TITLE: Workflow for proteomic analysis of purified lysosomes in cells lacking GRN 
AUTHORS: Sharan Swarup, J. Wade Harper

[DESCRIPTION]
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 HeLa cells lacking the GRN gene. Such cells can be produced using the following protocol: DOI: dx.doi.org/10.17504/protocols.io.4r3l2oxqqv1y/v1.

[GUIDELINES]
REFERENCES

1. Abu-Remaileh M, Wyant GA, Kim C, Laqtom NN, Abbasi M, Chan SH, Freinkman E, Sabatini DM. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.Science. 2017 Nov 10;358(6364):807-813. doi: 10.1126/science.aan6298. Epub 2017 Oct 26. PMID:29074583; PMCID: PMC5704967.
2. Wentao Dong, Nouf Laqtom, Monther Abu-Remaileh. Sample preparation protocol for lipidomics harvesting using lysosome immunoprecipitation (Lipidomics LysoIP, updated 02/09/21). protocols.io https://protocols.io/view/sample-preparation-protocol-for-lipidomics-harvest-br9ym97w
3. McAlister, G. C. et al. MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes. Analytical chemistry 86, 7150-7158 (2014).

[STEPS]
SECTION: Cell culture
1. Grow the appropriate cells (e.g. HeLa with or without the GRN gene created by gene editing, see DOI: dx.doi.org/10.17504/protocols.io.4r3l2oxqqv1y/v1) expressing TMEM192-3xHA in DMEM containing 10% FBS
 

SECTION: Lyso-IP
2. All buffers were supplemented with protease and phosphatase inhibitors (Roche).
SECTION: Lyso-IP
3. Cells at 80% confluency were harvested on ice by scraping and washed once with Phosphate buffered saline (PBS) containing protease inhibitors (Roche).
SECTION: Lyso-IP
4. The cells were pelleted at300 x g for 5 min at 4 °C .

SECTION: Lyso-IP
5. The cell pellet was resuspended in 1 mL KPBS buffer and lysed using 30 strokes in a2 mL Potter-Elvehjem homogenizer.

SECTION: Lyso-IP
6. Cells were washed once with KPBS buffer (136 mM KCL, 10 mM KH2PO4, pH 7.2 ).

SECTION: Lyso-IP
7. The lysed cells were spun down at 1000 x g for 5 min at 4 °C .


SECTION: Lyso-IP
8. The pellet was discarded and the protein concentration of the lysate was determined by Bradford assay.
SECTION: Lyso-IP
9. After normalizing the protein concentration to be equal across all replicates, 5% of the input sample was saved and 30-50 µL of anti-HA magnetic beads was added the remainder of the sample.

SECTION: Lyso-IP
10. The lysate/magnetic bead mixture was placed on gentle rotation for 60 min, at 4 °C  and beads were separated from the lysate using a magnetic stand.


SECTION: Lyso-IP
11. The beads were washed twice with KPBS containing 300 mM NaCl and once with KPBS buffer.

SECTION: Lyso-IP
12. 

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





SECTION: Trypsinization
13. Reduce lysates for 30 min at 25 °C (Room temperature) with 5 millimolar (mM) TCEP.

SECTION: Trypsinization
14. Alkylate cysteine residues with 20 millimolar (mM) Chloroacetamide for 30 min at Room temperature.

SECTION: Trypsinization
15. Add TCA to eluates to a final concentration of 20% and place on ice at 4 °C for at least 60 min.

SECTION: Trypsinization
16. Pellet the proteins for 30 min at maximum speed at 4 °C.

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

SECTION: Trypsinization
18. Resuspend the pellets in 4 volumes of ice cold 10% TCA and pellet by centrifugation at 4 °C for 10 min at maximum speed. Aspirate as before.

SECTION: Trypsinization
19. Resuspend the pellets in 4 volumes of ice cold methanol and pellet by centrifugation at 4 °C for 10 min at maximum speed. Aspirate as before.

SECTION: Trypsinization
20. Repeat the methanol wash.
SECTION: Trypsinization
21. Aspirate methanol as before and air dry the remaining 30 µL-40 µL of solution (speed-vac can also be used to dry sample).

SECTION: Trypsinization
22. Resuspend the dried pellets in 50 µL, 200 millimolar (mM) EPPS, pH 8.0.

SECTION: Trypsinization
23. Carry the peptide digestion out using LysC (0.25 µg) for 120 min at 37 °C followed by trypsin (0.5 µg) overnight at 37 °C.

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

SECTION: Labeling
25. Incubate for 60 min at 37 Room temperature.

SECTION: Labeling
26. Stop the reaction with 4 µL of hydroxylamine 5% for 15 min at 37 Room temperature.

SECTION: Labeling
27. Combine samples and dry in a speed-vac.
SECTION: Basic-pH RP peptide fractionation kit (follow manufacturer&#x27;s instructions)
28. Follow manufacturer’s instructions (Thermo Cat# 84868).
SECTION: Basic-pH RP peptide fractionation kit (follow manufacturer&#x27;s instructions)
29. Use elution: 17.5% ACN, 20% ACN, 22.5% ACN, 25% ACN, 27.5% ACN and 70% ACN.
SECTION: Basic-pH RP peptide fractionation kit (follow manufacturer&#x27;s instructions)
30. Speed vac individual samples to dryness.
SECTION: Basic-pH RP peptide fractionation kit (follow manufacturer&#x27;s instructions)
31. Proceed to stage-tip.
SECTION: Stage TiP
32. Resuspend samples in 100 µL of 5% FA, 5% ACN. Check to ensure that the pH of the samples is ~pH3 (or lower) using pH strips.

SECTION: Stage TiP
33. Perform C-18 cleanup:
SECTION: Stage TiP
33.1. a. Wash C-18 with 100 µL of 100% methanol.

SECTION: Stage TiP
33.2. b. Equilibrate C-18 with 50 µL of 50% ACN 5% FA.

SECTION: Stage TiP
33.3. c. Equilibrate C-18 with 100 µL of 5% ACN 5% FA.

SECTION: Stage TiP
33.4. d. Load sample on to C-18 to bind peptides.
SECTION: Stage TiP
33.5. e. Collect flow through and freeze.
SECTION: Stage TiP
33.6. f. Wash bound peptides on C-18 with 50 µL of 5% ACN 5% FA.

SECTION: Stage TiP
33.7. g. Elute peptides off C-18 with 50 µL of 75% ACN/5 % FA.

SECTION: Stage TiP
34. 3. Dry down eluted peptides in speed-vac.
SECTION: Stage TiP
35. 4. Re-constitute peptides in 10 µL of 5% ACN 5% FA.

SECTION: Mass spectrometry
36. 
 
Inject 3 µL for each LC–MS/MS analysis using available mass spectrometer with a 120-minute online LC separation.

SECTION: Mass spectrometry
37. 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
SECTION: Mass spectrometry
38. 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.
 

SECTION: Instrument settings
39. 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).
SECTION: Instrument settings
40. Separate the peptides on a 100 μm inner diameter microcapillary column packed in house with ~35 cm of Accucore150 resin (2.6 μm, 150 Å, ThermoFisher Scientific, San Jose, CA) with a gradient consisting of 5%–21% (ACN, 0.1% FA) over a total 150 min run at ~500 nL/min.
 


SECTION: Instrument settings
41. Select the precursors for MS2 analysis using a Top10 method.
 

SECTION: Instrument settings
42. 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.
SECTION: Instrument settings
43. 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.
SECTION: Instrument settings
44. 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).
SECTION: Data Analysis
45.