Lysosome immunopurification (LysoIP) protocol for subcellular metabolite profiling

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.

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.

Pool all required volumes together (100µL/ plate, e.g. 800 uL total for 8 plates, extra is not needed).

.

Shake bottle very well before removing as beads tend to sink to the bottom.

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.

Resuspend with KPBS with same amount of volume originally removed from bottle.

Aliquot 100µL into each 1.5 mL labeled tubes ➃.

Wash the first set of 15cm plates (each set has two plates) with 10mL of DMEM/plate (for HEKS, use no serum + no antibiotics).

Replace with 10mL of DMEM/plate (+ 40µL of 1:1000 diluted LysoTracker) for an hour.

10mL The second set of plates will be washed 0h 20m 0s later after the first set and so on.

One hour after DMEM wash, take the first set of plates from incubator to bench and place on ice.

Decant the media. Then Wash the cells twice by pouring ~5mL cold clean MS grade PBS on the edge of the plate, decant the first time and then aspirate the second time.

Add 950µL of cold KPBS to each 15-cm dish.

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.

Spin at 1000x g,0h 0m 0s for 0h 2m 0s at 4°C.

Aspirate the supernatant and resuspend the pellets with 950µL cold KPBS.

From this resuspended sample, take 25µL for whole cell in the 1.5 mL tube ➂.

Note: if pellet mixer is used instead of douncer, resuspend the pellets with 100µL cold KPBS in step 16, homogenize cells and then replenish to 950 uL and follow step 17.

Transfer the remainder (925µL) 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.

Use serological pipet to transfer sample from douncer into the 1.5 mL tubes ➁.

Spin 1,000g for 0h 2m 0s at 4°C.

a. Wash douncers during this spin for subsequent harvesting

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 800µL when using pellet mixer) .

Rock in cold room for 0h 3m 0s (everything from now on is in the cold room).

Put the ➃ tube on magnet. Count at least 0h 0m 25s to allow for beads to be pulled by magnets.

Wash the bound fraction 3 times with 1mLcold KPBS. Then aspirate all cold KPBS.

Resuspend the IP samples in 50µL of freezing cold 80% (v/v) MeOH with isotopically labeled amino acids (500 nM) as internal standards.

Place samples in ice and start Lyso IP for the next one (remember you are on a strict timed schedule).

At this point you should have WC samples (25µL from step 17 ) in the 1.5 mL tube ➂ and IP samples (50µL from step 26) in the 1.5 mL tube ➄ with beads still in it.

After 0h 10m 0s 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.

For WC samples, add 225µL freezing cold 80% (v/v) MeOH with isotopically labeled amino acids to tube ➂. Then vortex briefly.

For WC and LysoIP samples, centrifuge at top speed (15000rpm,0h 0m 0s, 0h 10m 0s, 4°C) and transfer the supernatant to a set of new tubes. Store WC an IP samples in these new tubes (from step 30) at -80°C. On the day of LC/MS measurement, vortex samples for 0h 10m 0s at 4°C and centrifuge at top speed (15000rpm,0h 0m 0s, 0h 10m 0s, 4°C). Then transfer supernatant to autosampler vials.