TITLE: Endosomal, lysosomal, mitochondrial, or Golgi immunoprecipitation for quantitative proteomics
AUTHORS: J. Wade Harper, Louis R R Hollingsworth, Chan Lee

[DESCRIPTION]
Previous studies have developed methods for the immunoisolation of lysosomes, mitochondria, EEA1-positive endosomes, Golgi, and other organelles from non-denaturing / non-detergent extracts. Here we describe our most up-to-date protocols for these approaches. Specifically, we describe methods to isolate lysosomes, mitochondria, EEA1-positive endosomes, Golgi, and the preparation of peptides for proteomics applications. We structure our protocol with a modular framework for the future addition of new cell types and organelle-IPs by anyone in the community.

[BEFORE_START]
Note that a significant amount of information is contained in the "Materials" section.

[STEPS]
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
8. Resuspend cell pellets in 1 mL of Lyso-KBPS buffer supplemented with protease and phosphatase inhibitor tablets and lyse with 25-30 strokes with a 2 mL Dounce homogenizer on ice. Collect with a Pasteur pipette. Harvest into low-bind microcentrifuge tubes.
 


 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
9. Centrifuge lysed cells at 1,000 xg for 5 min at 4 ºC, and transfer the post-nuclear supernatants (PNS) to new tubes on ice.
 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
10. Transfer 10 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; "INPUT sample". These amounts can be doubled if needed.
 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
6. Invert α-HA magnetic beads several times gently to resuspend them, and then aliquot (60-80 µL of bead slurry per replicate, plus 10% excess) into a low-bind tube. Rotate end-over-end at 4 ºC until use (below).

 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
12. Separate beads from the lysate with a magnetic stand, and collect the flow through ("FLOW THROUGH"). For Western blot analysis, combine 10 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 organellar proteomics
13. Using a magnetic stand, wash beads twice with 1 mL of ice-cold high salt Lyso-KPBS buffer (136mM KCL, 10 mM potassium phosphate, pH 7.25, 150 mM NaCl) with inhibitors. 

 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
15. Using a magnetic stand, pellet and aspirate the final Lyso-KPBS solution. Elute samples by addition of 90 µL 0.5% NP-40 in KBPS with inhibitors for 30 min at 4 ºC with gentle rotation. Separate beads from the lysate with a magnetic stand, and collect the flow through ("ELUTE").
 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
9.1. Spin the PNS from step 8 again at 1,000 xg for 5 min at 4 ºC, and transfer the final PNS to new tubes on ice.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
8.1. Add an additional 200 µL of ice-cold Lyso-KPBS to the homogenizer and collect with a Pasteur pipette. Wash the homogenizer with KPBS (without inhibitors) between replicates, and thoroughly wash between experimental conditions (with a final KPBS rinse).
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
11. Wash α-HA magnetic beads (60-80 µL of bead slurry per replicate) three times with 1 mL Lyso-KPBS buffer and then resuspend them in Lyso-KPBS with inhibitors using a wide-bore P1000 pipette. Add the resuspended bead slurry to each PNS, and incubate samples at 4 ºC for 15 min with gentle rotation.
SECTION: THP-1 differentiation and harvest for organelle-IP
6. Count suspension THP-1 monocytes, and seed 35 million cells in 15-cm plates per replicate. Bring to a final volume of 40 mL.
 

SECTION: THP-1 differentiation and harvest for organelle-IP
7. Add 10 mL of complete RPMI containing 100 ng/mL PMA to each plate for a final concentration of 20 ng/mL.

 

SECTION: THP-1 differentiation and harvest for organelle-IP
8. 24 hr - 48 hr later, replace PMA-containing medium with 50 mL complete RPMI (differentiated THP-1s should now be adherent). Rest for an additional 24 hr prior to any treatment(s) and cell harvesting.
SECTION: HEK cell plating for organelle-IP
6. Seed cells to be 80%-95% confluent on the day of the experiment. For HEK cells, we've found that a 1:8 split from a confluent 15 cm usually results in confluent cells in 2 days, but growth rates could be different due to clonal effects, genetic perturbations, etc.
 

5. The following steps are largely the same for Lyso, Mito, Golgi, and EndoIPs. Please be aware, however, that the buffer and magnetic beads are different for EndoIP, and that LysoIP contains a high salt wash different from Mito/Golgi IPs. We also recommend different incubation times for the different organelle isolations; however, folks in our lab have been successful in enriching organelles with a range of incubation times (e.g., 10 - 50 mins). Smaller bead binding times lead to more specific isolation, at the cost of less material.
 

4. Proceed to plate and harvest cells ("cell plating and harvesting", perform the appropriate OrganelleIP protocol ("OrganelleIP"), then prep peptides for proteomics ("proteomics prep", or stop with a western blot).
SECTION: Construct selection, cell line engineering, and validation
2. Validate tag successful tag incorporation by western blot.

For LysoIP, blot for the HA tag (1:1000, CST #2367S; AB_10691311) and endogenous TMEM192 (1:1000, Proteintech 28263-1-AP, AB_2881099).

For MitoIP, blot for the HA tag (1:1000, CST #2367S; AB_10691311).

For GolgiIP, blot for the HA tag (1:1000, CST #2367S; AB_10691311) and endogenous TMEM115 (1:500, Novus NBP1-80898; AB_11002107). Note that this antibody is fairly nonspecific (several nonspecific bands are more prominent than TMEM115 itself) and the reactivity of this antibody is untested in mouse cells.

For EndoIP, blot for the FLAG tag (1:1000, Sigma F1804; AB_262044) and endogenous EEA1 (1:1000, CST #3288S; AB_2096811).

SECTION: Construct selection, cell line engineering, and validation
1. For LysoIP: Express the LysoTag, TMEM192-3xHA, endogenously (Addgene#175777) or with stable lentiviral overexpression (e.g., with either Addgene#102930 or Addgene#134631). Keep the parental cell line as a negative control.

For EndoIP: Express the EndoTag, 3xFLAG-EEA1, endogenously (Addgene#214989 or Addgene#214990) or with stable lentiviral overexpression (e.g., with either Addgene#176491, Addgene#214994, Addgene#214995). Keep the parental cell line as a negative control.

For MitoIP: Express the MitoTag, 3xHA-OMP25, with stable lentiviral overexpression (e.g., with Addgene#83356). Keep the parental cell line, or a different epitope tag (e.g., Addgene#83355) as a negative control.

For GolgiIP: Express the GolgiTag, TMEM115-3xHA, endogenously (Addgene#214998, Addgene#214999, or Addgene#215000) or with stable lentiviral overexpression (e.g., with either Addgene#214996, Addgene#214997). Keep the parental cell line as a negative control.

 

SECTION: Cell plating and harvesting
5. The steps below are largely the same for HEK and other immortalized cell lines with appropriate morphology (e.g., HeLa cells, others). We have found that round cells are much tougher to lyse gently, and so certain cell types might require extensive optimization. We recommend attempting homogenization prior to the stable incorporation of tags to check feasibility. Additionally, these protocols should be tailored to the particular cell type prior to scaling up for a full proteomics experiment. Cells cannot be harvested and then frozen at any step for these protocols--the first pause point is after elution from the beads.
SECTION: THP-1 differentiation and harvest for organelle-IP
9. Harvest cells (or first add a treatment) on ice by scraping in 5 mL of cold DPBS. Harvest residual cells by adding an additional 2 mL DPBS to the plate. Pellet at 1,000 xg for 2 min at 4 ºC.
 

SECTION: HEK cell plating for organelle-IP
7. At 80%-95% confluency, harvest cells (or first add a treatment) on ice by scraping in 5 mL of cold DPBS. Harvest residual cells by adding an additional 2 mL DPBS to the plate. Pellet at 1,000 xg for 2 min at 4 ºC.
 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
7. Proceeding from "cell plating and harvesting":  Discard supernatants, wash pellets once with 1 mL of ice-cold Lyso-KPBS buffer (136mM KCL, 10 mM potassium phosphate, pH 7.25, protease and phosphatase inhibitors), and pellet again at 1,000 xg for 2 min at 4 ºC. Work quickly, because the cells will likely start to lyse.
 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
16. Resuspend beads in an additional 30 µL 0.5% NP-40 in KBPS with inhibitors to harvest any additional protein (collect the flow through). Combine with the eluate from step 14.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
17. 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. For MS processing, see the "Proteomics prep" step case above.
SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
14. Wash once with 1 mL ice-cold Lyso-KPBS (136mM KCl, 10 mM potassium phosphate, pH 7.25) with inhibitors. Transfer the samples to new tubes.
 


5. The proteomics prep follows from "OrganelleIP", and assumes 100 µL of eluate (20 µL taken for Western blot). If you have more (e.g., you didn't run the western blot) or less material, scale everything accordingly until after the TCA precipitation steps, then proceed as indicated.
SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
7. Proceeding from "cell plating and harvesting": Discard supernatants, wash pellets once with 1 mL of ice-cold Mito-KPBS buffer (136mM KCL, 10 mM potassium phosphate, pH 7.25, protease and phosphatase inhibitors), and pellet again at 1,000 xg for 2 min at 4 ºC. Work quickly, because the cells will likely start to lyse.
 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
8. Resuspend cell pellets in 1 mL of Mito-KBPS buffer supplemented with protease and phosphatase inhibitor tablets and lyse with 25-30 strokes with a 2 mL Dounce homogenizer on ice. Collect with a Pasteur pipette. Harvest into low-bind microcentrifuge tubes.

 


 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
8.1. Add an additional 200 µL of ice-cold Mito-KPBS to the homogenizer and collect with a Pasteur pipette. Wash the homogenizer with KPBS (without inhibitors) between replicates, and thoroughly wash between experimental conditions (with a final KPBS rinse).
SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
9. Centrifuge lysed cells at 1,000 xg for 5 min at 4 ºC, and transfer the post-nuclear supernatants (PNS) to new tubes on ice.
 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
6. Invert α-HA magnetic beads several times gently to resuspend them, and then aliquot (60-80 µL of bead slurry per dish, plus 10% excess) into a low-bind tube. Rotate end-over-end at 4 ºC until use (below).
 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
11. Wash α-HA magnetic beads (60-80 µL of bead slurry per dish) three times with 1 mL Mito-KPBS buffer and  then resuspend them in Mito-KPBS with inhibitors using a wide-bore P1000 pipette. Add the resuspended bead slurry to each PNS, and incubate samples at 4 ºC for 10 min with gentle rotation.
SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
10. Transfer 10 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; "INPUT sample". These amounts can be doubled if needed.
 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
13. Using a magnetic stand, wash beads twice with 1 mL of ice-cold Mito-KPBS buffer with inhibitors. 

 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
14. Wash once more with 1 mL ice-cold Mito-KPBS with inhibitors. Transfer the samples to new tubes.
 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
15. Using a magnetic stand, pellet and aspirate the final Mito-KPBS solution. Elute samples by addition of 90 µL 0.5% NP-40 in KBPS with inhibitors for 30 min at 4 ºC with gentle rotation. Separate beads from the lysate with a magnetic stand, and collect the flow through ("ELUTE").

 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
16. Resuspend beads in an additional 30 µL 0.5% NP-40 in KBPS with inhibitors to harvest any additional protein (collect the flow through). Combine with the eluate from step 14.
SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
17. 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. For MS processing, see the "Proteomics prep" step case above.
SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
9.1. Spin the PNS from step 8 again at 1,000xg for 5 min at 4 ºC, and transfer the final PNS to new tubes on ice.
SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
12. Separate beads from the lysate with a magnetic stand, and collect the flow through ("FLOW THROUGH"). For Western blot analysis, combine 10 µL of each flow through with 20 µL of RIPA lysis buffer and 10 µL of 4X LDS buffer with reducing agent.
SECTION: HEK cell plating for organelle-IP
8. Immediately proceed through the appropriate "OrganelleIP" protocol.
SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
6. Invert α-FLAG magnetic beads several times gently to resuspend them, and then aliquot (60-80 µL of bead slurry per dish, plus 10% excess) into a low-bind tube. Rotate end-over-end at 4 ºC until use (below).
 

SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
7. Proceeding from "cell plating and harvesting": Discard supernatants, wash pellets once with 1 mL of ice-cold Endo-KPBS buffer (25 mM KCl, 100 mM potassium phosphate, pH 7.2), and pellet again at 1,000 g for 2 min at 4 ºC. Work quickly, because the cells will likely start to lyse.
 

SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
8. Resuspend cell pellets in 1 mL of Endo-KBPS buffer supplemented with protease and phosphatase inhibitor tablets and lyse with 25-30 strokes with a 2 mL Dounce homogenizer on ice. Collect with a Pasteur pipette. Harvest into low-bind microcentrifuge tubes.

 

 

SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
8.1. Add an additional 200 µL of ice-cold Endo-KPBS to the homogenizer and collect with a Pasteur pipette. Wash the homogenizer with KPBS (without inhibitors) between replicates, and thoroughly wash between experimental conditions (with a final KPBS rinse).
SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
9. Centrifuge lysed cells at 1,000 xg for 5 min at 4 ºC, and transfer the post-nuclear supernatants (PNS) to new tubes on ice.
 


SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
9.1. Spin the PNS from step 8 again at 1,000 xg for 5 min at 4 ºC, and transfer the final PNS to new tubes on ice.
SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
10. Transfer 10 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; "INPUT sample". These amounts can be doubled if needed.
 

SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
11. Wash α-FLAG magnetic beads (~60 µL-80 µL of bead slurry per dish) three times with 1 mL Endo-KPBS buffer and then resuspend them in Endo-KPBS with inhibitors using a wide-bore P1000 pipette. Add the resuspended bead slurry to each PNS, and incubate samples at 4 ºC for 50 min with gentle rotation.
SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
12. Separate beads from the lysate with a magnetic stand, and collect the flow through ("FLOW THROUGH"). For Western blot analysis, combine 10 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 organellar proteomics
13. Using a magnetic stand, wash beads twice with 1 mL of ice-cold Endo-KPBS buffer with inhibitors. 

 

SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
14. Wash once more with 1 mL ice-cold Endo-KPBS with inhibitors. Use this wash to transfer the samples to new tubes.
 

SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
15. Using a magnetic stand, pellet and aspirate the final Endo-KPBS solution. Elute samples by addition of 90 µL 0.5% NP-40 in KBPS with inhibitors for 30 min at 4 ºC with gentle rotation. Separate beads from the lysate with a magnetic stand, and collect the flow through ("ELUTE").

 

SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
16. Resuspend beads in an additional 30 µL 0.5% NP-40 in KBPS with inhibitors to harvest any additional protein (collect the flow through). Combine with the eluate from step 14.
SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
17. 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. For MS processing, see the "Proteomics prep" step case above.
SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
6. Invert α-HA magnetic beads several times gently to resuspend them, and then aliquot (60-80 µL of bead slurry per dish, plus 10% excess) into a low-bind tube. Rotate end-over-end at 4 ºC until use (below).

 

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
7. Proceeding from "cell plating and harvesting":  Discard supernatants, wash pellets once with 1 mL of ice-cold Golgi-KPBS buffer (136mM KCL, 10 mM potassium phosphate, pH 7.25, protease and phosphatase inhibitors), and pellet again at 1,000 g for 2 min at 4 ºC. Work quickly, because the cells will likely start to lyse.
 

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
8. Resuspend cell pellets in 1 mL of Golgi-KBPS buffer supplemented with protease and phosphatase inhibitor tablets and lyse with 25-30 strokes with a 2 mL Dounce homogenizer on ice. Collect with a Pasteur pipette. Harvest into low-bind microcentrifuge tubes.

 

 

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
8.1. Add an additional 200 µL of ice-cold Golgi-KPBS to the homogenizer and collect with a Pasteur pipette. Wash the homogenizer with KPBS (without inhibitors) between replicates, and thoroughly wash between experimental conditions (with a final KPBS rinse).
SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
9.1. Spin the PNS from step 8 again at 1,000 xg for 5 min at 4 ºC, and transfer the final PNS to new tubes on ice.
SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
9. Centrifuge lysed cells at 1,000 xg for 5 min at 4 ºC, and transfer the post-nuclear supernatants (PNS) to new tubes on ice.
 

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
10. Transfer 10 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; "INPUT sample". These amounts can be doubled if needed.
 

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
11. Wash α-HA magnetic beads (60-80 µL of bead slurry per dish) three times with 1 mL Golgi-KPBS buffer and  then resuspend them in Golgi-KPBS with inhibitors using a wide-bore P1000 pipette. Add the resuspended bead slurry to each PNS, and incubate samples at 4 ºC for 15 min with gentle rotation.
SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
12. Separate beads from the lysate with a magnetic stand, and collect the flow through ("FLOW THROUGH"). For Western blot analysis, combine 10 µL of each flow through with 20 µL of RIPA lysis buffer and 10 µL of 4X LDS buffer with reducing agent.
SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
13. Using a magnetic stand, wash beads twice with 1 mL of ice-cold Golgi-KPBS buffer with inhibitors. 

 

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
14. Wash once with 1 mL ice-cold Golgi-KPBS with inhibitors. Use this wash to transfer the samples to new tubes.
 

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
15. Using a magnetic stand, pellet and aspirate the final Golgi-KPBS solution. Elute samples by addition of 90 µL 0.5% NP-40 in KBPS with inhibitors for 30 min at 4 ºC with gentle end-over-end rotation. Separate beads from the lysate with a magnetic stand, and collect the flow through ("ELUTE").

 

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
16. Resuspend beads in an additional 30 µL 0.5% NP-40 in KBPS with inhibitors to harvest any additional protein (collect the flow through). Combine with the eluate from step 14.
SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
17. 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. For MS processing, see the "Proteomics prep" step case above.
SECTION: THP-1 differentiation and harvest for organelle-IP
10. Immediately proceed through the appropriate "OrganelleIP" protocol.
6. Reduction: Add 1 µL of 500 mM TCEP and vortex (final 5 mM). Incubate at RT for 30 min.
10. Add 75 µL of TCA (Trichloroacetic acid). Vortex, pulse spin, then incubate flat on ice and in the dark/covered in foil, for > 1 h. Meanwhile, chill acetone on ice.
7. Alkylation: Add 3 µL of 500 mM IAA (Iodoacetamide; final 15 mM). Vortex, then incubate 30 min in the dark.
 

8. Quench: Add 2 µL of 1 M DTT. Vortex, then proceed immediately.
9. Add 200 µL of 50 mM EPPS. Vortex.
11. Centrifuge at 20,000 xg for 15 min.
12. Remove supernatant, leaving ~100 µL to avoid disturbing the pellet. Wash with 0.9 mL acetone. Centrifuge at 20,000 xg for 15 min.
12.1. Repeat acetone wash once more (step 11).
13. Dry the pellets with a speed vacuum.
14. Resuspend protein pellets with 10 µL of 8 M urea dissolved in 20 mM EPPS pH=8.5
 


15. Add 12 µL of LysC/EPPS solution (1 µL of 2 µg/µL LysC in 11 µL 200 mM EPPS pH=8.5) to each tube. Shake (600-800 RPM) for 2-4 h at 37 oC (ideally, with a heated lid to prevent evaporation).
16. Add 45 µL of Trypsin/EPPS solution (1 µL of 1 µg/µL Trypsin in 44 µL 200 mM EPPS pH=8.5) to each tube. Shake (600-800 RPM) for 12-24 h at 37 oC (ideally, with a heated lid to prevent evaporation).
17. Add 30 µL ACN (acetonitrile) to each sample and vortex to mix.
18. Add 6 μL TMT label reagent (12.5 μg/μl in ACN) to each vial. Vortex and then shake (600-800 RPM) for 2 h at RT.
19. In the last 30 min of the TMT labeling, prepare a stage tip (6 small cookies from Empore SPE C18 disks, or a commercial C18 column) and equilibrate it:

 

19.1. 200 μL 100% Methanol
19.2. 200 μL 80% ACN
19.3. 200 μL 5% ACN/1% FA (formic acid)
20. Place 160 μL of 1% FA in a microcentrifuge tube. Pipette 5 μL (~5 % of output) of each labeled sample into tube, vortex, and then proceed immediately to stage tip. Place remaining TMT-labeled peptides at -20 oC until after a ratio check.
21. After the sample flows through the stage tip, wash it with 200 μL 5% ACN/1% FA.

21.1. Elute the stage tip into an MS vial with 80 μL of 80% ACN
22. Dry the sample in a speed vacuum and then resuspend the dried peptides in 5% ACN/5% FA
23. Perform a ratio check search with MS. Ensure that TMT labeling is >95%, otherwise add more TMT labeling reagent to each tube and repeat the stage tip + ratio check.
24. Quench each tube with hydroxylamine at a final concentration of 0.5% (wt/v). With the volumes above, add ~1.1 μL of 50% (wt/v) hydroxylamine solution and then vortex. Incubate at RT for 15 min.
25. Pool equal amounts of peptides from each channel depending on the ratio check search. Note potential outliers from the ratio check search (e.g., one sample that was not represented, or was very low input).

 

26. Dry samples in the speed vacuum. This will likely take several hours (> 4 h).
27. Fractionate the sample with the High pH Reversed-Phase Peptide Fractionation Kit (Thermo 84868). Follow most of the manufacturer's instructions, but elute with the following 12 fractions:
27.1. 
 

28. Combine fractions 1 and 7; 2 and 8; 3 and 9; 4 and 10; 5 and 11; 6 and 12. This concatenates the sample space to 6 fractions.
29. Dry these 6 fractions on a speed vacuum. They will dry at different rates, and likely take between 4 and 8 h.
30. Resuspend samples in 200 μL of 3% ACN/1% FA. Check that pH is ~2, otherwise, further acidify with small amounts of 100% FA.
31. Prepare 6 stage tips (12 small cookies from Empore SPE C18 disks, or a commercial C18 column) and equilibrate them:
31.1. 200 μL 100% Methanol
31.2. 200 μL 80% ACN
31.3. 200 μL 5% ACN/1% FA
31.4. Load sample
31.5. 200 μL 5% ACN/1% Formic acid (FA)
31.6. Elute stage tips into an MS vials with 80 μL of 80% ACN
32. Dry these stage-tipped fractions on a speed vacuum. They will dry at different rates, and likely take between 2 and 4 h.
33. Resuspend the dried peptides in 8 μL of 5% ACN/5% FA, and proceed to MS.
SECTION: Construct selection, cell line engineering, and validation
3. Validate tag successful tag incorporation by immunoflourescence. See below (3.1) for a protocol link.

For LysoIP from human cells, check colocalization between the HA tag (1:400, Sigma 11867423001; AB_390918) and LAMP1 (1:200, CST # 9091; AB_2687579 or 1:200 CST #15665; AB_2798750). While PFA-TritonX fixation-permeabilization works, PFA-methanol resolves lysosomes better.

For MitoIP from human or mouse cells, check colocalization between the HA tag (1:400, Sigma 11867423001; AB_390918) and HSP60 (1:400, Abcam ab59457; AB_2121285). PFA-TritonX fixation-permeabilization works fine.

For GolgiIP from human cells, check colocalization between the HA tag (1:400 Sigma 11867423001; AB_390918) and GOLGA2 (Proteintech 11308-1-AP; AB_2115327). From mouse cells, check colocalization between the HA tag (1:400 Sigma 11867423001; AB_390918) and YIPF4 (1:200 Proteintech 15473-1-AP; AB_2217206). PFA-TritonX fixation-permeabilization works fine.

For EndoIP, check colocalization between the FLAG tag (1:100, Thermo MA1-91878; AB_1957945) and VPS35 (1:200, Santa Cruz sc-374372; AB_10988942) or RAB5 (1:200, Cell signalling #3547; RRID:AB_2300649). PFA-TritonX fixation-permeabilization works fine. When overexpressed using a lentivirus, EndoTag localization can be heterogeneous across cells (e.g., the EndoTag might be cytosolic in up to 50% of cells, even with a clonal population). We've found that organelle isolation from this mixed population still works, although you are likely losing material.

 

SECTION: Construct selection, cell line engineering, and validation
3.1. We often use the following protocol for IF: dx.doi.org/10.17504/protocols.io.kxygxyeeol8j/v1

 

SECTION: Endosomal immunoprecipitation (Endo-IP) for organellar proteomics
17.1. For Western blotting analysis, see antibodies in the "Materials" section for suggestions. Below is an example of an EndoIP optimization ("WT" is untagged background, and each # is a different clone that we were testing).
 

 

34. Validate that the intended organelle enriched over the background control IP. These graph based annotations (human gene names from source, and converted to mouse names) can be used to assess bulk enrichment.

List from:
https://www.biorxiv.org/content/10.1101/2023.12.18.572249v1
  

SECTION: Golgi immunoprecipitation (Golgi-IP) for organellar proteomics
17.1. See Fig. 1F for a representative Western Blot:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10193996/
 

SECTION: Mitochondrial immunoprecipitation (Mito-IP) for organellar proteomics
17.1. For a representative Western blot, see Figure 1D: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030821/

 

SECTION: Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
17.1. For a representative Western Blot, see Figure 1C: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704967/
 

34.1. Here is a user-friendly tool to explore proteomics data:
https://curtain.proteo.info/#/