May 11, 2023
Organelle isolation from Mouse Embryonic Fibroblasts (MEFs) stably expressing organelle tags for subsequent immunoblotting or proteomic analysis
- Matthew Taylor1,
- Pui Yiu Lam1,
- Francesca Tonelli1,
- Dario R Alessi1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Protocol Citation: Matthew Taylor, Pui Yiu Lam, Francesca Tonelli, Dario R Alessi 2023. Organelle isolation from Mouse Embryonic Fibroblasts (MEFs) stably expressing organelle tags for subsequent immunoblotting or proteomic analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1o627lr2/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: April 21, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 80869
Keywords: Organelle isolation from cells, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
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Abstract
We describe here a method to perform rapid isolation of intact organelles (including lysosomes and Golgi) from mouse embryonic fibroblasts stably expressing an organelle tag (TMEM192-3xHA, or LysoTag, and TMEM115-3xHA, or GolgiTag). First, cells are broken using a ball-bearing cell breaker, leading to plasma membrane rupture, while lysosomes and Golgi remain intact. Then, the cell homogenate is incubated with anti-HA magnetic beads to allow for immunopurification of HA-tagged lysosomes or Golgi in less than 15 minutes. The organelles purified using this method are highly enriched, intact, contaminant-free and, depending on solubilisation buffer, can be used for various downstream applications, including immunoblotting analysis and mass spectrometry proteomic analysis (as described here), but also metabolomic or lipidomic analysis. This protocol can be adapted to isolate organelles from commonly cultured cells, such as HEK293 and A549 cells, that express an organelle tag.
Attachments
meh7bhc4x.docx
46KB
Guidelines
Protocol overview:
- Organelle isolation from cells expressing organelle tags.
- Analysis of isolated organelles by immunoblotting
- Analysis of isolated organelles by mass spectrometry (proteomic analysis)
For the generation of MEFs stably expressing organelle tags, see dx.doi.org/10.17504/protocols.io.6qpvr456bgmk/v1
Materials
Reagents
- MEFs stably expressing organelle tag: GolgiTag (Tmem115-3xHA) or LysoTag (Tmem192-3xHA), or control empty tag (3xHA).
Note
For the generation of MEFs stably expressing organelle tags, see dx.doi.org/10.17504/protocols.io.6qpvr456bgmk/v1
- DPBS no calcium no magnesiumGibco - Thermo FisherCatalog #14190169
- KPBS Buffer:
| A | B | |
| KCL | 136 mM | |
| KH2PO4 in MS grade water | 10 mM |
Adjust to pH 7.25 with KOH.
- “Supplemented KPBS” (to be prepared immediately before harvesting the cells): KPBS buffer supplemented with 1X phosSTOP phosphatase inhibitor cocktail (PhosSTOP tablet: Roche, REF# 04906837001) and 1X protease inhibitor cocktail (cOmplete EDTA-free protease inhibitor cocktail tablet: Roche, REF# 11873580001)
- Thermo Scientific™ Pierce™ Anti-HA Magnetic Beads (Thermo Fisher Scientific, cat # 13474229)
- Lysis Buffer for Immunoblotting analysis:
| A | B | |
| Tris-HCl, pH 7.5 | 50 mM | |
| Triton X-100 | 1% (by volume) | |
| glycerol | 10% (by volume) | |
| NaCl | 150 mM | |
| sodium orthovanadate | 1 mM | |
| sodium fluoride | 50 mM | |
| sodium β-glycerophosphate | 10 mM | |
| sodium pyrophosphate | 5 mM | |
| microcystin-LR | 0.1 µg/ml | |
| cOmplete Mini (EDTA-free) protease inhibitor (Roche) | 1 tablet |
- Lysis Buffer for Mass spectrometry analysis:
| A | B | |
| SDS | 2% v/v | |
| HEPES pH 8 | 20 mM | |
| phosSTOP phosphatase inhibitor cocktail (Roche) | 1X | |
| protease inhibitor cocktail (completeEDTA-free, Roche) | 1X |
- STrap washing buffer:
| A | B | |
| MeOH | 90% | |
| TEABC at pH 7.2 | 10% |
- LC-MS grade H2O (LC-grade WaterFisher ScientificCatalog #10777404 )
- Triethylammonium bicarbonate bufferMerck MilliporeSigma (Sigma-Aldrich)Catalog #18597 – Make a 50 millimolar (mM) and 300 millimolar (mM) stock in LC-MS grade H2O, 8
- Pierce™ TCEP-HClThermo FisherCatalog #20491 –Make a 100 millimolar (mM) stock in 300 millimolar (mM) TEABC
- IodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149 – Make a 200 millimolar (mM) stock in 300 millimolar (mM) TEABC
- Trypsin/Lys-C Mix, Mass Spec Grade, 5 x 20ugPromegaCatalog #V5073 ) – 20 µg of trypsin/Lys-C reconstitute in 800 µL of 50 millimolar (mM) TEABC at a final concentration of 25 µg/mL
- SDS Micro-PelletsFormediumCatalog #SDS0500 – make a 20% solution in MilliQ water
- Phosphoric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #345245 – Make a 12.5% solution in MilliQ water
- Methanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5
- Acetonitrile LC-MS grade B&J BrandVWR InternationalCatalog #BJLC015-2.5
- Formic acid (Sigma; Cat # 56302)
- Trifluoroacetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #T6508
Equipment
Equipment
BELLY DANCER ORBITAL SHAKER
NAME
ORBITAL SHAKER
TYPE
IBI
BRAND
BDRAA115S
SKU
LINK
Equipment
DynaMag-2
NAME
Magnet
TYPE
Invitrogen
BRAND
12321D
SKU
LINK
- Isobiotec Cell-Breaker (isobiotec Vertriebs UG)
Equipment
Micro Star 17 / 17R, Microcentrifuges, Ventilated/Refrigerated
NAME
Microcentrifuge
TYPE
VWR®
BRAND
521-1647
SKU
LINK
- Bioruptor (Diagenode)
- Thermomixer (Eppendorf, UK)
- SpeedVac Vacuum Concentrator
- UltiMate 3000 RSLC nano-HPLC system (Thermo Fisher Scientific, UK) coupled to an Orbitrap ExplorisTM 480 mass spectrometer (Thermo Fisher Scientific, UK)
- Precolumn: Acclaim PepMapTM 100, C18, 100 µm x 2 cm, 5 µm, 100 Å
- Analytical column: PepMapTM RSLC C18, 75 µm x 50 cm, 2 µm, 100 Å
Consumables
- Corning® cell lifterMerck MilliporeSigma (Sigma-Aldrich)Catalog #CLS3008-100EA
- SafeSeal reaction tube 1.5 ml PP PCR Performance Tested Low protein-bindingSarstedtCatalog #72.706.600
- Greiner Bio-One™ Polypropylene Pipette TipFisher ScientificCatalog #686271 and PIPETTE TIP 10 - 100 µL SUITABLE FOR EPPENDORF 96 PIECES / ST RACKgreiner bio-oneCatalog #685261
- Stripetter/stripette gun and stripettes
- Set of Gilson pipettes P10, P200, P1000
- Terumo® Syringe 3-part SyringeTerumoCatalog #MDSS01SE
- Becton Dickinson Disposable needles 21G x 1 1/2 inch Becton-DickinsonCatalog #304432
- Syringe PP/PE without needleMerck MilliporeSigma (Sigma-Aldrich)Catalog #Z116866
- S-Trap™ micro columns (≤ 100 μg)ProtifiCatalog #C02-micro
Isobiotec cell-breaker assembly
Isobiotec cell-breaker assembly
Insert the metal ball of choice inside the cell breaker.
Note
Note: For MEFs, we recommend a 12 μm clearance.
Screw the lids on tightly.
Push 3 mL of KPBS through the cell breaker to wash it.
Carefully tap dry.
Place the cell-breaker on aluminium foil On ice until use (Step 28).
To clean the Isobiotec cell-breaker between samples and at the end of the experiment:
Open the cell-breaker from one side.
Take the metal ball out and rinse with MillIQ water.
Flush the cell breaker thoroughly with MilliQ water using 5-mL syringes through both syringe inlets whilst covering the opening on the side of the cell breaker.
Reassemble the cell-breaker by re-inserting the metal ball into the instrument and close the side panel tightly using the screws.
Flush the cell breaker through both syringe inlets with 5 mL of KPBS using 5-ml syringes.
Note
Note: There will be some residual KPBS left in the cell-breaker (approximately 200 µL ), this is optimal.
Proceed to homogenise the next sample.
Once finished, flush the cell breaker thoroughly with MilliQ water using 5-mL syringes through both syringe inlets whilst covering the opening on the side of the cell breaker.
Take all pieces apart (both side panels, panel screws and the metal ball).
Clean each part with a generous amount of 70% (v/v) ethanol in MilliQ water.
Wipe all parts dry and leave pieces apart to air-dry Overnight .
Note
Note: Packing up the cell-breaker before it is dry will lead to development of rust and colouring of the metal parts.
Anti-HA Magnetic beads preparation
Anti-HA Magnetic beads preparation
Transfer n x 100 µL of anti-HA Magnetic Beads (where n = number of samples) into a low binding Eppendorf tube.
Immobilize the beads by placing the tube into a Dyna-Mag tube holder for 00:00:30 .
30s
Remove the supernatant using a pipette.
Gently resuspend the beads in 1 mL of KPBS.
Repeat steps 8 to 10.
Immobilize the beads by placing the tube into a Dyna-Mag tube holder for 00:00:30 .
30s
Remove the supernatant using a pipette.
Gently resuspend the beads from step 13 in n x 100 µL of KPBS (where n = number of samples you have) to make a 1:1 slurry.
Aliquot the washed beads from step 14 into fresh low-binding Eppendorf tubes (100 µL of slurry for each sample).
Leave the tubes On ice until use (step 34).
Organelle isolation from cells expressing organelle tags
Organelle isolation from cells expressing organelle tags
For each experimental condition, seed cells into one 15 cm dish.
Note
Note: In parallel, seed cells transduced to express HA-empty as a control.
When cells have reached a confluency of ~ 90%, aspirate the culture medium.
Quickly wash once by adding 5 mL of PBS at Room temperature .
Completely aspirate the PBS.
Add 1 mL of ice-cold supplemented KPBS.
Place the cell dishes On ice .
Scrape the cells on the dish using a cell lifter to ensure all cells are detached from the dish.
Using a pipette, transfer the cell suspension to a low binding Eppendorf On ice .
Spin down at 1000 x g, 4°C, 00:02:00 .
2m
Discard the supernatant.
Resuspend the pellet in 1 mL of ice-cold supplemented KPBS.
Using a 1-ml syringe and 21G needle, transfer the cell suspension from step 27 into a KPBS rinsed, ice-cold Isobiotec cell-breaker (with gap-size of 12 µm ) kept On ice (Step 5).
Homogenise the cells with 10 passes through the cell breaker using 2 x 1-ml syringes.
Note
Note:
- One pass is defined by the cell suspension passing through both syringes.
- The homogenisation requires more force with more passes. Pay extra care to make sure the syringes are securely in their seals and that the sample doesn’t leak out. If you encounter too much pressure for passing the homogenate through the cell-breaker, consider using a ball that leaves a larger clearance gap.
Collect the homogenate from the cell breaker into a fresh Eppendorf tube using a 1-ml syringe.
Note
Note: To extract as much sample as possible from the cell-breaker post-homogenisation, push air into the cell-breaker using a syringe and collect from the other seal using another syringe.
Transfer the resulting homogenate to a low binding Eppendorf On ice .
Preclear the homogenate by centrifugation at 1000 x g, 4°C, 00:02:00 .
2m
For each sample, transfer 100 µL to a new low binding Eppendorf (= input) On ice .
Add the remaining homogenate to 100 µL of the pre-washed HA-Magnetic beads (Step 16).
Mix gently by flicking the bottom of the tube.
Incubate with agitation on a Belly Dancer orbital shaker for 00:05:00 at 4 °C .
5m
Note
The following steps should ideally be performed in a 4 °C cold room. If not available, then keep working On ice .
Place the tubes from Step 36 on a magnetic tube holder for 00:00:30 to immobilise the beads.
30s
Discard the supernatant or collect as a flow-through sample.
Resuspend the beads from Step 38 in 1 mL of supplemented KBPS.
Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 00:00:30 .
30s
Discard the supernatant.
Repeat steps 39 to 41 twice.
Resuspend the beads in 1 mL of supplemented KPBS and transfer to a new low binding Eppendorf tube On ice .
Place the tubes in a Dyna-Mag tube holder for 00:00:30 .
30s
Discard the supernatant.
The organelle IP beads (from step 45) and the input (from step 33) can now be processed for either 1) immunoblotting analysis, or 2) mass spectrometry analysis, as detailed below..
Sample analysis by immunoblotting
Sample analysis by immunoblotting
Input (from step 33)
Dilute in Lysis Buffer compatible for Immunoblotting analysis to a 1:1 ratio.
Incubate On ice for 00:10:00 .
10m
Clarify by centrifugation at 17000 x g, 4°C, 00:10:00 .
10m
Transfer the supernatant to a new low binding tube.
Organelle IP beads (from step 45).
Resuspend in 100 µL of lysis buffer compatible for immunoblot analysis.
Incubate On ice for 00:10:00 .
10m
Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 00:00:30 .
30s
Transfer the supernatant to a new low binding tube.
Quantify protein concentration by BCA assay.
Samples can be analysed by quantitative immunoblotting analysis as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6, ensuring an equal protein amount of both the input and IP is loaded (~ 2 µg ).
Sample analysis by Mass Spectrometry: Sample Processing
Sample analysis by Mass Spectrometry: Sample Processing
Input (from step 33):
Dilute in lysis buffer compatible for mass spectrometry analysis to a 1:1 ratio.
Sonicate using a Bioruptor (00:00:30 ON, 00:00:30 OFF for 15 cycles).
1m
Clarify by centrifugation at 17000 x g, 4°C, 00:10:00 .
10m
Transfer the supernatant to a clean low binding tube.
Organelle IP beads (from step 45):
Resuspend in 100 µL of lysis buffer compatible for mass spectrometry analysis.
Incubate at Room temperature for 00:10:00 .
10m
Sonicate using a Bioruptor (00:00:30 ON, 00:00:30 OFF for 15 cycles).
1m
Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 00:00:30 .
30s
Transfer the supernatant to a new low binding tube.
Reduction: Add TCEP to the samples from step 51.4 and 52.5 to a final concentration of 5 millimolar (mM) and place on a thermomixer at 1100 rpm, 60°C, 00:30:00 .
Cool the samples down to Room temperature .
Alkylation: Add IAA to the samples from step 54 to a final concentration of 20 millimolar (mM) and place on a thermomixer at 1100 rpm, 25°C, 00:30:00 , shielded from light.
Add sodium dodecyl sulfate (SDS) to a final concentration of 5% (v/v) and phosphoric acid to a final concentration of 1.2% (v/v) to the samples from step 55.
Dilute the sample with an additional volume of wash buffer (wash buffer volume equals to 6-fold of the sample volume) (90% MeOH, 10% TEABC at 7.2 ) and mix by vortexing.
Load each sample onto a S-TrapTM column.
Centrifuge at 1000 x g, 00:01:00 .
1m
Discard the flow-through.
Wash the S-TrapTM columns three times with 150 µL wash buffer (90% MeOH, 10% TEABC at 7.2 ). Discard the flowthrough after each wash.
Transfer the S-Trap column to a fresh 1.5-mL low binding tube.
Prepare a Trypsin/Lys-C Mix in 50 millimolar (mM) TEABC solution, 8 to a 25 µg/mL concentration.
On-column digestion: Add 60 µL (1.5 µg ) Trypsin/Lys-C Mix from step 63 to each S-Trap column from step 61 and incubate on a thermomixer at 47 °C for 01:00:00 with no agitation.
1h
Reduce the temperature on the thermomixer to 22 °C and incubate Overnight with no agitation.
1h
Peptide elution: Add 60 µL of 50 millimolar (mM) TEABC solution, pH 8 to each S-Trap column and centrifuge.
Add 60 µL of 0.15% (v/v) formic acid (FA) aqueous solution to each S-Trap column and centrifuge.
Add 60 µL of elution buffer (80% ACN with 0.15% FA in aqueous solution) to each S-Trap column and centrifuge.
Repeat step 68.
Discard the S-Trap columns.
Snap-freeze the samples on dry ice.
Dry the samples at 35 °C using a SpeedVac Vacuum Concentrator.
Resuspend the samples from step 72 in 60 µL solution containing 3% (v/v) ACN and 0.1% (v/v) FA in LC-MS grade H2O.
Incubate the samples on a thermomixer at 1200 rpm, 22°C, 00:30:00 .
Sonicate the samples for 00:30:00 in a water bath.
30m
Estimate peptide concentration of each sample using a NanoDrop instrument by measuring the solution absorbance A280 at 224 nm wavelength.
Sample analysis by Mass Spectrometry: Sample Injection onto Mass Spectrometer
Sample analysis by Mass Spectrometry: Sample Injection onto Mass Spectrometer
Note
Note: Liquid chromatography tandem mass spectrometry (LC-MS/MS) is performed using an UltiMate 3000 RSLC nano-HPLC system coupled to an Orbitrap ExplorisTM480 mass spectrometer.
For each sample, load 4 µg of digested protein sample onto the nano-HPLC system individually.
Trap the peptides using a precolumn (Acclaim PepMapTM 100, C18, 100 µm x 2 cm, 5 µm, 100 Å) using an aqueous solution containing 0.1% (v/v) TFA.
Separate the peptides using an analytical column (PepMapTM RSLC C18, 75 µm x 50 cm, 2 µm, 100 Å) at 45 °C using
- a linear gradient of 8 to 25% solvent B (an 80% ACN and 0.1% FA solution) for 01:38:00 ,
- 25 to 37% solvent B for 00:15:00 ,
- 37 to 95% solvent B for 00:02:00 ,
- 95% solvent B for 00:08:30 ,
- 95% to 3% solvent B for 00:00:30 , and
- 3% solvent B for 00:09:30 .
Set the flow rate at 250 nL/min.
2h 13m 30s
Acquire data in data-independent acquisition (DIA) mode containing 45 isolated m/z windows ranging from 350 to 1500.
Use a higher-energy collisional dissociation (HCD) with nitrogen for peptide fragmentation with the following isolation window:
| A | B | C | |
| m/z | z | Isolation Window | |
| 383.4 | 3 | 66.8 | |
| 423.0 | 3 | 13.5 | |
| 435.0 | 3 | 11.5 | |
| 446.5 | 3 | 12.5 | |
| 458.0 | 3 | 11.5 | |
| 469.0 | 3 | 11.5 | |
| 480.0 | 3 | 11.5 | |
| 490.5 | 3 | 10.5 | |
| 501.0 | 3 | 11.5 | |
| 512.0 | 3 | 11.5 | |
| 523.0 | 3 | 11.5 | |
| 533.5 | 3 | 10.5 | |
| 544.0 | 3 | 11.5 | |
| 554.5 | 3 | 10.5 | |
| 565.0 | 3 | 11.5 | |
| 575.5 | 3 | 10.5 | |
| 586.0 | 3 | 11.5 | |
| 597.5 | 3 | 12.5 | |
| 609.5 | 3 | 12.5 | |
| 621.5 | 3 | 12.5 | |
| 633.0 | 3 | 11.5 | |
| 645.0 | 3 | 13.5 | |
| 657.5 | 3 | 12.5 | |
| 670.5 | 3 | 14.5 | |
| 684.0 | 3 | 13.5 | |
| 697.0 | 3 | 13.5 | |
| 710.5 | 3 | 14.5 | |
| 725.5 | 3 | 16.5 | |
| 741.0 | 3 | 15.5 | |
| 756.5 | 3 | 16.5 | |
| 773.5 | 3 | 18.5 | |
| 791.0 | 3 | 17.5 | |
| 808.5 | 3 | 18.5 | |
| 827.0 | 3 | 19.5 | |
| 846.5 | 3 | 20.5 | |
| 866.5 | 3 | 20.5 | |
| 887.5 | 3 | 22.5 | |
| 910.5 | 3 | 24.5 | |
| 935.5 | 3 | 26.5 | |
| 962.5 | 3 | 28.5 | |
| 992.0 | 3 | 31.5 | |
| 1025.0 | 3 | 35.5 | |
| 1063.0 | 3 | 41.5 | |
| 1108.5 | 3 | 50.5 | |
| 1391.6 | 3 | 516.8 |
Sample analysis by Mass Spectrometry: Data analysis
Sample analysis by Mass Spectrometry: Data analysis
The DIA MS experiment's raw data were analysed using the DIA-NN software (Reference 1), employing a library-free search mode based on a reviewed Swiss-Prot database downloaded from UniProt.
Trypsin/P was selected as the digestive enzyme, and up to 2 missed cleavages were allowed. Carbamidomethylation at Cysteine residue was set as a fixed modification, while oxidation at methionine residue was included as a variable modification. The software automatically detected and adjusted the mass error (ppm).
A protein identification cut-off of 1% FDR was used, and a protein quantification required a minimum of 2 peptides in at least 75% samples.
The protein group search results generated from DIA-NN software were then imported into Perseus software (Reference 2) for statistical analysis.
For the organelle-IP samples, IP samples were first compared against the relevant mock IP samples to classify proteins significantly enriched, using a fold-change > 1.5 and p-value < 0.05.
The organelle enriched proteins were then compared against genotypes or treatments to investigate protein level changes at the targeted organelle.
For the whole cell lysate samples, proteins were directly compared against genotypes or treatments to determine the proteome changes in the cells.
Significant up-/down-regulated proteins (fold-change > |1.5| and p-value < 0.05) obtained from organelle-IP and whole cell lysate samples were then submitted to metascape (reference 3) for enrichment analysis.
The clustering analysis using metascape focuses on enrichment of GO biological processes pathway, GO molecular functions, and GO cellular components with p-value < 0.01.
The text files generated from Perseus software were imported into an in-house software, Curtain 2.0, for data visualisation.
Protocol references
References
1. Demichev, V.; Messner, C. B.; Vernardis, S. I.; Lilley, K. S.; Ralser, M., DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat Methods 2020,17 (1), 41-44.
2. Tyanova, S.; Temu, T.; Sinitcyn, P.; Carlson, A.; Hein, M. Y.; Geiger, T.; Mann, M.; Cox, J., The Perseus computational platform
for comprehensive analysis of (prote)omics data. Nature Methods 2016,13 (9), 731-740.
3. Zhou, Y.; Zhou, B.; Pache, L.; Chang, M.; Khodabakhshi, A. H.; Tanaseichuk, O.; Benner, C.; Chanda, S. K., Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 2019,10 (1), 1523.
4. dx.doi.org/10.17504/protocols.io.6qpvr456bgmk/v1
5. dx.doi.org/10.17504/protocols.io.bsgrnbv6