RapidAIM 2.0: a high-throughput assay to study functional response of human gut microbiome to xenobiotics

Aim: Our gut microbiome has its own functionalities which can be modulated by various xenobiotic and biotic components. The development and application of a high-throughput functional screening approach of individual gut microbiomes accelerates drug discovery and our understanding of microbiome-drug interactions. We previously developed the rapid assay of individual microbiome (RapidAIM), which combined an optimized culturing model with metaproteomics to study gut microbiome responses to xenobiotics. In this study, we aim to incorporate automation and multiplexing techniques into RapidAIM to develop a high-throughput protocol. Methods: To develop a 2.0 version of RapidAIM, we automated the protein analysis protocol, and introduced a tandem mass tag (TMT) multiplexing technique. To demonstrate the typical outcome of the protocol, we used RapidAIM 2.0 to evaluate the effect of prebiotic kestose on ex vivo individual human gut microbiomes biobanked with five different workflows. Results: We describe the protocol of RapidAIM 2.0 with extensive details on stool sample collection, biobanking, in vitro culturing and stimulation, sample processing, metaproteomics measurement, and data analysis. The analysis depth of 5,014 ± 142 protein groups per multiplexed sample was achieved. A test on five biobanking methods using RapidAIM 2.0 showed the minimal effect of sample processing on live microbiota functional responses to kestose. Conclusions: Depth and reproducibility of RapidAIM 2.0 are comparable to previous manual label-free metaproteomic analyses. In the meantime, the protocol realizes culturing and sample preparation of 320 samples in six days, opening the door to extensively understanding the effects of xenobiotic and biotic factors on our internal ecology.


Fecal Sample Collection
o CAUTION: Institutional ethical approval must be obtained ensuring all samples are obtained with informed written consent and in accordance with relevant guidelines.
o CAUTION: Human feces is a level 2 biohazard (risk group 2) that can contain pathogens such as bacteria, viruses, and parasites.Immunizations for those handling these samples may be available against some pathogens such as hepatitis.
Personal protective equipment includes googles, gloves and lab coat should be worn when handling feces.Biosafety cabinets and centrifuges with lids to contain aerosols and spills should be used.Samples, equipment, and waste materials need to be handled, decontaminated, and disposed of according to institutional biosafety guidelines.
o ADDITIONAL CONSIDERATIONS: De-identification of samples should be ensured by the study coordinator.Participants should be provided anonymity when provided kits for feces collection and for return of completed kits.Will your study require collection of dietary information and identification of feces consistency (e.g., Bristol stool sample identification chart)?

Materials and Reagents
Fecal sample collection kit -On site  A flow chart of all kit components with instructions for completion  Study specific documents (e.g., 24-hours dietary recall and Bristol stool identification chart)  Two leak-proof sterile return collection containers (e.g., Thermo Scientific Samco Wide Mouth Bio-Tight Specimen Container, Fisher Scientific cat.no.13-711-56) o Note: Container size should reflect the amount of feces needed for down-stream applications or storage.We mark a with a black to-fill line.Pre-weight dry collection container to calculate weight of feces following collection.Add label to be filled by participant with time and date of collection.Check that container lid is tight to ensure sample remains anaerobic.10.Fecal samples should be processed to a 20% (w/v) slurry in sterile 1X PBS (pH 7.4) and 1 mg/mL L-Cysteine for immediate RapiAIM or to a 20% (w/v) slurry in sterile 1X PBS (pH 7.4) containing 10% (v/v) glycerol (MilliporeSigma cat.no.G5516) and 1 mg/mL L-Cysteine for aliquoting and storage at -80 °C according to Method for Fecal Processing for RapidAIM and Storage in Supplementary Methods 2.

Fecal Sample Collection -Off Site
o CAUTION: Human feces is a level 2 biological reagent.Follow any regulations and requirements for proper packaging and shipment of level 2 biological materials.Place into an anerobic chamber with cap loosened for > 24-hours or until O2-level is < 0.5 mg/L

5.
Add 60 mL of deoxygenated feces stabilization buffer to leak-proof collection container.
Ensure cap is tightened to prevent oxygenation of buffer.
o NOTE: Feces stabilization buffer can be stored at 4 °C.Before aliquoting ensure dissolved O2-level is < 0.5 mg/L.Weigh container pre-and post-buffer addition to note weight of buffer added during collection.NOTE: For off-site collection we place a 2-week expiry date on these buffers and ask the participant request fresh buffer if sample not collected within two weeks of delivery.
Feces Sample Kit Assembly and Collection

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Assemble components listed above for sample collection kit.f.Using the scoop, collect feces from the middle of the specimen and directly put it into the dry collection container to the black fill line.
g. Add 60 mL of deoxygenated feces stabilization buffer.
h. Tightly close container.
i. Label the container with the time and date the sample was collected.j.Place specimen containers into secondary specimen bag with absorbent material and temperature recorder.
k. Throw all wastes into their garbage as appropriate.o NOTE: The oxygen level of the buffer + feces can be measured and recorded.We exclude samples from further processing if O2-level is >1 mg/L for samples collected 8-hours prior as exposure to higher O2-levels for extended periods indicated leaky containers and can affect microbiome viability/composition.Our experience shows variable O2-levels from 0.2-5 mg/L for samples collected on site or > 8 h from collection time.

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CAUTION: same as above  Sodium dodecyl sulfate (Millipore-Sigma -Sigma-Aldrich, cat.no.L3771) o CAUTION: Sodium dodecyl sulfate causes skin, eye and respiratory irritation.Use personal protective equipment. Acetone (Millipore-Sigma -Sigma-Aldrich, cat.no.179124) o CAUTION: Acetone is highly flammable liquid and vapor, causes serious eye irritation, and may cause drowsiness or dizziness.Keep away from open flames, hot surfaces and sources of ignition.Use only under a chemical fume hood.Use personal protective equipment. Acetic acid, glacial (HAc, Fisher Chemical, cat.no.A38-212) o CAUTION: Flammable liquid and vapor.Use personal protective equipment.Keep away from open flames, hot surfaces and sources of ignition.Use only under a chemical fume hood. Acetonitrile (Millipore-Sigma -Sigma-Aldrich, cat.no.34851) o CAUTION: Highly flammable and toxic.Keep away from open flames, hot surfaces and sources of ignition.Use only under a chemical fume hood.Use personal protective equipment.Ethyl alcohol, anhydrous (Commercial Alcohols, cat.no.P016EAAN) o CAUTION: Ethanol is highly flammable.Keep away from open flames, hot surfaces and sources of ignition. Formic acid (Millipore-Sigma -Sigma-Aldrich, cat.no.F0507) o CAUTION: Flammable liquid and vapor, causes severe skin burns and eye damage and toxic if inhaled.Keep away from open flames, hot surfaces and sources of ignition.Use only under a chemical fume hood.Use personal protective equipment. cOmplete™ protease inhibitor cocktail (Millipore-Sigma -Roche, cat.no.04693116001)  Dithiothreitol (Millipore-Sigma -Sigma-Aldrich, cat.no.43815)  Iodoacetamide (Millipore-Sigma -Sigma-Aldrich, cat.no.I1149)  Trypsin (Worthington Biochemical, cat.no.L5003740)  DC Protein Assay Reagents A, B and S (Bio-Rad Laboratories, cat. no.5000113, 5000114 and 5000115) o CAUTION: Reagent A causes severe skin burns and eye damage.

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NOTE: Feces stabilization buffer can be stored at 4 °C.Before aliquoting ensure dissolved O2-level is < 0.5 mg/L.Weigh container pre-and postbuffer addition to note weight of buffer added during collection.Feces Sample Kit Assembly and Collection 6. Assemble components listed above for sample collection kit. 7. Coordinator should add de-identifying number to the kit and deliver kit to participant pre-instructed on collection according to the following general flowchart for sample collection instructions.a. Wash hands thoroughly.NOTE: Gloves can be worn.b.Empty bladder completely.NOTE: The feces samples should not be contaminated by urine.c.Collect stool sample into the disposable container.NOTE: The sample must not contact the toilet water.d.Using the scoop, collect feces from the middle of the specimen and directly put it into the dry collection container to the black fill line.e. Add 60 mL of deoxygenated feces stabilization buffer.f.Tightly close container.g.Label the container with the time and date the sample was collected.h.Place in biohazard specimen transport bag with absorbent material.Seal.i. Throw all wastes into the biohazard bag and seal.j.Place specimen bag and waste bag into secondary transport box or bag.k.Wash hands thoroughly.l.Store the sample at room temperature and return within 1 hour to the study coordinator.The kit will contain a de-identifying number.m.Complete any study documents (e.g., 24 hours dietary recall form and Bristol stool identification page).Return to study coordinator.Laboratory Sample Deliver and Processing 8.The coordinator should deliver fecal sample to the laboratory.9.The fecal sample should be placed into the anerobic chamber immediately.NOTE: If sample cannot be processed immediately, sample can be placed at 4°C for up to 48-hours.

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A flow chart of all kit components with instructions for completion  Study specific documents (e.g., 24-hours dietary recall and Bristol stool identification chart) Two leak-proof sterile return collection containers (e.g., Thermo Scientific Samco Wide Mouth Bio-Tight Specimen Container, Fisher Scientific cat.no.13-711-56) o Note: Container size should reflect the amount of feces needed for down-stream applications or storage.We mark a with a black to-fill line.Pre-weight dry collection container to calculate weight of feces following collection.Add label to be filled by participant with time and date of collection. Commode for feces collection (eg: Commode Specimen Collection System; Fisher Scientific cat.no.02-544-208 or Sterile Collection Device, Therapak TM ; VWR cat.no.76230-712)  Disposable scoops for collection (e.g., Bel-Art TM Sterile Sampler Spoons; Fisher Scientific cat.no.14-429-D)  Wooden tongue depressors (Fisher Scientific cat.no.S80332)  Biohazard specimen transport bag (e.g., Fisher Scientific cat.no.22-310-094)  Absorbent material for packing of biological liquids (e.g., Therapak TM absorbent materials; Fisher Scientific cat.no.22-130-041)  Non-latex, chemically impermeable gloves  Pens and markers for completing documents  Leak proof refrigerant shippers; nontoxic and food-grade gel refriderants (Fisher Scientific cat.no.22-130-070).NOTE: We include 8 packs for large, insulated shipping box to maintain temperature at <8°C for up to 72-hours. Two boxes to send sample kit components out to participants.One box to house feces stabilization buffer to be stored at 4°C by participant and one box to house cold packs to be stored at -20°C by participant. Bubble wrap for packing (Fisher Scientific cat.no.NC0513328  Return tape (Find Tape.comLLC cat.no.JVCC RT 150/223 B)  Insulated Transport Box for refrigerated return of level 2 biological substances (e.g., Therapak TM Expanded Polystyrene Insulated Shippers; Fisher Scientific cat.no.22-130-407 Temperature recorder.NOTE: If temperature tracking is essential a recorder can be added to return package to monitor temperature throughout travel (e.g., WarmMark Fisher Scientific cat.no.22-111-029  Shipping labels including exempt human specimen shipping label Feces Stabilization Buffer -Off Site  1X phosphate buffered saline (pH 7.4; Wisent cat.no.311-010-CL)  Sodium thioglycolate (1g/L; MilliporeSigma cat.no.1066910500) or L-Cysteine (1mg/mL; MilliporeSigma cat.no.C7352)  Glycerol (10% (v/v); MilliporeSigma cat.no.G5516) Equipment  Serological pipettes and pipettor  Autoclave or Disposable Filter Units (0.20 µm; eg.; Fisher Scientific cat.no.FB12566504) Anerobic Chamber (e.g., Sheldon Maunfacturing cat.no.BAA30022)  Dissolved Oxygen Meter (e.g., Extech Instruments cat.no.DO210) Procedure Feces stabilization and storage buffer preparation 1. Prepare 1X PBS (pH 7.4) + 10% (v/v) glycerol and sterilize by autoclave or 0.20 µm vacuum filtering 2.

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Coordinator should add de-identifying number to the kit and deliver kit to participant preinstructed on collection according to the following general flow-chart for sample collection instructions.a. Upon receipt of kit, the participant is instructed to open the outer transport box.The participant should store feces stabilization buffer at 4°C in a refrigerator and box containing cold packs in their freezer.Other components can remain at room temperature.b.When participant is ready to collect feces sample, they should gather components of kit stored at room temperature, in refrigerator and in freezer.c.Wash hands thoroughly.NOTE: Gloves can be worn.d.Empty bladder completely.NOTE: The feces samples should not be contaminated by urine.e. Collect stool sample into the disposable container.NOTE: The sample must not contact the toilet water.

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Place into an anerobic chamber with cap loosened for >24-hours or until O2-level is <0.5 mg/l 3. Weigh L-Cysteine for addition to 1X PBS (pH7.4) + 10% (v/v) glycerol at 1 mg/mL.4.Mix well.oNOTE: Feces stabilization and storage buffer can be stored at 4°C.Before aliquoting ensure dissolved O2-level is < 0.5 mg/L.Anaerobic chamber Preparation 5. Bring materials and equipment into anaerobic chamber, including a. 15 mL Solid Glass beads -sterilized by autoclaving b. 1x 500 mL plastic bottles -sterile or sterilized by autoclaving c. 10x 50 mL conical centrifuge tubes -sterile or sterilized by autoclaving d. 10-and 25-mL serological pipettes and pipettor e. Wooden tongue depressors -sterilized by autoclaving f.Gauze -sterilized by autoclaving (for Processing Method 1) OR g.Sterile Centrifuge Filter Units 100 µm (for processing Method 2) Determining addition of stabilization and storage buffer for final 20% (w/v) fecal slurry 6. Amount of additional stabilization and storage buffer to be added to participant sample a. Weight of feces = (weight of feces sample + buffer + container) -buffer weightcontainer weight b.Total buffer needed for 20% fecal slurry = Weight of feces/0.20 c.Amount of additional buffer to add = total buffer needed -60 mL added by participant Processing Feces to 20% (w/v) slurry in stabilization and storage buffer 7. Place fecal sample into anaerobic chamber.

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Mix sample and buffer by vigorous shaking in original container.9.Pour sample into 500 mL container.Depending on consistency of fecal slurry, wooden tongue depressors can aid in transfer.10.Add glass beads and additional stabilization and storage buffer to make 20% (w/v) fecal slurry.Mix vigorously.Wooden tongue depressors can aid in mixing.Clarifying Fecal Slurryo NOTE: We have developed four alternative methods of clarifying fecal slurry of debris while maintaining microbiome composition.Each has pros and cons.The user can decide which method best suits their lab based upon these.o Method 1 -Gauze Filtration Pro -gauze is inexpensive; required no additional equipment Con -more time consuming o Method 2 -100 µm vacuum filtration Pro -fast-time efficient; very reproducible Con-filters ~4$/sample; requires clinical centrifuge; filter can clog requiring transfer to additional filter o Method 3 -100 µm spin tube filtration Pro -fast-time efficient; very reproducible Con-filters ~4$/sample; requires clinical centrifuge; spin tube can clog requiring transfer to additional spin tube o Method 4-100 g spin Pro -fast-time efficient; very reproducible Con-requires clinical centrifuge Method 1 -Gauze Filtration 11a.Place funnel onto 500 mL container and line funnel with 4-layers of gauze 12a.Pour a portion of fecal slurry into funnel.13a.Use wooden tongue depressor to press slurry through 14a.Repeat until all slurry is cleared through gauze; changing gauze as needed 15a.Aliquot gauze-filtered 20% (w/v) fecal slurry and store at -80 °C.Method 2 -100 µm Vacuum Filtration 11b.Aliquot fecal slurry into 50 mL centrifuge tubes 12b.Centrifuge at 100 g for 5 min 13b.Pour or pipette upper slurry into vacuum centrifuge tubes avoiding lower debris pellet and any floating debris.14b.Vacuum filter slurry through 100 µm filter.We use a manual vacuum pipettor.15b.Aliquot 100 µm vacuum-filtered 20% (w/v) fecal slurry and store at -80 °C.Method 3 -100 µm Spin Tube Filtration 11c.Aliquot 20 mL fecal slurry into spin tube containing a 100 µm filtering insert 12c.Centrifuge at 100 g for 5 min 13c.Aliquot 100 µm spin tube -filtered 20% (w/v) fecal slurry and store at -80 °C.
Weigh out 12.11 g Tris base and add 80 mL of ddH2O.While mixing on a magnetic stirrer, observe pH and slowly add HCl solution to reduce the pH to 8.0.Top up the solution to 100 mL using ddH2O and double check pH.Protein resuspension buffer6 M urea in 100 mM Tris-HCl, (pH 8) 0.25 M DTT solution Weigh 193 mg of DTT powder and add 5 mL ddH2O.Prepare freshly before use or prepare in advance and store solution at -80 o C. 0.5 M IAA solution Weigh 462 mg of IAA powder and add 5 mL ddH2O.Prepare freshly before use or prepare in advance and store solution at -80 o C.

Fecal Sample Processing for RapidAIM and for Long-term Storage at -80 °C
p. Seal outer box with return tape and call coordinator to ship back to site.Study coordinator will have return shipping labels already on shipping box.Materials and ReagentsSolid Glass beads 5mm (Fisher Scientific cat.no.11-312C)  500 mL plastic bottles (e.g., Nalgene TM Wide Mouth Packaging Bottles Leak Proof with Closure; Fisher Scientific cat.no.03-313-14E  50 mL conical centrifuge tubes (Fisher Scientific cat.no.14-959-49A)  Wooden tongue depressors (Fisher Scientific cat.no.S80332)  Gauze (Ultident cat.no.400-4122)  Funnels (Fisher Scientific cat.no.10-500)  Sterile Centifuge Filter Units 100 µm (e.g., Steriflip TM Sterile Centrifuge Filter Units; o NOTE: Ensure buffer preparation precedes sample delivery by at least 24-hours to ensure ample time for deoxygenation of storage buffer.Longer times will be necessary depending upon volume of buffer preparation.If using stored-buffers ensure they remained deoxygenated.o Make excess buffer based upon final feces storage at 20% (w/v) in storage buffer.1. Prepare 1X PBS (pH 7.4) + 10% (v/v) glycerol and sterilize by autoclave or 0.20 µm vacuum filtering.

packed desalting tip columns o
Trypsin solution100 mM Tris-HCl buffer containing 2 μg/mL trypsin, 1 mL is required per sample.Slice crosses into each cap of the Nunc™ 96 Well Caps.Cut the 20 μL filtered tips to the lower end of the filtering frits.Insert the cut 20 μL filtered tips into the opening of the well caps.Place the filter-tip-inserted well caps on a desalting plate.For each set of 96 well desalting tips, weigh 600 mg of ReproSil-Pur 120 C18-AQ, 10 µm beads and add 6,000 μL of 100% ACN.Immediately after sufficiently mixing to resuspend the C18 beads, use a 96-channel liquid handler or a multi-channel pipette to aliquot 50 μL of the resuspension into each desalting tip.20.The remainder can be kept as back-up or for label-free quantification in an LC-MS/MS.21.Use a SpeedVac with a plate adapter to dry the samples under room temperature.oPause point: Typically, dry peptides can be stored for ≤ 1 month at -20 °C or ≤ 6 months at -80 °C.