TITLE: Organelle isolation from Mouse Embryonic Fibroblasts (MEFs) stably expressing organelle tags for subsequent immunoblotting or proteomic analysis
AUTHORS: Matthew Taylor, Pui Yiu Lam, Francesca Tonelli, Dario R Alessi

[DESCRIPTION]
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.

[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

[STEPS]
SECTION: Isobiotec cell-breaker assembly
1. Insert the metal ball of choice inside the cell breaker. 
 

SECTION: Isobiotec cell-breaker assembly
2. Screw the lids on tightly.
SECTION: Isobiotec cell-breaker assembly
3. Push 3 mL of KPBS through the cell breaker to wash it.

SECTION: Isobiotec cell-breaker assembly
4. Carefully tap dry.
SECTION: Isobiotec cell-breaker assembly
5. Place the cell-breaker on aluminium foil on ice until use (Step 28).
SECTION: Isobiotec cell-breaker assembly
6. To clean the Isobiotec cell-breaker between samples and at the end of the experiment:
SECTION: Isobiotec cell-breaker assembly
6.1. Open the cell-breaker from one side.
SECTION: Isobiotec cell-breaker assembly
6.2. Take the metal ball out and rinse with MillIQ water.
SECTION: Isobiotec cell-breaker assembly
6.3. 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.
SECTION: Isobiotec cell-breaker assembly
6.4. Reassemble the cell-breaker by re-inserting the metal ball into the instrument and close the side panel tightly using the screws.
SECTION: Isobiotec cell-breaker assembly
6.5. Flush the cell breaker through both syringe inlets with 5 mL of KPBS using 5-ml syringes.
 


SECTION: Isobiotec cell-breaker assembly
6.6. Proceed to homogenise the next sample.
SECTION: Isobiotec cell-breaker assembly
6.7. 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.
SECTION: Isobiotec cell-breaker assembly
6.8. Take all pieces apart (both side panels, panel screws and the metal ball).
SECTION: Isobiotec cell-breaker assembly
6.9. Clean each part with a generous amount of 70% (v/v) ethanol in MilliQ water.
SECTION: Isobiotec cell-breaker assembly
6.10. Wipe all parts dry and leave pieces apart to air-dry  . 
 


SECTION: Anti-HA Magnetic beads preparation
7. Transfer n x 100 µL of anti-HA Magnetic Beads (where n = number of samples) into a low binding Eppendorf tube.
SECTION: Anti-HA Magnetic beads preparation
8. Immobilize the beads by placing the tube into a Dyna-Mag tube holder for 30 s.

SECTION: Anti-HA Magnetic beads preparation
9. Remove the supernatant using a pipette.
SECTION: Anti-HA Magnetic beads preparation
10. Gently resuspend the beads in 1 mL of KPBS.

SECTION: Anti-HA Magnetic beads preparation
11. Repeat steps 8 to 10.
SECTION: Anti-HA Magnetic beads preparation
12. Immobilize the beads by placing the tube into a Dyna-Mag tube holder for 30 s.

SECTION: Anti-HA Magnetic beads preparation
13. Remove the supernatant using a pipette.
SECTION: Anti-HA Magnetic beads preparation
14. 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.

SECTION: Anti-HA Magnetic beads preparation
15. Aliquot the washed beads from step 14 into fresh low-binding Eppendorf tubes (100 µL of slurry for each sample).
SECTION: Anti-HA Magnetic beads preparation
16. Leave the tubes on ice until use (step 34).

SECTION: Organelle isolation from cells expressing organelle tags
17. For each experimental condition, seed cells into one 15 cm dish. 
 

SECTION: Organelle isolation from cells expressing organelle tags
18. When cells have reached a confluency of ~ 90%, aspirate the culture medium.
SECTION: Organelle isolation from cells expressing organelle tags
19. Quickly wash once by adding 5 mL of PBS at Room temperature.

SECTION: Organelle isolation from cells expressing organelle tags
20. Completely aspirate the PBS.
SECTION: Organelle isolation from cells expressing organelle tags
21. Add 1 mL of ice-cold supplemented KPBS.

SECTION: Organelle isolation from cells expressing organelle tags
22. Place the cell dishes on ice.

SECTION: Organelle isolation from cells expressing organelle tags
23. Scrape the cells on the dish using a cell lifter to ensure all cells are detached from the dish.
SECTION: Organelle isolation from cells expressing organelle tags
24. Using a pipette, transfer the cell suspension to a low binding Eppendorf on ice.

SECTION: Organelle isolation from cells expressing organelle tags
25. Spin down at 1000 x g, 2 min, 4 °C.

SECTION: Organelle isolation from cells expressing organelle tags
26. Discard the supernatant.
SECTION: Organelle isolation from cells expressing organelle tags
27. Resuspend the pellet in 1 mL of ice-cold supplemented KPBS.

SECTION: Organelle isolation from cells expressing organelle tags
28. 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  ) kept on ice (Step 5).

SECTION: Organelle isolation from cells expressing organelle tags
29. Homogenise the cells with 10 passes through the cell breaker using 2 x 1-ml syringes. 
 

SECTION: Organelle isolation from cells expressing organelle tags
30. Collect the homogenate from the cell breaker into a fresh Eppendorf tube using a 1-ml syringe. 
 

SECTION: Organelle isolation from cells expressing organelle tags
31. Transfer the resulting homogenate to a low binding Eppendorf on ice.

SECTION: Organelle isolation from cells expressing organelle tags
32. Preclear the homogenate by centrifugation at 1000 x g, 2 min, 4 °C.

SECTION: Organelle isolation from cells expressing organelle tags
33. For each sample, transfer 100 µL to a new low binding Eppendorf (= input) on ice.

SECTION: Organelle isolation from cells expressing organelle tags
34. Add the remaining homogenate to 100 µL of the pre-washed HA-Magnetic beads (Step 16).

SECTION: Organelle isolation from cells expressing organelle tags
35. Mix gently by flicking the bottom of the tube.
SECTION: Organelle isolation from cells expressing organelle tags
36. Incubate with agitation on a Belly Dancer orbital shaker for 5 min at 4 °C. 

SECTION: Organelle isolation from cells expressing organelle tags
37. 
 
Place the tubes from Step 36 on a magnetic tube holder for 30 s to immobilise the beads.

SECTION: Organelle isolation from cells expressing organelle tags
38. Discard the supernatant or collect as a flow-through sample.
SECTION: Organelle isolation from cells expressing organelle tags
39. Resuspend the beads from Step 38 in 1 mL of supplemented KBPS.

SECTION: Organelle isolation from cells expressing organelle tags
40. Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 30 s.

SECTION: Organelle isolation from cells expressing organelle tags
41. Discard the supernatant.
SECTION: Organelle isolation from cells expressing organelle tags
42. Repeat steps 39 to 41 twice.
SECTION: Organelle isolation from cells expressing organelle tags
43. Resuspend the beads in 1 mL of supplemented KPBS and transfer to a new low binding Eppendorf tube on ice.

SECTION: Organelle isolation from cells expressing organelle tags
44. Place the tubes in a Dyna-Mag tube holder for 30 s.

SECTION: Organelle isolation from cells expressing organelle tags
45. Discard the supernatant.
SECTION: Organelle isolation from cells expressing organelle tags
46. 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..
SECTION: Sample analysis by immunoblotting
47. Input (from step 33)
SECTION: Sample analysis by immunoblotting
47.1. Dilute in Lysis Buffer compatible for Immunoblotting analysis to a 1:1 ratio. 
SECTION: Sample analysis by immunoblotting
47.2. Incubate on ice for 10 min.

SECTION: Sample analysis by immunoblotting
47.3. Clarify by centrifugation at 17000 x g, 10 min, 4 °C.

SECTION: Sample analysis by immunoblotting
47.4. Transfer the supernatant to a new low binding tube.
SECTION: Sample analysis by immunoblotting
48. Organelle IP beads (from step 45).
SECTION: Sample analysis by immunoblotting
48.1. Resuspend in 100 µL of lysis buffer compatible for immunoblot analysis.

SECTION: Sample analysis by immunoblotting
48.2. Incubate on ice for 10 min.

SECTION: Sample analysis by immunoblotting
48.3. Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 30 s.

SECTION: Sample analysis by immunoblotting
48.4. Transfer the supernatant to a new low binding tube.
SECTION: Sample analysis by immunoblotting
49. Quantify protein concentration by BCA assay.
SECTION: Sample analysis by immunoblotting
50. 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).
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
51. Input (from step 33): 
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
51.1. Dilute in lysis buffer compatible for mass spectrometry analysis to a 1:1 ratio.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
51.2. Sonicate using a Bioruptor (30 s ON, 30 s OFF for 15 cycles).

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
51.3. Clarify by centrifugation at 17000 x g, 10 min, 4 °C.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
51.4. Transfer the supernatant to a clean low binding tube.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
52. Organelle IP beads (from step 45):
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
52.1. Resuspend in 100 µL of lysis buffer compatible for mass spectrometry analysis.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
52.2. Incubate at Room temperature for 10 min.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
52.3. Sonicate using a Bioruptor (30 s ON, 30 s OFF for 15 cycles).

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
52.4. Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 30 s.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
52.5. Transfer the supernatant to a new low binding tube.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
53. 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  .

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
54. Cool the samples down to Room temperature.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
55. Alkylation: Add IAA to the samples from step 54 to a final concentration of 20 millimolar (mM) and place on a thermomixer at  , shielded from light.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
56. 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.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
57. 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 pH 7.2) and mix by vortexing.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
58. Load each sample onto a S-TrapTM column.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
59. Centrifuge at 1000 x g, 1 min.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
60. Discard the flow-through.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
61. Wash the S-TrapTM columns three times with 150 µL wash buffer (90% MeOH, 10% TEABC at pH 7.2). Discard the flowthrough after each wash.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
62. Transfer the S-Trap column to a fresh 1.5-mL low binding tube.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
63. Prepare a Trypsin/Lys-C Mix in 50 millimolar (mM) TEABC solution, pH 8 to a 25 µg/mL concentration.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
64. 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 60 min with no agitation.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
65. Reduce the temperature on the thermomixer to 22 °C and incubate 60 min with no agitation.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
66. Peptide elution: Add 60 µL of 50 millimolar (mM) TEABC solution, pH pH 8 to each S-Trap column and centrifuge.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
67. Add 60 µL of 0.15% (v/v) formic acid (FA) aqueous solution to each S-Trap column and centrifuge.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
68. Add 60 µL of elution buffer (80% ACN with 0.15% FA in aqueous solution) to each S-Trap column and centrifuge.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
69. Repeat step 68.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
70. Discard the S-Trap columns.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
71. Snap-freeze the samples on dry ice.
SECTION: Sample analysis by Mass Spectrometry: Sample Processing
72. Dry the samples at 35 °C using a SpeedVac Vacuum Concentrator.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
73. 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.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
74. Incubate the samples on a thermomixer at  .

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
75. Sonicate the samples for 30 min in a water bath.

SECTION: Sample analysis by Mass Spectrometry: Sample Processing
76. Estimate peptide concentration of each sample using a NanoDrop instrument by measuring the solution absorbance A280 at 224 nm wavelength.
SECTION: Sample analysis by Mass Spectrometry: Sample Injection onto Mass Spectrometer
77. 
 
For each sample, load 4 µg of digested protein sample onto the nano-HPLC system individually. 

SECTION: Sample analysis by Mass Spectrometry: Sample Injection onto Mass Spectrometer
78. 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.
SECTION: Sample analysis by Mass Spectrometry: Sample Injection onto Mass Spectrometer
79. 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 98 min, 
25 to 37% solvent B for 15 min, 
37 to 95% solvent B for 2 min, 
95% solvent B for 510 s, 
95% to 3% solvent B for 30 s, and 
3% solvent B for 570 s. 
Set the flow rate at 250 nL/min.

SECTION: Sample analysis by Mass Spectrometry: Sample Injection onto Mass Spectrometer
80. Acquire data in data-independent acquisition (DIA) mode containing 45 isolated m/z windows ranging from 350 to 1500.
SECTION: Sample analysis by Mass Spectrometry: Sample Injection onto Mass Spectrometer
81. Use a higher-energy collisional dissociation (HCD) with nitrogen for peptide fragmentation with the following isolation window: 
 
     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   
 
SECTION: Sample analysis by Mass Spectrometry: Data analysis
82. 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.
SECTION: Sample analysis by Mass Spectrometry: Data analysis
83. 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). 
SECTION: Sample analysis by Mass Spectrometry: Data analysis
84. 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.
SECTION: Sample analysis by Mass Spectrometry: Data analysis
85. The protein group search results generated from DIA-NN software were then imported into Perseus software (Reference 2) for statistical analysis.
SECTION: Sample analysis by Mass Spectrometry: Data analysis
86. 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.
SECTION: Sample analysis by Mass Spectrometry: Data analysis
90. 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.
SECTION: Sample analysis by Mass Spectrometry: Data analysis
91. The text files generated from Perseus software were imported into an in-house software, Curtain 2.0, for data visualisation.
SECTION: Sample analysis by Mass Spectrometry: Data analysis
87. The organelle enriched proteins were then compared against genotypes or treatments to investigate protein level changes at the targeted organelle.
SECTION: Sample analysis by Mass Spectrometry: Data analysis
88. For the whole cell lysate samples, proteins were directly compared against genotypes or treatments to determine the proteome changes in the cells.
SECTION: Sample analysis by Mass Spectrometry: Data analysis
89. 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.