Endosomal and lysosomal immunoprecipitation for proteomics, lipidomics, and TEM

Seed 293 cells or 293^EL cells expressing TMEM192-3xHA and 3xFLAG-EEA1 in 15-cm dishes, with one dish per replicate. Creation of the 293^EL cells is described in protocol dx.doi.org/10.17504/protocols.io.byi7puhn.

At 80% confluency, harvest cells on ice by scraping in 2 mL of cold DPBS. Pellet at 1,000xg for 2 min at 4 ºC.

Discard supernatants, wash pellets once with 1 mL of cold KPBS buffer (25 mM KCl, 100 mM potassium phosphate, pH 7.2), and pellet at 1,000xg for 2 min at 4 ºC.

Resuspend cell pellets in 1 mL of KBPS buffer supplemented with protease and phosphatase inhibitor tablets and lyse with 30 strokes with a 2 mL Dounce homogenizer on ice.

Centrifuge lysed cells at 1,000xg for 5 min at 4 ºC, and transfer the post-nuclear supernatants (PNS) to new tubes on ice.

If needed to remove excess nuclear components, spin the PNS from step 5 again at 1,000xg for 5 min at 4 ºC, and transfer the final PNS to new tubes on ice.

Determine total protein concentration by Bradford assay, and transfer 10-20 µL of each PNS to a new tube and combine with 20 µL of RIPA lysis buffer and 10 µL of 4X LDS buffer with reducing agent for later analysis by Western blot.

Wash α-HA magnetic beads (60 µL of bead slurry per dish) three times with 1 mL KPBS buffer with inhibtors and resuspend in KPBS with inhibitors. Add the resuspended bead slurry to each PNS, and incubate samples at 4 ºC for 50 min with gentle rotation.

Separate beads from the lysate with a magnetic stand, and collect the flow through. For Western blot analysis, combine 10-20 µL of each flow through with 20 µL of RIPA lysis buffer and 10 µL of 4X LDS buffer with reducing agent.

Using a magnetic stand, wash beads twice with 500 µL of high salt KPBS buffer (25 mM KCl, 100 mM potassium phosphate, 150 mM NaCl, pH 7.2) with protease and phosphatase inhibitors, then wash once with normal KPBS (25 mM KCl, 100 mM potassium phosphate, pH 7.2) with inhibitors.

Elute samples by addition of 120 µL 0.5% NP-40 in KBPS with inhibitors for 30 min at 4 ºC with gentle rotation. For Western blot analysis, combine 20 µL of each eluate with 6.7 µL of 4X LDS buffer with reducing agent. Immediately process remainder of eluates or snap freeze in liquid nitrogen and store at -80 ºC until processing for mass spectrometry.

Seed 293 or 293^EL cells in 15cm dishes with one dish per replicate.

If treating with DNM1/2 inhibitor Dyngo4a, treat 70-80% confluent dishes with either DMSO (0.4%) or Dyngo4a (20 µM final) in serum-free DMEM for 3h. After treatment, wash cells with DMEM with 10% serum and 0.4% DMSO.

Harvest cells at 70-80% confluency on ice by scraping in 2 mL DPBS and pelleting at 1,000xg for 2 min at 4 ºC.

Discard supernatants, and wash pellets once with 1 mL of KPBS buffer (25 mM KCl, 100 mM potassium phosphate, pH 7.2) and pellet at 1,000xg for 2 min at 4 ºC.

Resuspend cell pellets in 500 µL of KPBS supplemented with protease inhibitor cocktail and PhosSTOP tablets and lyse with 30 strokes with a 2 mL Dounce homogenizer on ice.

Centrifuge lysed cells at 1,000xg for 5 min at 4 ºC, and transfer the post-nuclear supernatants (PNS) to new tubes on ice.

If needed to remove excess nuclear components, spin the PNS from step 15 again at 1,000xg for 5 min at 4 ºC, and transfer the final PNS to new tubes on ice.

Determine total protein concentration of each lysate by Bradford assay, and transfer 10-20 µL of each PNS to a new tube and combine with 20 µL of RIPA lysis buffer and 10 µL of 4X LDS buffer with reducing agent for later analysis by Western blot (see protocol dx.doi.org/10.17504/protocols.io.byi8puhw).

Wash α-FLAG M2 magnetic beads (60 µL of bead slurry per dish) three times with 1 mL KPBS buffer with inhibitors, and resuspend in the same buffer. Add resuspended bead slurry to each PNS, and incubate at 4 ºC for 50 min with gentle rotation.

Separate beads from the lysate with a magnetic stand, and collect the flow through.

For Western blot analysis, combine 10-20 µL of each flow through with 20 µL of RIPA lysis buffer and 10 µL of 4X LDS buffer with reducing agent.

Using a magnetic stand, wash beads three times with 500 µL of KPBS buffer (25 mM KCl, 100 mM potassium phosphate, pH 7.2) with inhibitors.

The washed beads can be stored at -80 °C until being processed for lipidomics study.

Elution:

For analysis by negative stain transmission electron microscopy (TEM), elute samples by addition of 50 µL 3xFLAG peptide solution (500 µg/mL in KPBS) at 25 ºC for 45 min with gentle shaking. Transfer eluates to new tubes, and proceed to TEM analysis.

Alternatively, for organelle proteomics analysis by mass spectrometry, elute samples by addition of 120 µL 0.5% NP-40 in KBPS with inhibitors for 30 min at 4 ºC with gentle rotation. For Western blot analysis, combine 20 µL of each eluate with 6.7 µL of 4X LDS buffer with reducing agent. Immediately process the remainder of the eluates or snap freeze in liquid nitrogen and store at -80 ºC until processing for LC-MS.

For each replicate, seed 293^EL-APP* cells in 5x15cm dishes (2x15cm for Lyso-IP and 3x15cm for Endo-IP), and seed 293^EL-APP-/- cells in 5x15 cm dishes so that they will be approximately 60% confluent the next day and approximately 80-90% confluent two days later.

Generally, three replicates of each 293EL-APP* treatment group (e.g. DMSO or secretase inhibitors) and two replicates of 293EL-APP-/- should be processed simultaneously.

One day after seeding, treat cells with vehicle control (DMSO), GSI, GSM, or BSI to a final concentration of 2 µM and 0.2% DMSO. Incubate cells with the compounds for 15h.

The next day, harvest cells by discarding media and scraping in 2 mL KPBS buffer supplemented with DMSO, GSI, GSM, or BSI (note that the appropriate compound should be used in KPBS buffer throughout subsequent steps to continue inhibiting the desired enzyme).

Pellet cells at 1,000xg for 2 min at 4 ºC, discard supernatants, resuspend pellets in 5 mL KPBS, and pellet cells at 1,000xg for 2 min at 4 ºC.

Resuspend pellets in 5 mL of KPBS with the addition of protease and phosphatase inhibitors and lyse with 20 strokes with a 7-mL Dounce homogenizer and tight pestle.

Clarify lysate by centrifugation at 1,000xg for 5 min at 4 ºC. The lysate may be further clarified by transferring the PNS from the first spin to a new tube on ice, spinning again, and transferring the final PNS to a new tube.

Determine the protein concentration of each lysate by Bradford assay, and transfer 10-20 µL of each PNS to a new tube and combine with 20 µL of RIPA lysis buffer and 10 µL of 4x LDS buffer with reducing agent for later analysis by Western blot.

Combine 110 µL of each PNS with 183 µL of 8M urea/50mM NaCl/0.8% NP-40 buffer and store at -80 ºC for later analysis by mass spectrometry.

Prepare α-FLAG and α-HA magnetic beads (50 µL of bead slurry per dish) on a magnetic stand by washing three times with KPBS and resuspending in KPBS (25 µL per dish for α-FLAG beads and 50 µL per dish for α-HA beads). Add 150 µL of α-FLAG M2 beads per PNS (which came from 3x15cm dishes) and add 100 µL of α-HA beads per PNS (which came from 2x15cm dishes). Incubate samples for 45 min at 4 ºC with gentle rotation.

Separate beads from the flow through with a magnetic stand, and collect the flow through. For Western blot analysis, combine 10-20 µL of each flow through with 20 µL of RIPA lysis buffer and 10 µL of 4x LDS buffer with reducing agent.

Wash beads:

Wash α-FLAG beads twice with 500 µL KPBS containing the compound, and once with 1 mL KPBS without compounds.

Wash α-HA beads twice with 500 µL high-salt KPBS (KPBS with 155 mM NaCl) containing the compound, and once with 1 ml regular salt KPBS without compounds.

Elute samples with 5M urea/0.5% NP-40 KPBS buffer (180 µL for α-FLAG beads and 120 µL for α-HA beads) for 50 min at 30 ºC with shaking.

For Western blot analysis, combine 10 µL of each eluate with 3.3 µL of 4X LDS buffer with reducing agent.

Split the remainder of each eluate in two for future “Lyso” or “Endo” (20% of eluate) and “Lyso_LMW” or “Endo_LMW” (80% of eluate) samples, the latter of which are filtered as follows.

To detect low abundance Aβ peptides, filter samples with Amicon Ultra 0.5 mL 50 kDa centrifugal filters. Load 250 µL of each PNS onto a 50 kDa Amicon column, and reserve the remainder of the PNS to serve as the regular PNS sample.

Dilute Lyso_LMW samples with 112 µL of 5M urea/0.5% NP-40 buffer and load onto 50 kDa columns. Dilute Endo_LMW samples with 64 µL of 5 M urea/0.5% NP-40 buffer and load onto 50 kDa columns.

Centrifuge columns at 14,000 g at 10 ºC for 12 min or until residual column volume is approximately 50 µL. To increase the yield of filtered Aβ peptides, dilute residual retentate with 150 µL of 5 M urea/0.5% NP-40 buffer, and centrifuge the columns at 14,000xg at 10ºC for 12 min.

Measure the final filtrate volume and transfer to new Protein LoBind tubes. Dilute remaining, unfiltered PNS, Lyso, and Endo samples with 20 µL 5 M urea/0.5% NP-40.

Immediately process the remainder of the eluates or snap freeze in liquid nitrogen and store at -80 ºC until processing for proteomics study.