Organelle isolation from Mouse Embryonic Fibroblasts (MEFs) stably expressing organelle tags for subsequent immunoblotting or proteomic analysis
Francesca Tonelli, Dario R Alessi, Matthew Taylor, Pui Yiu Lam
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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
Steps
Isobiotec cell-breaker assembly
Insert the metal ball of choice inside the cell breaker.
Screw the lids on tightly.
Push 3mL 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.
Wipe all parts dry and leave pieces apart to air-dry .
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 5mL of KPBS using 5-ml syringes.
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.
Anti-HA Magnetic beads preparation
Transfer n x 100µL 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 0h 0m 30s.
Remove the supernatant using a pipette.
Gently resuspend the beads in 1mL of KPBS.
Repeat steps 8 to 10.
Immobilize the beads by placing the tube into a Dyna-Mag tube holder for 0h 0m 30s.
Remove the supernatant using a pipette.
Gently resuspend the beads from step 13 in n x 100µL 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 (100µL of slurry for each sample).
Leave the tubes On ice until use (step 34).
Organelle isolation from cells expressing organelle tags
For each experimental condition, seed cells into one 15 cm dish.
When cells have reached a confluency of ~ 90%, aspirate the culture medium.
Quickly wash once by adding 5mL of PBS at Room temperature.
Completely aspirate the PBS.
Add 1mL 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 1000x g,4°C.
Discard the supernatant.
Resuspend the pellet in 1mL 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.
Collect the homogenate from the cell breaker into a fresh Eppendorf tube using a 1-ml syringe.
Transfer the resulting homogenate to a low binding Eppendorf On ice.
Preclear the homogenate by centrifugation at 1000x g,4°C.
For each sample, transfer 100µL to a new low binding Eppendorf (= input) On ice.
Add the remaining homogenate to 100µL 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 0h 5m 0s at 4°C.
0h 0m 30s to immobilise the beads. Discard the supernatant or collect as a flow-through sample.
Resuspend the beads from Step 38 in 1mL of supplemented KBPS.
Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 0h 0m 30s.
Discard the supernatant.
Repeat steps 39 to 41 twice.
Resuspend the beads in 1mL of supplemented KPBS and transfer to a new low binding Eppendorf tube On ice.
Place the tubes in a Dyna-Mag tube holder for 0h 0m 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
Input (from step 33)
Dilute in Lysis Buffer compatible for Immunoblotting analysis to a 1:1 ratio.
Incubate On ice for 0h 10m 0s.
Clarify by centrifugation at 17000x g,4°C.
Transfer the supernatant to a new low binding tube.
Organelle IP beads (from step 45).
Resuspend in 100µL of lysis buffer compatible for immunoblot analysis.
Incubate On ice for 0h 10m 0s.
Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 0h 0m 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 (~ 2µg).
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 (0h 0m 30s ON, 0h 0m 30s OFF for 15 cycles).
Clarify by centrifugation at 17000x g,4°C.
Transfer the supernatant to a clean low binding tube.
Organelle IP beads (from step 45):
Resuspend in 100µL of lysis buffer compatible for mass spectrometry analysis.
Incubate at Room temperature for 0h 10m 0s.
Sonicate using a Bioruptor (0h 0m 30s ON, 0h 0m 30s OFF for 15 cycles).
Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 0h 0m 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 5millimolar (mM) and place on a thermomixer at 1100rpm.
Cool the samples down to Room temperature.
Alkylation : Add IAA to the samples from step 54 to a final concentration of 20millimolar (mM) and place on a thermomixer at 1100rpm, 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 1000x g.
Discard the flow-through.
Wash the S-TrapTM columns three times with 150µL 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 50millimolar (mM) TEABC solution, 8 to a 25µg/mL concentration.
On-column digestion: Add 60µL (1.5µg) Trypsin/Lys-C Mix from step 63 to each S-Trap column from step 61 and incubate on a thermomixer at 47°C for 1h 0m 0s with no agitation.
Reduce the temperature on the thermomixer to 22°C and incubate 1h 0m 0s with no agitation.
Peptide elution : Add 60µL of 50millimolar (mM) TEABC solution, pH 8 to each S-Trap column and centrifuge.
Add 60µL of 0.15% (v/v) formic acid (FA) aqueous solution to each S-Trap column and centrifuge.
Add 60µL 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 60µL solution containing 3% (v/v) ACN and 0.1% (v/v) FA in LC-MS grade H2O.
Incubate the samples on a thermomixer at 1200rpm.
Sonicate the samples for 0h 30m 0s in a water bath.
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
4µg 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
1h 38m 0s, - 25 to 37% solvent B for
0h 15m 0s, - 37 to 95% solvent B for
0h 2m 0s, - 95% solvent B for
0h 8m 30s, - 95% to 3% solvent B for
0h 0m 30s, and - 3% solvent B for
0h 9m 30s. Set the flow rate at 250 nL/min.
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
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.
