TITLE: Sample preparation protocol for total proteomic analysis of mouse tissues including brain
AUTHORS: Raja S. Nirujogi, Houjiang Zhou, Dario R Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mass  spectrometry-based proteomics has emerged as fundamental technique to  study functional changes of proteome including post translational  modifications. Sample preparation is key for an effective and  reproducible identification and quantification for proteomic analysis.  Here, we describe a step wise protocol for tissue specimens derived from  human/mice tissue and cell lines models. To maximize proteomic  coverage, we deploy a strong detergent (2% SDS), as well as high energy  sonication to ensure complete solubilization of tissue/cellular  proteins. We describe a facile protocol for straightforward capture of  solubilized protein samples on a S-trap column that allows removal of  SDS and other components that interfere with protease digestion. We  provide an optimized trypsin/Lys-C protease digestion protocol to  maximize protein digestion.  We also explain how to undertake tandem  mass tag labelling of digested proteins to multiplex up to 16 samples in  a single study. We also provide an notes of caution for each step of  the sample preparation as well as providing a comprehensive list of all  of the required reagents and apparatus. Using this protocol, we achieve a  high depth of proteome coverage by identifying >10,000 protein  groups reproducibly in all tissues and cells we have analyzed. We  believe our protocol can be easily adopted and implemented in the  routine large-scale proteomic analysis of cells/tissues extracts to  achieve high depth and reproducible protein analysis.</div></div>

[STEPS]
?. [Sample preparation and S-Trap Assisted Digestion]
Snap freeze mouse tissue immediately after isolation in liquid nitrogen and store at .
-80 °C
?. [Sample preparation and S-Trap Assisted Digestion]
Pulverize frozen tissue in liquid nitrogen to a fine powder and snap freeze immediately in 2 ml tubes. This is done using cell crusher kit.
Note: Maintain all Pulverizer units in liquid nitrogen including spatula. After use wash the pulveriser with a tap water and clean thoroughly several times with  .
?. [Sample preparation and S-Trap Assisted Digestion]
Weigh pulverized tissue powder on a weighing balance (5-7% of the weight should be protein i.e. ~ 5 to 7 mg protein/100 mg).
Note: Keep samples on dry ice during after weighing. Take a 1.5 ml tube, check the weight on weighing balance and subsequently tare the weight. Now take small scoop of sample using pre-chilled fine spatula to weigh the sample).
?. [Sample preparation and S-Trap Assisted Digestion]
Immediately add 10 times of the weight of the tissue of .
[(by mass) SDS lysis buffer]
Note: For   please add  .
?. [Sample preparation and S-Trap Assisted Digestion]
Put samples on an orbital shaker in cold room at .
33
?. [Sample preparation and S-Trap Assisted Digestion]
Boil samples at  for  and allow to cool.
95 °C
?. [Sample preparation and S-Trap Assisted Digestion]
Sonicate samples using a Diagenode Bioruptor (use it at high energy for 10 cycles (30Sec-ON/30Sec-Off).
?. [Sample preparation and S-Trap Assisted Digestion]
Clarify lysate by centrifuging at .
Centrifuge: 20800 34, 30 min, 4 10
?. [Sample preparation and S-Trap Assisted Digestion]
Measure protein amount using the Bicinchoninic acid assay (BCA) method in triplicate at 1 to 10 and 1 to 20 dilutions - repeat analysis if readings are not close.
?. [Sample preparation and S-Trap Assisted Digestion]
Take  for total proteomic analysis.
[protein]
?. [Sample preparation and S-Trap Assisted Digestion]
Perform reduction by adding a 1 in 10 dilution of a solution of  dissolved in  to bring final concentration of TCEP to .
[TCEP]
[TEABC]
?. [Sample preparation and S-Trap Assisted Digestion]
Incubate on a Thermomixer for  at  with a gentle agitation at .
60 °C
23 33
?. [Sample preparation and S-Trap Assisted Digestion]
Bring tubes to  and add a one in 10 dilution of freshly prepared  dissolved in water.
0 Room temperature
[iodoacetamide]
Note: It is critical that the samples are at   prior to addition of iodoacetamide.
?. [Sample preparation and S-Trap Assisted Digestion]
Incubate in dark on a Thermomixer at  for about  with a gentle agitation .
0 Room temperature
23 33
?. [Sample preparation and S-Trap Assisted Digestion]
Quench alkylation by addition of a 1 in 10 dilution of  dissolved in  to bring final concentration of TCEP to .
[TCEP]
[TEABC]
?. [Sample preparation and S-Trap Assisted Digestion]
Incubate on a Thermomixer for  at  with a gentle agitation .
0 Room temperature
33
?. [Sample preparation and S-Trap Assisted Digestion]
Add SDS to a final concentration of  from .
[(by mass)]
[ (by mass) SDS stock]
Note: The lysate is already in   so supplement with a stock of   in order to bring the final SDS concentration to  . A final concentration of 5% SDS is recommended for complete soulblization of proteins for S-Trap assisted protocol.
?. [Sample preparation and S-Trap Assisted Digestion]
Add a 1 in 10 dilution of  into the sample to make a final concentration of ~.
[(by vol) phosphoric acid]
[(by vol) phosphoric acid]
Note: For example,   to  .
?. [Sample preparation and S-Trap Assisted Digestion]
Dilute the sample to in 7 times the current volume of the mixture in of S-Trap wash buffer ( in  v/v) (for examples if sample volume is  add  (( in v/v)).
[(by vol) methanol]
[TEAB]
50 µl
[S-Trap wash buffer ]
[(by vol) methanol]
[TEAB ]
Note: If the lysate volume is now more than  , scale S-Trap wash buffer accordingly i.e. 7 times the volume of the lysate.
?. [Sample preparation and S-Trap Assisted Digestion]
Prepare an S-Trap mini column in a 2 ml tube.
?. [Sample preparation and S-Trap Assisted Digestion]
Add the diluted protein mixture to the column.
?. [Sample preparation and S-Trap Assisted Digestion]
Centrifuge briefly to capture the protein particles.
Centrifuge: 1000 34, 23 10
Note: All subsequent wash steps needs to be done at room temperature (23oC). If you observe clogging or if the sample doesn't pass through the column then increase centrifugation speed up to a maximum of 4,000g.
?. [Sample preparation and S-Trap Assisted Digestion]
Wash column with  a total of 4 times (spin  between washes).
[S-Trap buffer]
Centrifuge: 1000 34, 1 min
Note: The protein remains bound on the column and SDS and buffer components that effect trypsin digestion are removed.
?. [Sample preparation and S-Trap Assisted Digestion]
Move the S-Trap column to a clean 2 ml tube for digestion.
?. [Sample preparation and S-Trap Assisted Digestion]
Add a  containing  and  freshly dissolved in  (1:15).
[solution of freshly dissolved trypsin]
[trypsin]
[Lys-C]
[TEAB]
Note: We use   and   per  .
?. [Sample preparation and S-Trap Assisted Digestion]
Centrifuge briefly at .
Centrifuge: 200 34, 1 min
?. [Sample preparation and S-Trap Assisted Digestion]
Collect flowthrough and reapply the trypsin solution back onto the column being careful to avoid air bubbles.
?. [Sample preparation and S-Trap Assisted Digestion]
Cap the tubes and incubate at  without shaking for .
47 °C
Note: Do not shake as this causes bubbles and damages the column.
?. [Sample preparation and S-Trap Assisted Digestion]
Incubate for another  at .
0 Room temperature
?. [Sample preparation and S-Trap Assisted Digestion]
Add  then spin to elute and place the eluate in a new 1.5 ml Eppendorf tube termed "eluate tube".
[50 mM TEAB]
?. [Sample preparation and S-Trap Assisted Digestion]
Next, add  and spin to elute. Also add this eluate to the "eluate tube".
[0.15% (by vol) Formic Acid]
?. [Sample preparation and S-Trap Assisted Digestion]
Finally, add  in  and spin to elute. Also add this eluate to the "eluate tube".
[50% (by vol) Acetonitrile]
[(by vol) formic acid]
Note: 3 eluates should have been added to the eluate tube.
?. [Sample preparation and S-Trap Assisted Digestion]
Take  - , vacuum dry and inject on MS to verify the digestion efficiency.
1 µl
[combined eluate]
Note: Analyse data with a   gradient run on QE HF-X or Orbitrap Lumos mass spectrometer in a FT-FT-HCD mode. Search data with Proteome Discoverer 2.1 or 2.4 version. Determine the digestion efficiency by plotting number of missed cleavages. Zero missed cleavages should be >75% and single missed cleavages should be between 20-23%.
?. [Sample preparation and S-Trap Assisted Digestion]
Vacuum dry the remaining peptide amount and store in  deep freezer until ready to undertake TMT labelling.
-80 °C
?. [Tandem Mass Tags Labelling]
Dissolve  within the 11 or 16-plex TMT reagent kit with  to obtain a  concentration for each TMT reporter tag.
[of each of the TMT mass tag reagents]
[100% by vol anhydrous acetonitrile]
?. [Tandem Mass Tags Labelling]
Leave them at  for . Following, vortex and briefly spin .
0 Room temperature
Centrifuge: 2000 34, 2 min
Note: Dissolved TMT reagents are prone to hydrolysis so immediately after aliquoting store remainder reagent in   deep freezer for long-term storage up to six months and try to avoid multiple freeze thaw cycles.
?. [Tandem Mass Tags Labelling]
Dissolve lyophilized peptides in  of a mixture containing  + .
50 µl
[50 mM TEAB buffer]
[100% (by vol) anhydrous acetonitrile]
Note: It is important to maintain a final   for an effective TMT reaction.
?. [Tandem Mass Tags Labelling]
Keep samples on a floater and place it on a water bath sonicator for .
?. [Tandem Mass Tags Labelling]
Centrifuge samples at high speed .
Centrifuge: 20800 34, 10 min
?. [Tandem Mass Tags Labelling]
Transfer dissolved peptides into a 1.5 ml protein low-binding eppendorf tube.
?. [Tandem Mass Tags Labelling]
Add  i.e.  aiming for a 1:1 mass ratio of peptide:TMT reagent.
[20 µg/µl TMT reagent]
200 µg
?. [Tandem Mass Tags Labelling]
Give a gentle vortex and brief spin .
Centrifuge: 2000 34, 1 min
?. [Tandem Mass Tags Labelling]
Place samples on a Thermomixer and incubate with a gentle agitation at .
33
?. [Tandem Mass Tags Labelling]
Add another  to make a final  reaction. Vortex, brief spin at  and incubate on a Thermomixer for .
[50 mM TEAB buffer]
100 µl
Centrifuge: 2000 34, 1 min
Note: It is a good practice to maintain the total volume to   final reaction as it helps in reducing pipetting error when aliquoting   of sample for label check efficiency).
?. [Tandem Mass Tags Labelling]
In order to verify the TMT labelling efficiency of each TMT mass tag, take a  from each of the TMT samples and pool this in a single tube and vacuum dry immediately using a SpeedVac.
[aliquot]
Note: It is important to verify the labelling efficiency of each TMT mass tag and it should label > 98%, by analysing on Mass spec. We recommend doing this employing a   FT-FT-MS2 study. This will establish that each reporter tag is efficiently labelled and ensure that an equal level of each peptide is labelled with each of the TMT tags. Search MS raw data with Proteome Discoverer 2.2 or 2.4 by enabling TMTreporter tag mass (+229.163 Da) on Lysine residue and Peptide N-terminus as dynamic modifications. Filter TMT labelled Peptide spectral matches (PSMs) in the modification tab to calculate the number of labelled and unlabelled PSMs to determine the labelling efficiency. Also, export PSM abundance in txt.file, to plot a Boxplot using R-software to determine the ~1:1 abundance within and between replicates.
?. [Tandem Mass Tags Labelling]
Place remaining  of the reaction in  freezer. If the labelling efficiency is >98% and levels of each labelled peptide appear to be close to 1:1 then proceed with the below steps.
95 µl
-80 °C
?. [Tandem Mass Tags Labelling]
Thaw stored TMT labelled samples from step 46 to .
0 Room temperature
?. [Tandem Mass Tags Labelling]
Prepare  by dissolving in water from a .
[(by vol) final Hydroxyl amine solution]
[(by vol) stock solution]
?. [Tandem Mass Tags Labelling]
Add  to each sample to quench TMT reaction by incubating the reaction at  on a Thermomixer for .
[5% (by vol) Hydroxylamine]
0 Room temperature
?. [Tandem Mass Tags Labelling]
Pool all samples into a single tube.
?. [Tandem Mass Tags Labelling]
Take 20% of the reaction i.e.  to as a backup, snap freeze on dry ice and vacuum dry.
220 µl
Note: This is important because if there is a sample loss during the downstream analysis or to further validate the findings.
?. [Tandem Mass Tags Labelling]
Snap freeze the remaining  and vacuum dry using Speed vac, for high pH fractionation.
[reaction]
?. [Tandem Mass Tags Labelling]
Desalt sample using tC18 50 mg cartridge by following the protocol described .
[73]
?. [Tandem Mass Tags Labelling]
After following C18 clean-up protocol, submit desalted sample to MS facility for high pH fractionation and request to fractionate to 96 fractions and concatenate by pooling distant fractions e.g A1+D1, A2+D2.. B1+E1, B2+E2 and so on to a total of 48 fractions for LC-MS/MS analysis.
Note: Deep fractionation significantly reduces ion interference and ratio distortion caused by co-elution of peptides in turn and improves TMT-reporter ion quantification thus increasing the reliability of peptide and protein quantifications. Also, deep fractionation greatly improves the dynamic range of peptides/proteins and increases the coverage. We generally identify between 10,000 to 12,000 quantified protein groups.
?. [Tandem Mass Tags Labelling]
For each of the 48 fractions measure peptide concentrations using a Nanodrop 1000 spectrophotometer.
?. [Tandem Mass Tags Labelling]
Prepare  in  and submit each fraction to the mass spectrometry facility.
[of each fraction]
[LC buffer (0.1% (by vol) formic acid in 3% (by vol) Acetonitrile)]
?. [Tandem Mass Tags Labelling]
Analyse each fraction by acquiring data in FT-FT-FT (MS3) HCD mode on a Orbitrap Fusion Lumos Mass spectrometer for  run for each fraction.
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Prepare single layer of C18 stage-tip using 16-gauge syringe needle.
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Dissolve the vacuum dried peptides sample derived from  in .
[ solvent-A1 (0.1% (by vol) TFA)]
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Add  to the C18 stage-tip.
[100% (by vol) Acetonitrile]
Note: This is required to activate the C18 resin.
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Centrifuge at . Discard flow through.
Centrifuge: 2000 34, 2 min
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Add .
[Solvent-A1 (0.1% (by vol) TFA)]
This is required to equilibrate the C18 resin.
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Centrifuge at . Discard flow-through and repeat this step.
Centrifuge: 2000 34, 2 min
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Load the acidified peptide digest to Stage-tip.
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Centrifuge at .
Centrifuge: 1500 34, 5 min
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Reapply the flow through to the C18 stage-tip column and repeat this step.
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Add , and centrifuge at . Discard flow through. Repeat again.
[solvent-A1 (0.1% (by vol) TFA v/v)]
Centrifuge: 2000 34, 2 min
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Elute peptides absorbed to C18 column by placing the stage-tips into new 1.5 ml low binding tubes.
Note: Using new tubes is important to avoid contamination.
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Add .
[Elution buffer (Solvent-B1 50% (by vol) acetonitrile in 0.1% (by vol) TFA)]
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Centrifuge at . Add another  and repeat the centrifugation.
Centrifuge: 1500 33, 2 min
[Elution buffer (Solvent-B1 50% (by vol) acetonitrile in 0.1% (by vol) TFA)]
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Immediately snap freeze on dry ice the eluates and vacuum dry the samples using a SpeedVac.
?. [C18 Stage-tip Protocol to desalt TMT labelled samples prior to MS analysis-Important step before final pooling of samples]
Submit to Mass spectrometry analysis as described in .
?. [Sep-Pak Purification Protocol]
Dissolve vacuum dried pooled TMT sample from  in .
[solvent-A1 (0.1% (by vol) TFA)]
?. [Sep-Pak Purification Protocol]
Place sample on a floater and place it in on a water bath sonicator and sonicate it for .
?. [Sep-Pak Purification Protocol]
Centrifuge sample at  at room temperature and transfer sample into a new 1.5 ml Eppendorf tube.
Centrifuge: 20800 34, 10 min
?. [Sep-Pak Purification Protocol]
Check that the pH of the sample is acid >2, by pipetting  onto a pH strip.
[sample]
Note: This is important because Stage-tips are equilibrated to acidic pH and this aid in proper binding of acidified peptides to C18 resin, if sample appears to be not acidic increase TFA concentration to   and recheck pH.
?. [Sep-Pak Purification Protocol]
Prepare single tC18 50 mg Sep-Pak cartridge and place this column in 15 ml falcon tube.
?. [Sep-Pak Purification Protocol]
Add . Centrifuge on an Eppendorf centrifuge at  and discard flowthrough. Repeat this step again.
[100% (by vol) Acetonitrile]
Centrifuge: 60 34, 1 min
Note: This step is required to activate the C18 resin.
?. [Sep-Pak Purification Protocol]
Equilibrate the tC18 50 mg Sep-Pak cartridge, by adding . Centrifuge on an Eppendorf centrifuge at  and discard flowthrough. Repeat this step two more times.
[solvent-A1 (0.1% (by vol) TFA v/v)]
Centrifuge: 60 34, 1 min
?. [Sep-Pak Purification Protocol]
Load acidified sample onto the tC18 50 mg Sep-Pak cartridge.
Note: Do not centrifuge and let the sample pass through by gravity or if needed push sample using rubber bulb (Fisher brand™ Rubber Pipette Bulb #12446180).
?. [Sep-Pak Purification Protocol]
Collect the flow through and reapply to the column and save the flow through.
Note: If you don’t detect any peptides in MS, the flow through should have the peptides and this has been caused due to poor equilibration of the column.
?. [Sep-Pak Purification Protocol]
Wash the tC18 50 mg Sep-Pak cartridge by addition of . Centrifuge on an Eppendorf centrifuge at  and discard flowthrough. Repeat this step two more times.
[solvent-A2 (0.1% (by vol) Formic acid)]
Centrifuge: 60 34, 1 min
?. [Sep-Pak Purification Protocol]
Place column on a new 1.5 ml Eppendorf tube.
Note: Using new tubes is important to avoid contamination.
?. [Sep-Pak Purification Protocol]
Elute the peptides from the tC18 50 mg Sep-Pak cartridge by addition of .
[solventB2 (60% (by vol) Acetonitrile in 0.1% (by vol) Formic acid)]
Note: Do not centrifuge and let the sample pass through by gravity or if needed push sample using rubber bulb (Fisher brand™ Rubber Pipette Bulb #12446180).
?. [Sep-Pak Purification Protocol]
Repeat elution step (step 84): Elute the peptides from the tC18 50 mg Sep-Pak cartridge by addition of .
[solventB2 (60% (by vol) Acetonitrile in 0.1% (by vol) Formic acid)]
Note: Do not centrifuge and let the sample pass through by gravity or if needed push sample using rubber bulb (Fisher brand™ Rubber Pipette Bulb #12446180).
?. [Sep-Pak Purification Protocol]
Repeat elution step (step 84):Elute the peptides from the tC18 50 mg Sep-Pak cartridge by addition of .
[solventB2 (60% (by vol) Acetonitrile in 0.1% (by vol) Formic acid)]
Note: Do not centrifuge and let the sample pass through by gravity or if needed push sample using rubber bulb (Fisher brand™ Rubber Pipette Bulb #12446180).
?. [Sep-Pak Purification Protocol]
Combine the  and snap freeze and vacuum dry using SpeedVac.
[eluate]
?. [Sep-Pak Purification Protocol]
Store sample in  freezer or submit to mass spectrometry facility for high pH fractionation as described in .
-80 °C