Apr 13, 2024

Public workspaceEndosomal, lysosomal, mitochondrial, or Golgi immunoprecipitation for quantitative proteomics

 Forked from Endosomal and lysosomal immunoprecipitation for proteomics, lipidomics, and TEM
DOI
dx.doi.org/10.17504/protocols.io.261ge5p57g47/v1
  • 1Department of Cell Biology, Harvard Medical School Boston, MA 02115, USA;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Louis R R Hollingsworth
Open access
DOI: dx.doi.org/10.17504/protocols.io.261ge5p57g47/v1
Protocol Citation: J. Wade Harper, Louis R R Hollingsworth, Chan Lee 2024. Endosomal, lysosomal, mitochondrial, or Golgi immunoprecipitation for quantitative proteomics. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5p57g47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 19, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 96954
Keywords: ASAPCRN
Funders Acknowledgement:
National Institutes of Health
Grant ID: R01NS083524
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Abstract
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.
Materials
Buffers (see table below for chemicals)
PBS/DPBS: 137 mM NaCl , 2.7 mM KCl, 10 mM Phosphate, pH 7.4 (MedChemExpress HY-K1023)

KPBS Buffer (also called Mito-KPBS, Golgi-KPBS, and Lyso-KPBS): 136mM KCL, 10 mM KH2PO4. Adjust to pH 7.25 with KOH. (Note On the day of use, add Roche cOmplete protease inhibitor cocktail tablet (REF# 11873580001) and Roche PhosSTOP tablet (REF# 04906837001)
--> To make 500 mL: Dissolve 5.07 g KCl and 0.68 g KH2PO4 in 450 mL water. Adjust to pH 7.25 with KOH and then bring up the volume to 500 mL.

High-Salt-KPBS (For LysoIP): KPBS + 150 mM NaCl

EndoIP-KPBS (For EndoIP): 25 mM KCl, 100 mM potassium phosphate, pH 7.2
--> To make 500 mL, dissolve 0.93 g KCl, 5.366 g K2HPO4, 2.61 g KH2PO4 in 450 mL water, adjust to pH 7.2 with KOH, then top off volume with water.

Antibodies and chemicals:
ABCDEF
REAGENT or RESOURCE SOURCE IDENTIFIER ORGANELLEWB dilutionIF dilution
Antibodies
anti-EEA1 (C45B10) rabbit mAb Cell Signaling Technology 3288 Early/sorting endosome1:10001:200
anti-RAB5 (C8B1) rabbit mAb Cell Signaling Technology 3547 Early endosome1:10001:200
anti-PSEN1 (D39D1) rabbit mAb Cell Signaling Technology 5643 ER/endosome/Golgi1:1000
anti-PSEN2/AD5 (EP1515Y) rabbit mAb Abcam ab51249 ER/Golgi/endosome1:1000
anti-LAMP1 (D2D11) rabbit mAb Cell Signaling Technology 9091 Lysosome/endosome1:10001:200
anti-LAMP1 (D4O1S) Mouse mAb Cell Signaling Technology 15665Lysosome/endosome-1:200 (better)
anti-LAMP2 (D5C2P) rabbit mAb Cell Signaling Technology 49067 Lysosome1:1000
anti-TMEM192 rabbit pAb Proteintech 28263-1-AP Lysosome1:1000
anti-HA (6E2) mouse mAb Cell Signaling Technology 2367 LysoTag, GolgiTag, or MitoTag1:1000
anti-HA (3F10) rat mAbSigma1186742300LysoTag, GolgiTag, or MitoTag-1:400
anti-FLAG M2 mouse mAb Sigma-Aldrich F1804 Epitope (EndoTag)1:1000-
anti-DYKDDDK mouse mAbThermoMA1-91878Epitope (EndoTag)-1:100
anti-ZO-1 rabbit pAb Proteintech 21773-1-AP Plasma membrane1:1000
anti-Giantin (GOLGB1)Abcamab37266Golgi1:10001:100
anti-YIPF4Proteintech15473-1-APGolgi1:10001:200
anti-Golga1 rabbit pAb Proteintech 12640-1-AP Golgi1:1000
anti-TMEM115Novus80898Golgi1:5001:100
anti-GOLGA2/GM130 rabbit pABProteintech11308-1-APGolgi1:10001:400
anti-Golgin97 (CDF4) mouse mAbThermo# A-21270Golgi1:10001:50
anti-Calreticulin rabbit pAb Proteintech 10292-1-AP Endoplasmic reticulum1:1000
anti-S6K rabbit pAb Proteintech 14485-1-AP Ribosome (cytoplasm)1:1000
anti-RAB11 (D4F5) rabbit mAb Cell Signaling Technology 5589 Recycling endosome1:500
anti-Lamin A/C (4C11) mouse mAb Cell Signaling Technology 4777 Nucleus1:1000
anti-VDAC1/Porin rabbit pAb Proteintech 55259-1-AP Mitochondrion1:1000
anti-RAB7 (D95F2) rabbit mAb Proteintech 9367 Late endosome/lysosome1:1000
anti-VPS35Santa Cruz374372Endosome1:10001:200
anti-GAPDH (D16H11) XP rabbit mAb Cell Signaling Technology 5174 Loading control1:1000
anti-PEX19 rabbit pAb Proteintech 14713-1-AP Peroxosome1:1000
anti-CD71/TFR1 (D7G9X) rabbit mAb Cell Signaling Technology 13113 Endosome/plasma membrane1:1000
anti-HSP90 (3F11C1) mouse mAb Proteintech 60318-1-Ig Cytoplasm1:1000
anti-HSP60 (LK-1) mouse mAbAbcamab59467Mitochondria1:400
IRDye 680RD Goat anti-Rabbit IgG secondary antibody Li-Cor 926-68071 -1:10,000-
IRDye 680RD Goat anti-Mouse IgG secondary antibody Li-Cor 926-68070 -1:10,000-
IRDye 800CW Goat anti-Rabbit IgG secondary antibody Li-Cor 926-32211 -1:10,000-
IRDye 800CW Goat anti-Mouse IgG secondary antibody Li-Cor 926-32210 -1:10,000-
Goat anti-Rabbit IgG, HRP-linked antibody Cell Signaling Technology 7474P2 -1:10,000-
Goat anti-Rabbit IgG HRP conjugate Bio-Rad 1706515 -1:10,000-
Goat anti-Mouse IgG HRP conjugate Bio-Rad 1706516 -1:10,000-
Anti-mouse 488Thermo A-11029--1:200
Anti-mouse 568ThermoA-11029--1:200
Anti-mouse 647ThermoA-11029--1:200
Anti-rat 488ThermoA-11029--1:200
Anti-rat 647ThermoA-11029--1:200
Anti-Rabbit 488ThermoA-11034--1:200
Anti-Rabbit 568Thermo A-11029--1:200
Anti-Rabbit 647ThermoA-11029--1:200
Chemicals, peptides, and recombinant proteins
anti-FLAG M2 magnetic beads Sigma-Aldrich M8823
Pierce anti-HA magnetic beads Thermo Fisher Scientific 88837
TMTProTM 18Plex Label Reagent set Thermo Fisher Scientific A52045
Pierce™ High pH Reversed-Phase Peptide Fractionation Kit Thermo Fisher Scientific 84868
HyClone Fetal bovine serum GE Healthcare SB30910
Puromycin Sigma-Aldrich P9620
G418 (Geneticin) Invivogen ant-gn-2
Dulbecco’s MEM (DMEM), high glucose, pyruvate GIBCO / Invitrogen 11995
Penicillin-Streptomycin (10,000 U/mL)Thermo15140163
GlutaMAX™ SupplementThermo35050061
RPMIThermo11875119
PhosSTOP Roche 04906845001
Complete EDTA-free protease inhibitor cocktail Sigma-Aldrich 11873580001
Tris(2-carboxyethyl)phosphine hydrochloride solution Sigma-Aldrich 646547
Iodoacetamide Sigma-Aldrich I1149
Trichloroacetic acid solution 6.1 N (TCA) Sigma-Aldrich T0699
Trifluoroacetic acid (TFA) fisher scientific A11650
Acetonitrile (ACN)Sigma34851-4X4L
HPLC WaterSigma270733
Hydroxylamine solution 50 wt. % Sigma-Aldrich 438227
MethanolSigma34860-2L-R
EthanolVWRTX89125172HU
Formic Acid (FA) Sigma-Aldrich 5330020050
Pierce Trypsin Protease, MS grade Thermo Fisher Scientific 90305
Lysyl endopeptidaseR (Lys-C) Wako 129-02541
REVERT 700 total protein stain kit Li-Cor 926-11016
NuPAGE LDS sample buffer (4X) Thermo Fisher Scientific NP0007
NuPAGE sample reducing agent (10X) Thermo Fisher Scientific NP0009
NuPAGE MES SDS Running Buffer (20X) Thermo Fisher Scientific NP0002
Immobilon-FL PVDF Membrane Millipore IPFL00010
WHEATON Dounce Tissue Grinder, 7 mL DWK Life Sciences 357542
KIMBLE KONTES Dounce Tissue Grinder, 2 mL DWK Life Sciences 885300-0002
Nonidet P40 substitute Sigma-Aldrich 74385
Urea Sigma-Aldrich U5378
EPPS 0.2M buffer solution, pH 8.5 Alfa Aesar J61476.AE
Empore C18 47 mm Extraction Disc, Model 2215 3M 98060402173
Sep-Pak C18 1 cc Vac Cartridge Waters WAT054955
RIPA lysis and extraction buffer Thermo Fisher Scientific 89900
Experimental models: Cell lines
293 cells ATCC CRL-1573
293T cells ATCC CRL-3216
THP-1 cells ATCC TIB-202
Before start
Note that a significant amount of information is contained in the "Materials" section.
Construct selection, cell line engineering, and validation
Construct selection, cell line engineering, and validation
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.

Note
For stable overexpression with lentivirus, we recommend FACS sorting the cells for medium/low expressors to preserve organellar integrity/identity and localization of the IP handles. Antibiotic selection after introducing a transgene with lentivirus can lead to orders of magnitude differences in expression levels between cells. Check that the cells do not lose the transgene during scale-up (this has not been a problem for us when FACS sorting cells with "purity" or "ultra purity" mode enabled).

We recommend checking gentle lysis feasibility for a given cell type prior to incorporating these tags and further validation/optimization.

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).

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.

Note
For each organelleIP handle, ensure that the tag localization does not change in response to treatment, genetic perturbation, etc., so that the identities of the isolated organelles are the same between control and experimental condition(s).

We often use the following protocol for IF: dx.doi.org/10.17504/protocols.io.kxygxyeeol8j/v1

Note
See our pubpub publication for representative confocal images.

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).
Step case

OrganelleIP
16 steps

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.
Note
Include a set of negative controls (a cell line without the appropriate epitope tag) within every organelle-IP experiment. It's otherwise very difficult to tell whether proteins that appear "significantly changing" in response to a treatment, genotype, etc. do so because a difference in the background, rather than a real localization change. We discuss how to use this control to check organelle enrichment at the end of the "Proteomics prep" section.

Cells cannot be harvested and then frozen at any step for these protocols--the first pause point is after elution from the beads.

Step case

LysoIP
15 steps

Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
Lysosomal immunoprecipitation (Lyso-IP) for organellar proteomics
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).

Note
We've successfully used various amounts of beads. More beads (e.g., 80-100 µL slurry per replicate) are ideal for proteomics experiments, but bead volume will quickly increase the cost of the experiment.

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.
Note
Include phosphatase inhibitors even if downstream analysis only includes proteomics/WB. Peripheral membrane-associated proteins depend on lipid identity.

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.
Video


Note
Dounce homogenization should be optimized prior to scale-up, as it depends heavily on the user and the cell line. We tend towards the upper end (30 strokes) for HEK/HeLa cells, and less (25-30) for differentiated THP-1s. It's likely that more extended cell lines rupture faster than cells with more spherical morphology. One way to check lysis efficiency is to Dounce 1-10 cm plate in 1-mL lysis buffer, dilute an aliquot of the lysate in KPBS, and then stain with trypan blue. We've found that automated counting can be inconsistent, so it's best to also look yourself. Don't expect more than 40-60 % of cells to be lysed, and never attempt to reach 100%.

Importantly, to Dounce, slowly and deliberately stroke the pestle up and down without introducing bubbles. Bring the pestle up such that it doesn't quite leave the liquid level to avoid bubbles.

Critical
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).
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.
Note
From here on out, we use P1000 tips for transfer and washes. While we've found that normal tips work fine, wide-bore tips minimize the chance of organelle damage and are therefore preferable.

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.
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.
Note
Antibodies that work well for proteins that localize to particular organelles are listed in the "Materials" tab.

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.
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.
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.

Note
To wash the beads, aspirate the solution, remove the beads from the magnetic rack, and gently resuspend them 2-3 times with a wide bore P1000 pipette before putting them back on the magnetic rack. Work as quickly as possible.

Allow at least 30 s for beads to adhere to the magnet.

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.
Note
Transferring the beads to new tube reduces nonspecific background that binds to the tube during the PNS bead-binding step.


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").
Note
Alternative elution methods can be used for different downstream applications. Detergent solubilization works well for western blot/MS. Note that the LysoTag will mostly remain associated with the beads.

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.
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.
Pause
For a representative Western Blot, see Figure 1C: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704967/
Note
A negative control IP (or set of IPs) must be included to check enrichment properly.

Protocol references