety of each sample in one large well per sample on a 12.5% SDS-PAGE gel and perform electrophoresis only until the migration front is at ~1 cm in the gel.
8.2.Cut gel bands containing the proteins, including migration front with bromophenol blue, using sterile razor blades and transfer the bands to 1.7 mL tubes. The gel bands can then be kept at −20oC until used further.
8.3.Incubate the gel bands with de-staining solution, minimal amount just to cover them, on a vortex mixer at low speed for 1 hour. Repeat with fresh de-staining solution as necessary to remove all staining, changing the solution by pelleting the gel bands by brief centrifugation in a mini centrifuge and pipetting the supernatant out.
8.4.Incubate the gel bands with the reducing solution at 60oC for 45 minutes. Pellet the gel bands and discard supernatant.
8.5.Incubate the gel bands with the alkylating solution in the dark at room temperature for 30 minutes. Pellet the gel bands and discard supernatant.
8.6.Wash the gel bands 3 times with 250 µL of 50 mM ammonium bicarbonate buffer on a vortex mixer at low speed, for 30 minutes each wash.
8.7.Transfer the gel bands to a clean glass plate (previously cleaned with methanol or isopropanol) and cut them into small pieces with a sterile razor blade.
8.8.Place the gel pieces in a 1.7 mL tube and dehydrate by incubating in acetonitrile, just enough to cover the gel pieces, for 10 minutes, vortexing a few times. Gel pieces will become white and stick together.
8.9.Pellet the gel pieces by brief centrifugation in a mini centrifuge, discard acetonitrile and dry in a Speed Vacuum Concentrator (~30 minutes).
8.10.Rehydrate the gel pieces in trypsin digestion solution, just enough to cover the gel pieces, for 15 minutes at room temperature. Vortex to mix.
8.11.Digest the proteins in the trypsin digestion solution at 37oC using block heater filled with water, for 16 hours.
8.12.Add 10% formic acid final to the in-gel protein digestion mixture and vortex to mix. Verify the pH with pH test strips, which should be between 2‒3. If needed, adjust pH with formic acid.
8.13.Pellet the gel pieces by brief centrifugation in a mini centrifuge, transfer the supernatant to a new 1.7 mL tube, cool on ice and centrifuge briefly to bring down condensation.
8.14.Extract all peptides from the gel pieces by adding peptide extraction solution, just enough to cover the gel pieces, and sonicate 10 minutes. Pellet the gel pieces by brief centrifugation in a mini centrifuge. Transfer the supernatant, combining with that from Step 8.13. Repeat the extraction procedure a second time and combine all supernatants in a 1.7 mL tube.
8.15.Clarify the peptide solution by centrifugation at 16,000 xg in a microcentrifuge, at room temperature for 10 minutes. Transfer the supernatant to a new 1.7 mL tube.
8.16.Reduce to ~20 µL using a Speed Vacuum Concentrator.
8.17.The peptides are then separated by standard liquid chromatography followed by tandem mass spectrometry.
8.18.To identify the proteins, mass spectra are searched against the SwissProt Dictyostelium discoideum protein database and an additional common contaminant database (e.g. trypsin, keratins).
|2||Low APEX2 fusion protein expression levels||-Low transformation efficiency -Unstable fusion protein due to APEX2 cysteine residue 32 (C32) that can affect folding of some proteins ||-Generate stable, clonal cell lines -Use an APEX2-C32S mutant |
|4||No or little biotin labeling||-Reagents not fresh -Reagents not fully dissolved in their solvent -Cell lysis||-It is important to prepare many of the reagents fresh, where indicated, the day of the experiment and even within ~2 hours. of utilization. -Make sure that digitonin and BP are completely dissolved by sonicating the solutions in a bath sonicator. -Be very careful and gentle when permeabilizing the cells with digitonin and washing them after the treatment, rough manipulation of the cells at these steps will lead to cell lysis.|
|6||Inefficient purification||-Left over BP in lysates, competing for binding the beads -Low binding of the biotin-labeled proteins to the streptavidin beads -The biotin-labeled proteins did not elute||-Increase the quenching solution volume and number of washes -Make sure that the pH of the RIPA buffer is ~7.5 and to use 60 μL or more of 6% streptavidin-agarose slurry for each 360 µg of lysate proteins. Also try longer incubations of lysates with the streptavidin beads. -Make sure that biotin in the 100 mM stock solution is completely dissolved after thawing an aliquot to prepare the 3X biotin elution sample buffer, and warm up the 6X protein sample buffer + ddH2O to at least 37oC before adding the biotin to prevent precipitation. This will ensure adequate concentration of biotin and optimal elution.|