TITLE: High-Throughput Stool Metaproteome Extraction
AUTHORS: Carlos Gonzalez, Josh Elias

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The gut microbiome is strongly associated various disease related periods. In order to better understand how the host and microbiome interact during these periods, it is necessary to survey the proteomic response of both the host and microbes in a large portion of a population. However to date, techniques used to isolate stool proteins have been low-throughput and due to their lack of purity can foul columns and increase mass spectrometer downtime. To address these shortcomings, this protocol was developed. It allows for the processing of 96 samples in parallel and is compatible with common multiplexing labeling technology. It has thus far been used to process anywhere between 10 and 400 samples, with very reproducible results.</div></div>

[STEPS]
?. [Lysis and solubilization]
Spin down bead beater plates for 1 minute to pull beads into well at  . Aliquot approximately   of stool into 96 well beads plate. This protocol will work with less stool at the risk of increasing preparative replicate bias. The samples should be arrayed in a logical way, especially if the end goal is to use multiplexing reagents. Make sure samples are pushed to the bottom of the plate or the lysis buffer may overflow into neighboring wells.
Centrifuge: 1000 34
200 mg
?. [Lysis and solubilization]
To each sample, add   750 uL of lysis buffer:UreaTris-BaseSDSUse a multichannel pipette or get carpal tunnel syndrome instantly.
750 µl
?. [Lysis and solubilization]
Seal plate with provided top. Plate the sealed plate into 96-well bead beater. 20 Hz for  . Spin down samples in plate centrifuge at  for  at  .
Centrifuge: 3000 34
4 °C
?. [Lysis and solubilization]
Observe the samples and make sure they are sufficiently homogenated/solubilized. Aliquot   of the homogenated sample into a new Waters 2 mL deep well plate. Spin the new plate for   at and take supernatant into new 2 mL Waters plate, this will be the sample stock.
500 µl
Centrifuge: 3000 34
?. [Lysis and solubilization]
To each well, add 10 uL   DTT. Reseal with same mat. Place samples in incubator at  for
47 °C
?. [Lysis and solubilization]
Remove plates from incubator and let cool to room temperature. Spin plates down for  at   After plate has cooled, remove sealing mat and add  of  iodoacetamide. Place into totally dark environment (e.g. closed drawer) for
Centrifuge: 1000 34
30 µl
?. [Protifi Plate Digestion]
Aliquot   of sample into new Thermo 1.2 mL plate (Waters plate works as well but is harder to pipette from). Add   of   phosphoric acid. Add  Protifi Sample Binding Buffer:  methanol  triethylammonium bicarbonate (TEAB)Mix each sample by pipetting up and down for  .
50 µl
5 µl
300 µl
You will often see a cloudy colloidal suspension after adding the binding buffer.
?. [Protifi Plate Digestion]
Add the mixture from step 7 to the Protifi S-trap 96-well plate. Place S-trap on top of Waters 96 well 2 mL plate. This will be waste plate. Use the positive pressure machine to press sample through. Our experience suggests that 6 psi on the main dial and 15 on the secondary control dial will push through a majority of samples. There will be clogging and this can be corrected by gently scraping the top of the well with a pipette tip.
?. [Protifi Plate Digestion]
Wash samples with  fresh Protifi Binding Buffer 5 times. You may have a few clogs at this point as well, clear them as before. Be sure to empty waste plate as needed. After washes are done, place S-trap plate on fresh 1.2 mL Thermo plate.
500 µl
?. [Protifi Plate Digestion]
To each well, add  of trypsin dissolved in  of TEAB. Place Protifi S-trap lid back on plate (should be loose fitting). Place in incubator for  . After  have passed, check that digestion mixutre has been taken up by wells. If it is still sitting on top, place 96 well plate and it's collection plate into the positive pressure machine for approximately and check if liquid drained into collection plate. If any liquid came out (it is normal that it does), place back into appropriate well and continue with digestion.
5 µg
125 µl
?. [Lysis and solubilization]
Add 3  benzonase to each sample and place in incubator at  for
3 µl
37 °C
?. [Protifi Plate Digestion]
After digestion, elute peptides using positive pressure. Once initial digestion volume has been eluted, add   of TEAB and use positive pressure to wash directly into previously eluted peptides. Repeat this with  of formic acid (FA), then   of a mixture of  acetonitrile and   FA. This should be approximately  of digested peptides and wash. Speedvac to dryness.
100 µl
100 µl
100 µl
425 µl
?. [RPS or C18 cleanup]
Resuspend the dry peptides in   of FA. Pipetting up and down as well as washing well walls is critical in getting maximum peptide recovery. Spin plate at  for  . Carefully avoiding any debris left in the well, move peptide suspension into fresh plate.
150 µl
Centrifuge: 3000 34
?. [RPS or C18 cleanup]
Consult ownder of Assaymap Bravo to use Peptide Cleanup V2 app using either RPS or C18 tips. These tips have a capacity of approximately   but you will likely lose quite a bit of it during this step. Dry down samples in speedvac
100 µg
?. [RPS or C18 cleanup]
Resuspend dried down peptides in   of  FA.
30 µl
?. [RPS or C18 cleanup]
If samples will not be labeled (eg TMT, see below) use nanodrop to adjust concentration that is appropriate for your mass spectrometer (usually  to is sufficient for nano-flow LC systems.
0.5 µg
1 µg
?. [OPTIONAL - TMT labeling]
From step 15, nanodrop samples and record concentration. If everything was properly done and you started with enough sample, each well should have upwards of of unlabeled peptide. We will use  of this for TMT labeling. Resuspend each well at a concentration of  per  . Transfer  of peptide from each well into a new plate. Dry down both plates. Leave your original plate dryand store in  .
40 µg
20 µg
0.5 µg
1 µl
20 µg
-20 °C
?. [OPTIONAL - TMT labeling]
Resuspend  plate in   of  Na-HEPES at  (should be around that pH already). Move peptide suspension into fresh Eppendorf 150 uL plate
20 µg
50 µl
?. [OPTIONAL - TMT labeling]
Take TMT kit out of freezer and let warm to room temperature for approximately , do not open lids to avoid condensation . Spin down each reagent tube to collect residue on top on benchtop micro centrifuge. For each sample to be labeled with a specific channel, add   of acetonitrile (ACN). EXAMPLE: if you have a single 96 well plate and a TMT-11plex, and every sample in column 1 will be labeled with channel 126, add (n+1)*20 uL of ACN, where n is the number of samples in column 1. After each reagent has been resuspended, close lid and quickly vortex then wait  for reagent to completely dissolve, with another quick vortex  . After reagent is dissolved, spin down and aliquot into fresh 96 well plate first row (up to TMT-11), you will use a multichannel in next step to redistribute into each sample.
20 µl
[into 5 minute period]
?. [OPTIONAL - TMT labeling]
From the concentrations obtained in  , calculate the ratio of TMT reagent to peptide. Thermo suggests that you have at least 8:1 TMT to peptide, however YMMV. For the example below our ratio was approximately 6.5:1 TMT to peptide.
Kuster paper showing TMT ratio as low as 1:1 if concentrations are kept at least 4ug/uL.:https://www.mcponline.org/content/early/2019/04/09/mcp.TIR119.001385
?. [OPTIONAL - TMT labeling]
With a multichannel pipette,  carefully redistribute   of the appropriate TMT reagent into each well. Cover loosely with Axygen silicone cover to avoid ACN loss. Incubate for
20 µl
?. [OPTIONAL - TMT labeling]
Quench reaction with  of  hydroxylamine solution in  HEPES-Na. Incubate for 15 minutes at room temperature
10 µl
?. [OPTIONAL - TMT labeling]
Acidify each well down to approximately  with   formic acid. This should be done on a dummy sample with no peptides in it. In general this may be up to  .
15 µl
?. [OPTIONAL - TMT labeling]
Resuspend sample in   FA and check concentration on nanodrop. Dilute to 0.5 ug/uL. Bottle up samples and shoot on mass spec. Quant ratios
10 µl
?. [OPTIONAL - TMT labeling]
You now need to construct your ratio check samples. Each ratio check should have a total of   peptide. This means that each channel needs to contribute equally to that amount so the total taken from each well will depend on what TMT kit is used. For example if a TMT-11 kit is used, each sample/channel would contribute 10 ug/11 = 0.9 ug. Transfer appropriate amount from each sample/channel into its ratio check tubeIf you are doing a bridge channel, each bridge will need a total of 20 ug so make sure you calculate that too.
10 µg
?. [OPTIONAL - TMT labeling]
Run resulting raw files through Proteome Discoverer 2.2. Calculate ratios from each channel using PSM quant values. Use these ratio values to adjust each channel on original plate for a 1:1 ratio. Compile new sample and reshoot.