TITLE: Endosomal and lysosomal immunoprecipitation for proteomics, lipidomics, and TEM
AUTHORS: Hankum Park, Frances V Hundley, J. Wade Harper

[DESCRIPTION]
Previous studies have developed methods for isolation of lysosomes, mitochondria, and peroxisomes from non-denaturing extracts. Here we describe an approach for purification of early/sorting endosomes, providing a means by which to examine early aspects of the endolysosomal system and to combine this with lysosome purification using Lyso-IP.  We refer to this new method as Endo-IP. This allows us to examine the proteome and lipidome of endosomes, and to perform electron microscopy imaging of endosomes. 

[STEPS]
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
1. Seed 293 cells or 293EL cells expressing TMEM192-3xHA and 3xFLAG-EEA1 in 15-cm dishes, with one dish per replicate. Creation of the 293EL cells is described in protocol dx.doi.org/10.17504/protocols.io.byi7puhn.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
2. 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.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
3. 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.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
4. 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.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
5. Centrifuge lysed cells at 1,000xg for 5 min at 4 ºC, and transfer the post-nuclear supernatants (PNS) to new tubes on ice.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
6. 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.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
7. 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.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
8. 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.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
9. 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.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
10. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
11. Seed 293 or 293EL cells in 15cm dishes with one dish per replicate.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
11.1. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
12. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
13. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
14. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
15. Centrifuge lysed cells at 1,000xg for 5 min at 4 ºC, and transfer the post-nuclear supernatants (PNS) to new tubes on ice.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
16. 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).
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
17. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
18. Separate beads from the lysate with a magnetic stand, and collect the flow through.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
18.1. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
19. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
19.1. The washed beads can be stored at -80 °C until being processed for lipidomics study.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
20. Elution:
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
20.1. 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.
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
20.2. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
21. For each replicate, seed 293EL-APP* cells in 5x15cm dishes (2x15cm for Lyso-IP and 3x15cm for Endo-IP), and seed 293EL-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. 
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
21.1. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
22. 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.  
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
23. 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).
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
24. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
25. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
26. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
27. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
28. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
29. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
30. 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. 
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
31. Wash beads:
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
31.1. Wash α-FLAG beads twice with 500 µL KPBS containing the compound, and once with 1 mL KPBS without compounds.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
31.2. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
32. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
33. For Western blot analysis, combine 10 µL of each eluate with 3.3 µL of 4X LDS buffer with reducing agent. 
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
34. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
34.1. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
34.2. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
34.3. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
34.4. 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.
SECTION: Endosomal and lysosomal enrichment for APP/Aβ TOMAHAQ proteomics
35. Immediately process the remainder of the eluates or snap freeze in liquid nitrogen and store at -80 ºC until processing for proteomics study.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organelle proteomics
5.1. 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. 
SECTION: Endosomal immunoprecipitation (Endo-IP) for proteomics, TEM, and lipidomics
15.1. 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.