TITLE: Lysosome immunopurification (LysoIP) protocol for subcellular metabolite profiling
AUTHORS: Wentao Dong, Nouf Laqtom, Monther Abu-Remaileh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lysosomes function as metabolic hub in the cell by degrading and recycling biomolecules. Despite its critical cellular function, there has been challenges to quantitatively assess lysosomal metabolite profiles. To overcome this hurdle, we developed a rapid harvesting and purification method using immunoprecipitation (LysoIP). This protocol provides details for preparing LysoIP samples for metabolite profiling.</div></div>

[STEPS]
?. [Preparation of homogenizers and sample tubes]
Wash the glass vessel homogenizer with MilliQ Water, 10 times each. wash the tissue grinder homogenizer thoroughly with DI Water and MilliQ Water, especially the gap between the white parts, don’t touch the part that goes into the glass vessel. Then dry upside-down using paper towels. Carefully place the glass vessels against something to prevent falling down. Minimize any contact between the grinder and anything else.
?. [Preparation of homogenizers and sample tubes]
Prepare microcentrifuge tubes as follows on a metal rack on ice (for each sample, from left to right): ➀ 2 mL tube for cell suspension from harvesting; ➁ 1.5 mL tube for post-homogenization cell suspension (organelles in supernatant, membranes in pellet); ➂ 1.5 mL tube for whole cell sample; ➃ 1.5 mL tube for beads; ➄5 mL tube for post-magnetic samples; ➅ 1.5 mL tube for final lysoIP samples. Carefully label tubes ➂ and ➅ with detailed samples and experiments names.
?. [Preparation of Anti-HA beads]
Pool all required volumes together (/ plate, e.g. 800 uL total for 8 plates, extra is not needed)..
100 µl
?. [Preparation of Anti-HA beads]
Shake bottle very well before removing as beads tend to sink to the bottom.
?. [Preparation of Anti-HA beads]
Wash 3 x with the same volume cold clean MS grade KPBS, after settling on magnet. Remove the holder from the magnet itself before dispending washing KPBS to avoid wetting the magnet.
?. [Preparation of Anti-HA beads]
Resuspend with KPBS with same amount of volume originally removed from bottle.
?. [Preparation of Anti-HA beads]
Aliquot  into each 1.5 mL labeled tubes ➃.
100 µl
?. [Cell preparation before harvesting]
Wash the first set of 15cm plates (each set has two plates) with  of DMEM/plate (for HEKS, use no serum + no antibiotics).
10 mL
?. [Cell preparation before harvesting]
Replace with  of DMEM/plate (+   of 1:1000 diluted LysoTracker) for an hour.
10 mL
40 µl
?. [Cell preparation before harvesting]
The second set of plates will be washed  later after the first set and so on.
10 mL
Note: it is very important to maintain the schedule of the plate washing --> Lyso IP. If there are any deviations to the schedule for any reason, note them as it may affect results of experiment.
?. [Cell preparation before harvesting]
One hour after DMEM wash, take the first set of plates from incubator to bench and place on ice.
?. [Cell preparation before harvesting]
Decant the media.  Then Wash the cells twice by pouring ~ cold clean MS grade PBS on the edge of the plate, decant the first time and then aspirate the second time.
5 mL
?. [Cell harvesting]
Add  of cold KPBS to each 15-cm dish.
950 µl
?. [Cell harvesting]
Scrape the cells down to the bottom of your plates with a cell lifter and transfer the cell suspension into the 2ml tube ➀.  Note: this step should be carefully accounted for and done the same between plates. Visually check (with an angle) that all cells have been harvested. We are using a 2mL tube since 950 uL KPBS + cells gives around 2mL volume.
?. [Cell harvesting]
Spin at  for  at .
Centrifuge: 1000 34
4 °C
Note: centrifuge must already be cooled to   at this point.
?. [Cell harvesting]
Aspirate the supernatant and resuspend the pellets with   cold KPBS.
950 µl
?. [Cell harvesting]
From this resuspended sample, take  for whole cell  in the 1.5 mL tube ➂.   Note: if pellet mixer is used instead of douncer, resuspend the pellets with   cold KPBS in step 16, homogenize cells and then replenish to 950 uL and follow step 17.
25 µl
100 µl
?. [Homogenization and LysoIP]
Transfer the remainder () of cells into a clean and pre-chill douncer. Dounce the cells 25 times (for 293 T cells, other cells need to be optimized) gently on ice and avoid making bubbles.
925 µl
Note: count and repeat for each of the samples (both in number and speed).
?. [Homogenization and LysoIP]
Use serological pipet to transfer sample from douncer into the 1.5 mL tubes ➁.
?. [Homogenization and LysoIP]
Spin 1,000g for  at .
4 °C
?. [Homogenization and LysoIP]
a.      Wash douncers during this spin for subsequent harvesting
?. [Homogenization and LysoIP]
Put the remaining supernatant (it contains the organelles) on the 1.5 ml tube ➃ with beads and resuspend by pipetting up and down ONE TIME (take  when using pellet mixer) .
800 µl
Note: leave the pellet and make sure not to accidently suck up any of the pellet as that can negatively affect experiment.
?. [Homogenization and LysoIP]
Rock in cold room for  (everything from now on is in the cold room).
Note: Max speed. Set timer for 3 mins.
?. [Homogenization and LysoIP]
Put the ➃ tube on magnet. Count at least  to allow for beads to be pulled by magnets.
Note:  it is important to keep this count the same between each wash and each sample for consistency I.e. 25 seconds each time .
?. [Homogenization and LysoIP]
Wash the bound fraction 3 times with cold KPBS. Then aspirate all cold KPBS.
1 mL
Note: during the first wash, make sure to aspirate any liquid trapped on the inner side of the cap. Pipet up and down 2 or 3 times and keep consistent each wash, each sample. After the second wash, resuspend and then switch to the clean 1.5 mL tube ➄ for the third wash (this step helps give cleaner results) .
?. [Processing of polar metabolite samples]
Resuspend the IP samples in  of freezing cold  MeOH with isotopically labeled amino acids (500 nM) as internal standards.
50 µl
Note: it is difficult to resuspend. Can begin flushing on the side of beads stuck on the tube, then gradually move inward.
?. [Processing of polar metabolite samples]
Place samples in ice and start Lyso IP for the next one (remember you are on a strict timed schedule).
?. [Processing of polar metabolite samples]
At this point you should have WC  samples ( from step 17 ) in the 1.5 mL tube ➂ and IP samples ( from step 26) in the 1.5 mL tube ➄ with beads still in it.
25 µl
50 µl
?. [Processing of polar metabolite samples]
After   finishing the last IP, place IP samples in the tube ➄ on the magnet, collect supernatant, and transfer to the 1.5 mL tube ➅.
?. [Processing of polar metabolite samples]
For WC samples, add  freezing cold  MeOH with isotopically labeled amino acids to  tube ➂. Then vortex briefly.
225 µl
?. [Processing of polar metabolite samples]
For WC and LysoIP samples,  centrifuge  at top speed (, , ) and transfer the supernatant to a set of new tubes. Store  WC an IP samples in these new tubes (from step 30) at . On the day of LC/MS measurement, vortex samples for   at  and  centrifuge  at top speed (, , ). Then transfer supernatant to autosampler vials.
Centrifuge: 15000 33
4 °C
-80 °C
4 °C
Centrifuge: 15000 33
4 °C