TITLE: Whole-cell proteomics and Analysis with or without nutrient stress by Tandem Mass Tagging-based proteomics V2
AUTHORS: Sharan Swarup, J. Wade Harper wade_harper@hms.harvard.edu, Kelsey Hickey

[DESCRIPTION]
The analysis of relative protein abundance has emerged as an important tool in cell biology. Typically, it is possible to quantify >8000 proteins under standard conditions. Tandem Mass Tags (TMT) are isobaric reagents that contain a set of isotopically distinct reporter ions, which can be used to quantify individual peptides in distinct samples through multiplexing(McAlister et al., 2014). Because the TMT analysis is performed in multiplexed format (up to 18 plex), it is possible to examine the effect of different perturbations (treatments, time courses, etc) on the total abundance of the proteome and include replicate samples as desired. This protocol is applicable to many different cell types, although the number of proteins quantified may differ, depending on the complexity of the proteomes in individual cell types. The small amount of protein needed (50-100 μg) makes application of this approach simple for many different types of cells. This protocol explicitly is used to examine the effects of nutrient stress on protein abundance in cell lines with or without autophagy.

[BEFORE_START]
WORKFLOW:
  


[GUIDELINES]
Mass spectrometry:
The analysis of TMT-labelled 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.

Inject 3 μl 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) and di-glycine (K) 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.

Instrument settings:
Mass spectrometry data were collected 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). Peptides were separated 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 3%–26% (0-100 min), 26-32% (100-110min) (ACN, 0.1% FA) over a total 120 min run at ~400 nL/min.For analysis, we loaded 1/3 of each fraction onto the column. Each analysis used the Multi-Notch MS3-based TMT method(McAlister et al., 2014). The scan sequence began with an MS1spectrum (Orbitrap analysis; resolution 120,000 at 200 Th; mass range 400−1250 m/z; automatic gain control (AGC) target 1×106; maximum injection time 100 ms). Precursors for MS2analysis were selected using a Top 4 sec method. MS2analysis consisted of collision-induced dissociation (quadrupole Orbitrap analysis; AGC 1×105; isolation window 0.7 Th; normalized collision energy (NCE) 35; maximum injection time 300 ms resolution was 7,500 at 200 Th).Monoisotopic peak assignment was used, and previously interrogated precursors were excluded using a dynamic window (120 s ± 7 ppm).Following acquisition of each MS2spectrum, a synchronous-precursor-selection (SPS) MS3scan was collected on the top 10 most intense ions in the MS2spectrum(McAlister et al., 2014). MS3precursors were fragmented by high energy collision-induced dissociation (HCD) and analyzed using the Orbitrap (NCE 65; AGC 2×105;maximum injection time 500 ms, resolution was 50,000 at 200 Th).

[STEPS]
SECTION: Harvest, precipitation and digestion
3. For whole proteome analysis, 50 µg  is required for each replicate.  Cells from step 2 are washed with PBS  three times. Cells were lysed by in UREA denaturing buffer (8M Urea, 150mM NaCl, 50mM EPPS pH8.0,  containing mammalian protease inhibitor cocktail (Sigma), and Phos-STOP) Cell lysates were collected by cell scrapers and sonicated on ice for 10 seconds. 
SECTION: Harvest, precipitation and digestion
4. Centrifugate suspensions at 13000 rpm, 4 °C for 10 min  and collect the supernatant.
SECTION: Harvest, precipitation and digestion
5. Transfer quantified protein lysate concentration and 50 µg for each sample to a clean 1.5 mL protein Lo-Bind Eppendorf tubes. Reduce lysates for 20 min at Room temperature with 5 millimolar (mM) , and alkylate cysteine residues with 20 millimolar (mM)(Room temperature, 30 min).

SECTION: Harvest, precipitation and digestion
6. Extract protein content by methanol-chloroform precipitation and subsequent MeOH washes.
SECTION: Harvest, precipitation and digestion
6.1. Add 4x volumes of MeOH and vortex.

SECTION: Harvest, precipitation and digestion
7. Resuspend protein pellets  100 µL of 200 millimolar (mM)  (pH 8.5).

SECTION: Harvest, precipitation and digestion
8. Digest samples at 37 °C for 120 min with endoproteinase Lys-C (Wako, Japan) at a 1/200 enzyme/protein ratio.

SECTION: Harvest, precipitation and digestion
9. Digest with Trypsin (1:100) for 360 minat 37 °C.

SECTION: TMT-Labeling of samples
10.  Add 5 µL to each sample. Solubilize TMT reagents are in ACN as per manufacturer’s instructions and 5 µL  is used for every 50 µg. Performe TMT labeling in a final concentration of 20-25% ACN. Add 20 µL  to bring the reaction volume at 125 µL.The number of samples, and hence the number of individual TMT reagents, will depend upon the design of the experiment.

SECTION: TMT-Labeling of samples
11. Incubate for 60 minat 37 Room temperature to label the samples.
SECTION: Harvest, precipitation and digestion
6.2. Add 1x volume of chloroform and vortex.
SECTION: Harvest, precipitation and digestion
6.3. Add 3x volume of water and vortex.
SECTION: Harvest, precipitation and digestion
6.4. Spin down at Room temperature  for  5 min at high speed.

SECTION: Harvest, precipitation and digestion
6.5. Aspirate and discard the upper aqueous phase. Do not disturb the protein disc at the interface of the aqueous phase (top) and organic phase (bottom).
SECTION: Harvest, precipitation and digestion
6.6. Add 4x volumes of MeOH and vortex.
SECTION: Harvest, precipitation and digestion
6.7. Spin down at Room temperature  for 5 min  at high speed.

SECTION: Harvest, precipitation and digestion
6.10. Air dry (or speed-vac) protein pellet down to remove all traces of MeOH.
SECTION: Harvest, precipitation and digestion
6.9. Repeat MeOH wash.
SECTION: Harvest, precipitation and digestion
6.8. Aspirate and discard supernatant. Do not disturb the protein pellet at the bottom of the tube.
SECTION: TMT-Labeling of samples
12. Combine 1% of each labeled sample together in a tube, quench the reaction with 4 µL  for 15 min at 37 Room temperature, and dry down using the speed-vac. This combined sample is used to perform the ratio check to test labeling efficiency. The remaining amount of each sample can be stored in the freezer.
SECTION: Stage tip for Ratio Check
13. Resuspend the dried sample in 100 µL . Check to ensure that the pH of the sample is ~pH 3 (or lower) using pH strip.

SECTION: Stage tip for Ratio Check
14. Make stage tip by placing 6-8 “cookies” of C-18 embedded membranes in 200 µL.

SECTION: Stage tip for Ratio Check
15. Perform C-18 cleanup:
SECTION: Stage tip for Ratio Check
15.1. Equilibrate C-18 with 100 µL .

SECTION: Stage tip for Ratio Check
16. Dry down eluted peptides in speed-vac.
SECTION: Stage tip for Ratio Check
15.2. Wash C-18 with 50 µL .

SECTION: Stage tip for Ratio Check
15.3. Wash C-18 with 100 µL .

SECTION: Stage tip for Ratio Check
15.4. Load sample on to C-18 to bind peptides.
SECTION: Stage tip for Ratio Check
15.5. Wash bound peptides on C-18 with 50 µL .
SECTION: Stage tip for Ratio Check
15.6. Elute peptides off C-18 with 50 µL  into a mass spec vial.

SECTION: Stage tip for Ratio Check
17. Reconstitute peptides in 10 µL.

SECTION: Stage tip for Ratio Check
18. Perform ratio check by analyzing the total amount of reporter ions present, as measured by mass spectrometry, for each TMT reporter ion channel.
SECTION: Stage tip for Ratio Check
19. Quench the entire volume of each sample using 8 µL  for 15 min at 37 Room temperature. Combine samples in 2 ml Eppendorf tube to equal amounts, based on normalization values you obtain from the ratio check.

SECTION: Stage tip for Ratio Check
20. Dry down labeled, combined sample in speed-vac.
SECTION: Stage tip for Ratio Check
21. Re-constitute the sample in 750 µL.

SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
22. Place SepPak column into vacuum slot on a vacuum manifold.
SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
24. Start the vacuum, gently, try to ensure that the pressure gauge is below 10. This can be achieved by opening one of the valves gently. The fluid should pass through slowly, drop wise.
SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
25. Fill SepPak with 1 mL, Flow Liquid through.

SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
26. Fill SepPak with 1 mL , Flow Liquid through, repeat 2 more times.

SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
27. Replace tube with 2 ml collection tube.
SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
28. Add Peptides, Flow Liquid through (~ 750 µl).
SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
29. Wash with 1 mL, Flow Liquid through, 2 times.

SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
30. Replace 15 ml collection tube with 2 ml collection tube.
SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
31. Elute with 750 µL. Since there is residual liquid left in the SepPak, ensure that all the liquid flows through SepPak.

SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
32. Dry down in SpeedVac 37 Room temperature 15 min or 30 °C.

SECTION: SepPak clean-up (C18 solid-phase extraction (SPE))
23. Fill SepPak with 1 mL.
SECTION: Fractionation
33. Resuspend sample in 100 µL pH 8.

SECTION: Fractionation
34. Fractionate using pH reverse-phase HPLC:
SECTION: Fractionation
34.1. Fractionate samples by high-pH reverse-phase HPLC (Agilent LC1260) into 96 fractions over a 90 min run.

SECTION: Fractionation
34.2. Fractions are run through an Aeris peptide XB-c18 column (Phenomenex; 250 mm x 3.6 mm), with mobile phase A containing 5 1425 and 10 millimolar (mM) pH 8  and phase B containing 90 1425 and 10 millimolar (mM) pH 8  (all inLC-MS grade H20).

SECTION: Fractionation
35. Combine fractionated samples (either 12 or 24 fractions) in a non-continuous manner into individual 1.5 ml Eppendorf tube (see outline below form Paulo et al., 2016).
  

SECTION: Fractionation
36. Dry down in SpeedVac.
SECTION: Fractionation
37. Resuspend peptides in 100 µL.

SECTION: Fractionation
38. Check pH (~3.5) with pH indicator strips.
SECTION: Stage tip for proteomics sample
39. Stage tip each fraction.
SECTION: Stage tip for proteomics sample
39.1. Make stage tips and equilibrate. Spin down at 3000 rpm, 3 min .

SECTION: Stage tip for proteomics sample
39.2. Perform C-18 cleanup:
SECTION: Stage tip for proteomics sample
39.3. Wash with 50 µL.

SECTION: Stage tip for proteomics sample
39.4. Wash with 100 µL .

SECTION: Stage tip for proteomics sample
39.5. Load sample
SECTION: Stage tip for proteomics sample
39.6. Collect flow through and freeze.
SECTION: Stage tip for proteomics sample
39.7. Wash with 50 µL.
SECTION: Stage tip for proteomics sample
39.8. Elute with 50 µL in mass spec vial.

SECTION: Stage tip for proteomics sample
40. Dry down in SpeedVac.
SECTION: Stage tip for proteomics sample
41. Reconstitute pellet in 10 µL .

SECTION: Stage tip for proteomics sample
42. Freeze sample at --20 °C until ready to run proteomics.

SECTION: Cell culture and nutrient stress treatments
1. Wild-type HEK293 (human embryonic kidney, fetus, ATCC CRL-1573, RRID: CVCL_0045) cells or the analogous cells lacking either ATG7 or FIP200 (see DOI: XXX) were grown in Dulbecco’s modified Eagle’s medium (DMEM, high glucose and pyruvate) supplemented with 10% fetal calf serum and maintained in a 5% CO2 incubator at 37oC. Cells were maintained at <80% confluency throughout the course of experiments.  

SECTION: Cell culture and nutrient stress treatments
2. DMEM was removed and cells were washed 3 times with PBS followed by resuspending cells in EBSS or DMEM lacking amino acids prepared according to H. Chino, et al Mol Cell 74, 909-921 e906 (2019). The investigator can select the length of time for starvation but this is typically 10-18 hours.