Preparation of Tissue Sections for Proteomic Analysis

Begin with tissue cryosections in eppendorf tubes. Add 100µL-500µL lysis buffer to tissue.

Place tubes in dry ice for 0h 5m 0s

Defrost tubes on wet ice for0h 5m 0s and then vortex briefly.

Add ice to water in the sonicator to make an icy slurry.

Sonicate samples in ice bath for 0h 10m 0s and vortex.

Spin tubes in microcentrifuge for 0h 5m 0s at 14000 rpm.

Pipet supernatant into new labeled Eppendorf tube. Discard pelleted tissue.

Determine protein concentration of samples via Pierce BCA Protein Assay kit:

1. Prepare BSA standard curve with lysis buffer following BCA kit instructions.
2. Pipette 25µL of standards into the “curve” wells in a clear flat bottom plate.
3. Pipette 20µL of lysis buffer into the sample wells.
4. Pipette 5µL of sample into each sample well and mix 5x.
5. Prepare working reagent as instructed in BCA protocol.
6. Add 200µL working reagent to each curve/sample well.
7. Incubate samples for 0h 30m 0s at 37°C.
8. Add template to Softmax Pro during 0h 30m 0s incubation, with 5x dilution for samples.
9. Read plate at an absorbance of 562 nm.
10. Export results into BCA excel workbook to determine volume for 100ug of protein for the precipitation.

Add 100µg of protein sample to a 1.5mL labeled eppendorf tube.

Add lysis buffer to the sample to equal 100µL.

Add 300µL ice cold 75:25 acetone:methanol to the sample.

Vortex sample and incubate for 2h 0m 0s at -80°C.

Alternatively, incubate overnight at -20°C.

Place tube rotor in cold centrifuge at 4°C.

Remove tubes from freezer and centrifuge samples for 0h 15m 0s at 4000 RPM. When removing from centrifuge, place on ice or cold block to prevent pellet from dislodging.

Carefully remove and discard supernatent.

Add 300µL of ice cold acetone to all samples and spin for 0h 15m 0s at 4000 RPM.

Remove and discard supernatent. Briefly allow residual acetone to evaporate from the tubes 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.

Resuspend the 100 µg pellet in 10µL of neat TFE and 10µL of 100mM Tris (pH 8.0). Vortex to dissolve pellet.

Reduce samples with 1µL premade TCEP (0.5M) at room temperature for 0h 30m 0s .

Prepare 0.5M IAA:

Dilute 1 vial of pre-weighed IAA with 100µL of Rapid Trypsin Digestion Buffer.

Keep IAA in a drawer (light sensitive).

Alkylate samples with by adding 2µL of 0.5M IAA to each sample.

Incubate in a dark drawer at room temperature for 0h 30m 0s .

Add 73µL of Rapid Trypsin Buffer to each sample.

Prepare Promega Rapid Trypsin:

Add 100µL of Promega Resuspension Buffer to 1 bottle of rapid trypsin.

Add 4µL prepared 1ug/ul Rapid Trypsin to each sample (1:25 enzyme:protein).

Gently vortex each sample.

Incubate plate at 55°C for 0h 45m 0s .

Remove samples from incubator and pulse in centrifuge to to push any condensation out of the cap.

Add 5µL 60% Formic Acid to each well sample to stop the digestion.

Place samples on ice to prepare for desalting or place them in -80°C for future use.

Prepare tip soaking plate by placing 100µL of 1-propanol into the wells of a 96 well plate with MTP adaptor. Only fill the wells that tips will be placed in.

Prepare tip rack by only placing the amount you need for digested samples and quality control samples.

Wash

Add 20µL of Solvent B to each tip.

Place the Evotips box in a centrifuge with appropriate counter balance.

Centrifuge at 700g for 700x g,0h 0m 0s for 0h 1m 0s.

Condition

Place Evotip adaptor rack on top of the MTP plate with 1-propanol.

Soak for 0h 1m 0s.

Inspect the tips to make sure that all tips are pale white. If not, soak longer.

Equilibrate

Add 20µL of Solvent A to each tip.

Place the Evotips box in a centrifuge with appropriate counter balance.

Centrifuge at 700g for 700x g,0h 0m 0s for 0h 1m 0s.

Sample Load

Add of sample or control to each tip, for a total of 20µL .

For samples, add 500ng of digested sample.

For controls, add 100ng of HeLa digest.

Place the Evotips box in a centrifuge with appropriate counter balance.

Centrifuge at 700g for 700x g,0h 0m 0s for 0h 1m 0s.

Wash

Add 20µL of Solvent A to each tip.

Place the Evotips box in a centrifuge with appropriate counter balance.

Centrifuge at 700g for 700x g,0h 0m 0s for 0h 1m 0s.

Preservation

Add 100µL of Solvent A to each tip.

Place the Evotips box in a centrifuge with appropriate counter balance.

Pulse the centrifuge at 700g for 700x g,0h 0m 0s for 0h 0m 10s.

This will keep the tips wet.

Samples are now ready to for LCMS analysis.