Workflow for proteomic analysis of purified lysosomes with or without damage

Grow the appropriate cells (e.g. HEK293T) expressing TMEM192-3xHA in DMEM containing 5% FBS

To damage lysosomes, add GPN 0.2mM) or LLoMe (0.5millimolar (mM)-1.0millimolar (mM)) to cells for 0h 15m 0s to 1h 0m 0s.

All buffers were supplemented with protease inhibitors.

After treatment of cells at 80% confluency with or without lysosomal damage, cells were harvested on ice by scraping and washed once with Phosphate buffered saline (PBS) containing protease inhibitors (Roche).

The cells were pelleted at300x g,0h 0m 0s for 0h 5m 0s at 4°C .

Cells were washed once with KPBS buffer (136mM KCL, 10mM KH2PO4, 50mM Sucrose, 7.2 ).

The cell pellet was resuspended in 1mL KPBS and lysed using 30 strokes in a2mL Potter-Elvehjem homogenizer.

The lysed cells were spun down at 1000x g,0h 0m 0s for 0h 5m 0s at 4°C .

The pellet was discarded and the protein concentration of the lysate was determined by Bradford assay.

After normalizing the protein concentration to be equal across all replicates, 5% of the input sample was saved and 50-100µL of anti-HA magnetic beads was added the remainder of the sample.

The lysate/magnetic bead mixture was placed on gentle rotation for 0h 20m 0s, at 4°C and beads were separated from the lysate using a magnetic stand.

The beads were washed twice with KPBS containing 300mM NaCl and once with KPBS buffer.

Elute each sample with 100µL KPBS containing 0.5% (v/v) NP-40 in thermo mixer at 4°C for 0h 30m 0s.

Reduce lysates for 0h 30m 0s at 25°C (Room temperature) with 5millimolar (mM) TCEP.

Alkylate cysteine residues with 20millimolar (mM) Chloroacetamide for 0h 30m 0s at Room temperature.

Add TCA to eluates to a final concentration of 20% and place On ice at 4°C for at least 1h 0m 0s.

Pellet the proteins for 0h 30m 0s at maximum speed at 4°C.

Aspirate supernatant carefully and leave ~30µL-40µL of solution so as to not disturb the pellet.

Resuspend the pellets in 4 volumes of ice cold 10% TCA and pellet by centrifugation at 4°C for 0h 10m 0s at maximum speed. Aspirate as before.

Resuspend the pellets in 4 volumes of ice cold methanol and pellet by centrifugation at 4°C for 0h 10m 0s at maximum speed. Aspirate as before.

Repeat the methanol wash.

Aspirate methanol as before and air dry the remaining 30µL-40µL of solution (speed-vac can also be used to dry sample).

Resuspend the dried pellets in 50µL, 200millimolar (mM) EPPS, 8.0.

Carry the peptide digestion out using LysC (0.25µg) for 2h 0m 0s at 37°C followed by trypsin (0.5µg) overnight at 37°C.

Add 3µL-4µL of the TMT reagent and 15µL of 100% ACN to each 50µL sample.

Incubate for 1h 0m 0s at 37Room temperature.

Stop the reaction with 4µL of hydroxylamine 5% for 0h 15m 0s at 37Room temperature.

Combine samples and dry in a speed-vac.

Follow manufacturer’s instructions (Thermo Cat# 84868).

Use elution: 17.5% ACN, 20% ACN, 22.5% ACN, 25% ACN, 27.5% ACN and 70% ACN.

Speed vac individual samples to dryness.

Proceed to stage-tip.

Resuspend samples in 100µL of 5% FA, 5% ACN. Check to ensure that the pH of the samples is ~pH3 (or lower) using pH strips.

Perform C-18 cleanup:

a. Wash C-18 with 100µL of 100% methanol.

b. Equilibrate C-18 with 50µL of 50% ACN 5% FA.

c. Equilibrate C-18 with 100µL of 5% ACN 5% FA.

d. Load sample on to C-18 to bind peptides.

e. Collect flow through and freeze.

f. Wash bound peptides on C-18 with 50µL of 5% ACN 5% FA.

g. Elute peptides off C-18 with 50µL of 75% ACN/5 % FA.

3. Dry down eluted peptides in speed-vac.

4. Re-constitute peptides in 10µL of 5% ACN 5% FA.

Inject 3µL for each LC–MS/MS analysis using available mass spectrometer with a 120-minute online LC separation.

Search raw data against UniProt human protein database using any proteomic analysis software with the following parameters:

• Up to 3 missed cleavages allowed for trypsin/LysC digestion
• Carbamidomethyl (C), TMT (N-term peptide and K) set as a fixed modification
• Oxidation (M) set as variable modifications

Extract signal to noise intensity values of each TMT reporter and identified proteins, and further calculate the ratio of each condition to the control sample’s intensity.

Collect mass spectrometry data using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a Proxeon EASY-nLC1200 liquid chromatography (LC) pump (Thermo Fisher Scientific).

Separate the peptides on a 100μm inner diameter microcapillary column packed in house with ~35cm of Accucore150 resin (2.6μm, 150 Å, ThermoFisher Scientific, San Jose, CA) with a gradient consisting of 5%–21% (ACN, 0.1% FA) over a total 2h 30m 0s run at ~500nL/min.

Select the precursors for MS2 analysis using a Top10 method.

Use the monoisotopic peak assignment and exclude the previously interrogated precursors using a dynamic window (150 s ± 7898 ppm) and perform the dependent scans on a single charge state per precursor.

Following acquisition of each MS2 spectrum, collect a synchronous-precursor-selection (SPS) MS3 scan on the top 10 most intense ions in the MS2 spectrum.

Fragment the MS3 precursors by high energy collision-induced dissociation (HCD) and analyze using the Orbitrap (NCE 65; AGC 3Å~105; maximum injection time 150 ms, resolution was 50,000 at 200 Th).