Sep 18, 2021
Lysosome immunopurification (LysoIP) protocol for subcellular metabolite profiling
- Wentao Dong1,
- Nouf Laqtom1,
- Monther Abu-Remaileh1
- 1Department of Chemical Engineering, Department of Genetics, The Institute for Chemistry, Engineering & Medicine for Human Health (ChEM-H), Stanford University, Stanford, CA 94305, USA.
- Metabolomics Protocols & WorkflowsTech. support email: bbmisraccb@gmail.com
External link: https://science.sciencemag.org/content/358/6364/807/tab-pdf
Protocol Citation: Wentao Dong, Nouf Laqtom, Monther Abu-Remaileh 2021. Lysosome immunopurification (LysoIP) protocol for subcellular metabolite profiling. protocols.io https://dx.doi.org/10.17504/protocols.io.bybjpskn
Manuscript citation:
Eapen VV, Swarup S, Hoyer MJ, Paulo JA, Harper JW, Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy. eLife doi: 10.7554/eLife.72328
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: September 16, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 53323
Keywords: lysosomes, immunoprecipitation, metabolomics, lipidomics, ASAPCRN
Abstract
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.
Materials
Consumables
- Marker pen
- Pipette set (1000 μl)
- PPE kit (Lab coat, gloves, safety glasses)
- Ice and ice bucket
- 1.5 ml Eppendorf tubes rack
Reagents
- Anti-HA magnetic beads (Thermo Fisher Scientific, cat. no. 88837)
- Optima LC/MS water (Fisher, cat. no. W6-4)
- Optima LC/MS methanol (Fisher, cat. no. A456-4)
- KPBS (136 mM KCl, 10 mM KH2PO4, pH 7.25 using KOH in Optima LC/MS water)
- Isotopically labeled amino acids (Cambridge Isotope Laboratories, cat. no. MSK-A2-S)
- LysoTracker Red DND-99 (ThermoFisher Scientific, cat. no. L7528)
Equipment
- Glass Vessel: (VWR, cat no. 89026-386)
- Tissue Grinder: (VWR, cat no. 89026-398)
- Benchtop centrifuge (VWR)
- Milli-Q water system
- ID-X Orbitrap Tribrid Mass Spectrometer
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
Preparation of homogenizers and sample tubes
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.
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
Preparation of Anti-HA beads
Pool all required volumes together (100 µL / 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 100 µL into each 1.5 mL labeled tubes ➃.
Cell preparation before harvesting
Cell preparation before harvesting
Wash the first set of 15cm plates (each set has two plates) with 10 mL of DMEM/plate (for HEKS, use no serum + no antibiotics).
Replace with 10 mL of DMEM/plate (+ 40 µL of 1:1000 diluted LysoTracker) for an hour.
10 mL The second set of plates will be washed 00:20:00 later after the first set and so on.
Note
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.
20m
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 ~5 mL cold clean MS grade PBS on the edge of the plate, decant the first time and then aspirate the second time.
Cell harvesting
Cell harvesting
Add 950 µL 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 1000 x g for 00:02:00 at 4 °C .
Note
Note: centrifuge must already be cooled to 4 °C at this point.
2m
Aspirate the supernatant and resuspend the pellets with 950 µL cold KPBS.
From this resuspended sample, take 25 µL for whole cell in the 1.5 mL tube ➂.
Note: if pellet mixer is used instead of douncer, resuspend the pellets with 100 µL cold KPBS in step 16, homogenize cells and then replenish to 950 uL and follow step 17.
Homogenization and LysoIP
Homogenization and LysoIP
Transfer the remainder (925 µL ) 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.
Note
Note: count and repeat for each of the samples (both in number and speed).
Use serological pipet to transfer sample from douncer into the 1.5 mL tubes ➁.
Spin 1,000g for 00:02:00 at 4 °C .
2m
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 800 µL when using pellet mixer) .
Note
Note: leave the pellet and make sure not to accidently suck up any of the pellet as that can negatively affect experiment.
Rock in cold room for 00:03:00 (everything from now on is in the cold room).
Note
Note: Max speed. Set timer for 3 mins.
3m
Put the ➃ tube on magnet. Count at least 00:00:25 to allow for beads to be pulled by magnets.
Note
Note: it is important to keep this count the same between each wash and each sample for consistency I.e. 25 seconds each time .
25s
Wash the bound fraction 3 times with 1 mL cold KPBS. Then aspirate all cold KPBS.
Note
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
Processing of polar metabolite samples
20m
20m
Resuspend the IP samples in 50 µL of freezing cold 80 % (v/v) MeOH with isotopically labeled amino acids (500 nM) as internal standards.
Note
Note: it is difficult to resuspend. Can begin flushing on the side of beads stuck on the tube, then gradually move inward.
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 (25 µL from step 17 ) in the 1.5 mL tube ➂ and IP samples (50 µL from step 26) in the 1.5 mL tube ➄ with beads still in it.
After 00:10:00 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.
10m
For WC samples, add 225 µL 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 (15000 rpm , 00:10:00 , 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 00:10:00 at 4 °C and centrifuge at top speed (15000 rpm , 00:10:00 , 4 °C ). Then transfer supernatant to autosampler vials.
30m