TITLE: Whole-cell proteomics and Analysis by Tandem Mass Tagging-based proteomics
AUTHORS: Felix Kraus, Sharan Swarup, Vinay V. Eapen, J. Wade Harper wade_harper@hms.harvard.edu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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(</span><a href="applewebdata://8AC23749-8314-4097-9D47-838582FD0B6E#_ENREF_40" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">McAlister et al., 2014</span></a><span>). 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 ug) makes application of this approach simple for many different types of cells</span></div></div>

[STEPS]
?. [Harvest, precipitation and digestion]
For whole proteome analysis,   is required for each replicate. Lyse cells in lysis buffer and pass them through a 21G needle 10 times. Alternatively, lyse cells bysonication as per manufactures instructions.
[of protein lysate]
?. [Harvest, precipitation and digestion]
Centrifugate suspensions at  for   and collect the supernatant.
Centrifuge: 13000 33, 4 10
?. [Harvest, precipitation and digestion]
Transfer quantified protein lysate concentration and  for each sample to a clean 1.5 mL protein Lo-Bind Eppendorf tubes. Reduce lysates for  at  with  , and alkylate cysteine residues with (, ).
[of lysate ]
0 Room temperature
[TCEP]
[Chloroacetamide ]
0 Room temperature
?. [Harvest, precipitation and digestion]
Extract protein content by methanol-chloroform precipitation and subsequent MeOH washes.
?. [Harvest, precipitation and digestion]
Add 4x volumes of MeOH and vortex.
?. [Harvest, precipitation and digestion]
Resuspend protein pellets   of   ().
100 µl
[EPPS buffer]
?. [Harvest, precipitation and digestion]
Digest samples at  for  with endoproteinase Lys-C (Wako, Japan) at a 1/200 enzyme/protein ratio.
37 °C
?. [Harvest, precipitation and digestion]
Digest with Trypsin (1:100) for at .
37 °C
?. [TMT-Labeling of samples]
Add  to each sample. Solubilize TMT reagents are in ACN as per manufacturer’s instructions and   is used for every . Performe TMT labeling in a final concentration of 20-25% ACN. Add   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.
[of the TMT reagent]
[of TMT label]
[of protein lysate]
[of ACN]
?. [TMT-Labeling of samples]
Incubate for at  to label the samples.
37 Room temperature
?. [Harvest, precipitation and digestion]
Add 1x volume of chloroform and vortex.
?. [Harvest, precipitation and digestion]
Add 3x volume of water and vortex.
?. [Harvest, precipitation and digestion]
Spin down at   for   at high speed.
0 Room temperature
?. [Harvest, precipitation and digestion]
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).
?. [Harvest, precipitation and digestion]
Add 4x volumes of MeOH and vortex.
?. [Harvest, precipitation and digestion]
Spin down at   for   at high speed.
0 Room temperature
?. [Harvest, precipitation and digestion]
Air dry (or speed-vac) protein pellet down to remove all traces of MeOH.
?. [Harvest, precipitation and digestion]
Repeat MeOH wash.
?. [Harvest, precipitation and digestion]
Aspirate and discard supernatant. Do not disturb the protein pellet at the bottom of the tube.
?. [TMT-Labeling of samples]
Combine 1% of each labeled sample together in a tube, quench the reaction with   for  at , 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.
[of 5% hydroxylamine]
37 Room temperature
?. [Stage tip for Ratio Check]
Resuspend the dried sample in  . Check to ensure that the pH of the sample is ~ (or lower) using pH strip.
[of 5% FA/5% ACN]
?. [Stage tip for Ratio Check]
Make stage tip by placing 6-8 “cookies” of C-18 embedded membranes in .
[Rainin tip]
?. [Stage tip for Ratio Check]
Perform C-18 cleanup:
?. [Stage tip for Ratio Check]
Equilibrate C-18 with  .
[of 100% methanol]
?. [Stage tip for Ratio Check]
Dry down eluted peptides in speed-vac.
?. [Stage tip for Ratio Check]
Wash C-18 with  .
[of 50% ACN/5% FA]
?. [Stage tip for Ratio Check]
Wash C-18 with  .
[of 5% ACN/5% FA]
?. [Stage tip for Ratio Check]
Load sample on to C-18 to bind peptides.
?. [Stage tip for Ratio Check]
Wash bound peptides on C-18 with  .
[of 5% ACN/5% FA]
?. [Stage tip for Ratio Check]
Elute peptides off C-18 with   into a mass spec vial.
[75% ACN/ 5% FA]
?. [Stage tip for Ratio Check]
Reconstitute peptides in .
[5% ACN/5% FA]
?. [Stage tip for Ratio Check]
Perform ratio check by analyzing the total amount of reporter ions present, as measured by mass spectrometry, for each TMT reporter ion channel.
?. [Stage tip for Ratio Check]
Quench the entire volume of each sample using   for  at . Combine samples in 2 ml Eppendorf tube to equal amounts, based on normalization values you obtain from the ratio check.
[of 5% hydroxylamine]
37 Room temperature
?. [Stage tip for Ratio Check]
Dry down labeled, combined sample in speed-vac.
?. [Stage tip for Ratio Check]
Re-constitute the sample in .
[of 5% ACN/5% FA]
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Place SepPak column into vacuum slot on a vacuum manifold.
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
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.
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Fill SepPak with , Flow Liquid through.
[5% FA/50% ACN]
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Fill SepPak with  , Flow Liquid through, repeat 2 more times.
[5% FA/5% ACN]
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Replace tube with 2 ml collection tube.
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Add Peptides, Flow Liquid through (~ 750 µl).
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Wash with , Flow Liquid through, 2 times.
[5% FA/5% ACN]
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Replace 15 ml collection tube with 2 ml collection tube.
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Elute with . Since there is residual liquid left in the SepPak, ensure that all the liquid flows through SepPak.
[75% ACN/5% FA]
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Dry down in SpeedVac  or .
37 Room temperature
30 °C
?. [SepPak clean-up (C18 solid-phase extraction (SPE))]
Fill SepPak with .
[100% MeOH]
?. [Fractionation]
Resuspend sample in .
[of 10 mM NH4HCO3]
?. [Fractionation]
Fractionate using pH reverse-phase HPLC:
?. [Fractionation]
Fractionate samples by high-pH reverse-phase HPLC (Agilent LC1260) into 96 fractions over a  run.
?. [Fractionation]
Fractions are run through an Aeris peptide XB-c18 column (Phenomenex; 250 mm x 3.6 mm), with mobile phase A containing  and   and phase B containing  and   (all inLC-MS grade H20).
[ACN]
[NH4HCO3]
[ACN]
[NH4HCO3]
?. [Fractionation]
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).
?. [Fractionation]
Dry down in SpeedVac.
?. [Fractionation]
Resuspend peptides in .
[5%FA /5% ACN]
?. [Fractionation]
Check pH (~3.5) with pH indicator strips.
?. [Stage tip for proteomics sample]
Stage tip each fraction.
?. [Stage tip for proteomics sample]
Make stage tips and equilibrate. Spin down at  .
Centrifuge: 3000 33, 3 min
?. [Stage tip for proteomics sample]
Perform C-18 cleanup:
?. [Stage tip for proteomics sample]
Wash with .
[50% ACN /5% FA]
?. [Stage tip for proteomics sample]
Wash with  .
[5% ACN /5% FA]
?. [Stage tip for proteomics sample]
Load sample
?. [Stage tip for proteomics sample]
Collect flow through and freeze.
?. [Stage tip for proteomics sample]
Wash with .
[50% ACN /5% FA]
?. [Stage tip for proteomics sample]
Elute with  in mass spec vial.
[75% ACN/1% FA ]
?. [Stage tip for proteomics sample]
Dry down in SpeedVac.
?. [Stage tip for proteomics sample]
Reconstitute pellet in  .
[5% FA / 5% ACN]
?. [Stage tip for proteomics sample]
Freeze sample at - until ready to run proteomics.
-20 °C