TITLE: Workflow for proteomic analysis of purified lysosomes with or without damage
AUTHORS: Sharan Swarup, J. Wade Harper

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lysosomes are a major degradative organelle within eukaryotic cells. Previous work has developed a method wherein the TMEM192 protein is tagged on its C-terminus with an epitope tag in order to immunopurify (IP) lysosomes from cell extracts.1 This process is referred to as Lyso-IP. Such lysosomes can be used for proteomic analysis or for metabolomic analysis. The Lyso-IP is adapted from a previous reported method (Wyant et al., 2018). Here we also describe processing steps using proteomics after lysosome purification in the context of lysosomal damaging agents. Agents such as L-Leucyl-L-Leucine methyl ester (hydrochloride) (LLoMe) and Gly-Phe-β-naphthylamide (GPN) induce lysosomal damage, leading to the degradation of damaged lysosomes by lysophagy. This adaptation of Lyso-IP provides a route to identify proteins that are recruited to damaged lysosomes using quantitative proteomics.</div></div>

[STEPS]
?. [Cell culture]
Grow the appropriate cells (e.g. HEK293T) expressing TMEM192-3xHA in DMEM containing 5% FBS
One 15 cm plate of cells (80% confluence) is used per replicate.
?. [Cell culture]
To damage lysosomes, add GPN ) or LLoMe (-) to cells for  to .
0.2
The length of time employed depends on the desired level of lysosomal damage desired.
?. [Lyso-IP]
Elute each sample with  KPBS containing  NP-40 in thermo mixer at  for .
100 µl
4 °C
Elutes were snap frozen in liquid nitrogen and stored in  until further processing.
?. [Trypsinization]
Reduce lysates for  at  () with  TCEP.
25 °C
0 Room temperature
?. [Trypsinization]
Alkylate cysteine residues with  Chloroacetamide for  at .
0 Room temperature
?. [Trypsinization]
Add TCA to eluates to a final concentration of 20% and place  at  for at least .
on ice
4 °C
?. [Trypsinization]
Pellet the proteins for  at maximum speed at .
4 °C
?. [Trypsinization]
Aspirate supernatant carefully and leave ~- of solution so as to not disturb the pellet.
30 µl
40 µl
Note: It is common not to observe a visible pellet.
?. [Trypsinization]
Resuspend the pellets in 4 volumes of ice cold 10% TCA and pellet by centrifugation at  for  at maximum speed. Aspirate as before.
4 °C
?. [Trypsinization]
Resuspend the pellets in 4 volumes of ice cold methanol and pellet by centrifugation at  for  at maximum speed. Aspirate as before.
4 °C
?. [Trypsinization]
Repeat the methanol wash.
?. [Trypsinization]
Aspirate methanol as before and air dry the remaining - of solution (speed-vac can also be used to dry sample).
30 µl
40 µl
?. [Trypsinization]
Resuspend the dried pellets in ,  EPPS, .
50 µl
?. [Trypsinization]
Carry the peptide digestion out using LysC () for  at  followed by trypsin () overnight at .
0.25 µg
37 °C
0.5 µg
37 °C
?. [Labeling]
Add - of the TMT reagent and  of 100% ACN to each  sample.
3 µl
4 µl
15 µl
50 µl
?. [Labeling]
Incubate for  at .
37 Room temperature
?. [Labeling]
Stop the reaction with  of hydroxylamine 5% for  at .
4 µl
37 Room temperature
?. [Labeling]
Combine samples and dry in a speed-vac.
?. [Basic-pH RP peptide fractionation kit (follow manufacturer's instructions)]
Follow manufacturer’s instructions (Thermo Cat# 84868).
?. [Basic-pH RP peptide fractionation kit (follow manufacturer's instructions)]
Use elution: 17.5% ACN, 20% ACN, 22.5% ACN, 25% ACN, 27.5% ACN and 70% ACN.
?. [Basic-pH RP peptide fractionation kit (follow manufacturer's instructions)]
Speed vac individual samples to dryness.
?. [Basic-pH RP peptide fractionation kit (follow manufacturer's instructions)]
Proceed to stage-tip.
?. [Stage TiP]
Resuspend samples in  of 5% FA, 5% ACN. Check to ensure that the pH of the samples is ~pH3 (or lower) using pH strips.
100 µl
?. [Stage TiP]
Perform C-18 cleanup:
?. [Stage TiP]
a. Wash C-18 with  of 100% methanol.
100 µl
?. [Stage TiP]
b. Equilibrate C-18 with  of 50% ACN 5% FA.
50 µl
?. [Stage TiP]
c. Equilibrate C-18 with  of 5% ACN 5% FA.
100 µl
?. [Stage TiP]
d. Load sample on to C-18 to bind peptides.
?. [Stage TiP]
e. Collect flow through and freeze.
?. [Stage TiP]
f. Wash bound peptides on C-18 with  of 5% ACN 5% FA.
50 µl
?. [Stage TiP]
g. Elute peptides off C-18 with  of 75% ACN/5 % FA.
50 µl
?. [Stage TiP]
3. Dry down eluted peptides in speed-vac.
?. [Stage TiP]
4. Re-constitute peptides in  of 5% ACN 5% FA.
10 µl
?. [Mass spectrometry]
Inject  for each LC–MS/MS analysis using available mass spectrometer with a 120-minute online LC separation.
The analysis of peptides by mass spectrometry will depend on the type of instrument/platform used. Typical instrument settings for analysis on a Thermo Fusion Lumos instrument are provided in the following section.
3 µl
?. [Mass spectrometry]
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 digestionCarbamidomethyl (C), TMT (N-term peptide and K) set as a fixed modificationOxidation (M) set as variable modifications
?. [Mass spectrometry]
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.
This process will depend on the type of analysis software employed with the specific MS platform being used.
?. [Instrument settings]
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).
?. [Instrument settings]
Separate the peptides on a  inner diameter microcapillary column packed in house with ~ of Accucore150 resin (, 150 Å, ThermoFisher Scientific, San Jose, CA) with a gradient consisting of 5%–21% (ACN, 0.1% FA) over a total  run at ~.
100
35
2.6
500
Details of typical instrument parameters are provided below. For Multi-Notch MS3-based TMT analysis3, the scan sequence began with an MS1 spectrum (Orbitrap analysis; resolution 60,000 at 200 Th; mass range 375−1500 m/z; automatic gain control (AGC) target 5Å~105; maximum injection time 50 ms) unless otherwise stated in the instrument parameters in each supplemental table.
?. [Instrument settings]
Select the precursors for MS2 analysis using a Top10 method.
MS2 analysis consisted of collision-induced dissociation (quadrupole ion trap analysis; Turbo scan rate; AGC 2.0Å~104; isolation window 0.7 Th; normalized collision energy (NCE) 35; maximum injection time 90 ms).
?. [Instrument settings]
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.
?. [Instrument settings]
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.
?. [Instrument settings]
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).
?. [Data Analysis]
Data analysis will be platform and purpose specific.
?. [Lyso-IP]
All buffers were supplemented with protease inhibitors.
?. [Lyso-IP]
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).
?. [Lyso-IP]
The cells were pelleted at for  at  .
Centrifuge: 300 34
4 °C
?. [Lyso-IP]
Cells were washed once with KPBS buffer ( KCL,  KH2PO4,  Sucrose,  ).
136
10
50
?. [Lyso-IP]
The cell pellet was resuspended in  KPBS and lysed using 30 strokes in a Potter-Elvehjem homogenizer.
1 mL
2 mL
?. [Lyso-IP]
The lysed cells were spun down at  for  at  .
Centrifuge: 1000 34
4 °C
?. [Lyso-IP]
The pellet was discarded and the protein concentration of the lysate was determined by Bradford assay.
?. [Lyso-IP]
After normalizing the protein concentration to be equal across all replicates, 5% of the input sample was saved and  of anti-HA magnetic beads was added the remainder of the sample.
50 µl
?. [Lyso-IP]
The lysate/magnetic bead mixture was placed on gentle rotation for , at   and beads were separated from the lysate using a magnetic stand.
4 °C
?. [Lyso-IP]
The beads were washed twice with KPBS containing  NaCl and once with KPBS buffer.
300