TITLE: Automated, Rapid Preparation of Tissue Sections for Proteomic Analysis
AUTHORS: Jamie Allen, Jeff Spraggins, Danielle Gutierrez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">To describe the procedure for the lysis, reduction/alkylation, trypsin digestion, and clean-up of tissue.  Lysis will cover the lysing of tissue and protein concentration.  Acetone precipitation will cover the precipitation of proteins.  Digestion will cover the process for digesting 100 µg of protein using the Agilent AssayMap Bravo Robot and Promega Rapid Trypsin/LysC.  The clean-up of the cells will cover the desalting process on the Agilent AssayMap Bravo and the subsequent preparation of the samples for LC/MS peptide analysis.</div><div class = "text-block">Expected Outcome/Data:</div><div class = "text-block">Cell samples lysed, digested, and desalted for analysis on MS instrument.  Samples to be analyzed within one or two days of desalting.</div></div>

[STEPS]
?. [Lysis/Concentration Assay]
Place 5-10 sections of tissue in an Eppendorf tube and keep on ice.
?. Add  lysis buffer to tubes and vortex for 30-60 seconds.
200 µl
?. Place tubes in dry ice for
?. Defrost tubes on wet ice for and then vortex briefly.
?. Add ice to water in the sonicator to make an icy slurry.
?. Sonicate samples in ice bath for  and vortex.
?. Spin tubes in microcentrifuge for  at 14000rpm.
?. Pipet supernatant into new labeled Eppendorf tube.  Discard pelleted tissue.
?. Determine protein concentration of samples via Pierce BCA Protein Assay kit:Prepare BSA standard curve with lysis buffer following BCA kit instructions.Pipette   of standards into the “curve” wells in a clear flat bottom plate.Pipette   of lysis buffer into the sample wells.Pipette   of sample into each sample well and mix 5x.Prepare working reagent as instructed in BCA protocol.Add   working reagent to each curve/sample well.Incubate samples for   at  .Add template to Softmax Pro during   incubation, with 5x dilution for samples.Read plate at an absorbance of 562 nm.Export results into BCA excel workbook to determine volume for 100ug of protein for the precipitation.Pipette  of standards into the “curve” wells in a clear flat bottom plate.Pipette  of lysis buffer into the sample wells.Pipette  of sample into each sample well and mix 5x.Prepare working reagent as instructed in BCA protocol.Add   working reagent to each curve/sample well.Incubate samples for   at  .Add template to Softmax Pro during   incubation, with 5x dilution for samples.Read plate at an absorbance of 562 nm.Export results into BCA excel workbook to determine volume for 100ug of protein for the precipitation.Add  working reagent to each curve/sample well.Incubate samples for  at .Add template to Softmax Pro during  incubation, with 5x dilution for samples.Read plate at an absorbance of 562 nm.Export results into BCA excel workbook to determine volume for 100ug of protein for the precipitation.
25 µl
20 µl
5 µl
200 µl
37 °C
?. [Acetone Precipitation]
Add  of protein sample to 1.2mL square Abgene plate.
100 µg
?. [Acetone Precipitation]
Add lysis buffer to the sample to equal .
100 µl
?. [Acetone Precipitation]
Add  ice cold 75:25 acetone:methanol to the sample.
300 µl
?. [Acetone Precipitation]
Seal plate with sealing mat and agitate gently by hand.  Incubate for  at .  Alternatively, incubate overnight at .  Place plate rotor in cold centrifuge at .
-80 °C
-20 °C
4 °C
?. [Acetone Precipitation]
Remove plate from freezer and centrifuge samples for  at 4000 RPM.  When removing from centrifuge, place on ice or cold block to prevent pellet from dislodging.
?. [Acetone Precipitation]
Add  of ice cold acetone to all samples and spin for  at 4000 RPM.
300 µl
?. [Acetone Precipitation]
Remove and discard supernatent.  Briefly allow residual acetone to evaporate from the plate at room temperature.  The drying should only be as long as it takes to get TFE and Tris ready to add.  Do not over-dry the pellet, or it may not dissolve properly.
?. [100ug Digestion]
Prepare (items) for use on Bravo:1.    Prepare 100nM TCEP (Make the following for each column used on Bravo)       Add  of 0.5M TCEP +  of Rapid Trypsin Digestion Buffer       Add  to each well in a Greiner clear U bottom plate2.    Prepare 200mM IAA (Make the following for each column used on Bravo)        Dilute 1 vial of pre-weighed IAA with  of Rapid Trypsin Digestion Buffer        Add  of 0.5M IAA +  of Rapid Trypsin Digestion Buffer        Add  to each well in a Greiner clear U bottom plate3.    Add  of Rapid Trypsin Digestion Buffer to each well in a 1.2mL Abgene plate.
42 µl
168 µl
25 µl
100 µl
84 µl
126 µl
25 µl
100 µl
?. [100ug Digestion]
Resuspend the 100ug pellet in  of neat TFE and  of 100mM Tris (pH 8.0).  Vortex gently or use T-shake on Bravo at max speed for .
10 µl
10 µl
?. [100ug Digestion]
Set up automation with Bravo In-Solution Digestion: Single Plate v1.0:1.    Browse for a Method:        VWorks Workspace→Methods→AM In Solution Digestion Single Plate v1.0→DARPA in Solution Digestion           (or most current digestion method) 2.    Load Values 3.    Place plates/reservoirs with lids on appropriate spots on robot deck.
?. [100ug Digestion]
Reduce with  of 100mM TCEP at room temperature for .  Bravo will pause after addition.  Place IAA plate on Bravo deck and restart.
5 µl
?. [100ug Digestion]
Akylate with  of 200mM IAA in the dark at room temperature for  (place in drawer below Bravo).  Bravo will pause after addition.
5 µl
?. [100ug Digestion]
Add  of Rapid Trypsin Digestion Buffer to sample with Bravo.
65 µl
?. [100ug Digestion]
While Bravo is adding diluent, prepare Promega Rapid Trypsin by adding  of Promega Resuspension Buffer to 1 bottle of rapid trypsin.
100 µl
?. [100ug Digestion]
Add  prepared 1ug/ul Rapid Trypsin manually to each well (1:25 enzyme:protein).
4 µl
?. [100ug Digestion]
Replace sealing mat or seal plate with PlateLoc and gently shake.
?. [100ug Digestion]
Incubate plate at  for  .
55 °C
?. [100ug Digestion]
Remove plate from incubator and pulse in centrifuge for  to move any condensation back into the wells.
?. [100ug Digestion]
Add  60% Formic Acid manually to each well.
5 µl
?. [100ug Digestion]
Seal plate with sealing mat or aluminum seal using PlateLoc and either prepare for desalting or place plate in for future use.
-80 °C
?. [Desalting Samples]
Prepare items for use on Bravo and place on plate deck.* Columns filled in 12 well reservoir plates will correspond to sample columns filled in PCR plate:Prepare Equilibration plate by adding  Equilibration buffer to wellsPrepare Priming and Syringe plate by adding  Priming and Syringe wash buffer to wells.Prepare Elution plate by adding  Elution buffer to each column. Make sure that water wash bottles are full to the top.Place C18 cartridges on tip deck corresponding to number of samples.Add  of sample to Eppendorf PCR plate (this includes 10 uL overage).Centrifuge plate at 4000g for  to pellet any debris and remove bubbles.
4.2 ml
4.2 ml
4.2 ml
30 µl
?. [Desalting Samples]
Open Peptide Cleanup v.2.0 in App Library:Set columns for the amount of samples.Load   of sample.Elute in   of sample.Run Program.Load  of sample.Elute in  of sample.
21 µl
20 µl
?. [Desalting Samples]
Place plate in SpeedVac for approximately .  Continue checking until samples are completely dried.
?. [Desalting Samples]
If finished, seal plate with aluminum seal or PCR cap strips and place in  for future use.
-80 °C
?. [Preparing Samples for Instrument - Reconstitute Dried Sample]
Label Eppendorf tubes and place vial inserts into each one.
?. [Preparing Samples for Instrument - Reconstitute Dried Sample]
Label LC autosampler vials and set aside.
?. [Preparing Samples for Instrument - Reconstitute Dried Sample]
Add  of 0.1% Formic Acid to each well, pipetting up and down 10x.  This will make a 1ug/uL solution for analysis.
20 µl
?. [Preparing Samples for Instrument - Reconstitute Dried Sample]
Pipette sample into respective vial insert/tube.
?. [Preparing Samples for Instrument - Reconstitute Dried Sample]
Briefly spin tube to remove any air bubbles in insert.
?. [Preparing Samples for Instrument - Reconstitute Dried Sample]
Use forceps to move into labeled LC vial and cap.
?. [Preparing Samples for Instrument - Reconstitute Dried Sample]
Store in  freezer until ready for instrument.
-20 °C
?. [Acetone Precipitation]
Carefully remove and discard supernatent.