TITLE: Preparation of Tissue Sections for Proteomic Analysis
AUTHORS: Jamie Allen, Angela R.S.  Kruse, Audramjudd, Melissa Farrow, Jeff Spraggins

[DESCRIPTION]
Scope:
To describe the procedure for the lysis, reduction/alkylation, trypsin digestion, and clean-up of protein extracts from tissue sections.  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 Promega Rapid Trypsin/LysC. Clean-up will cover the desalting and sample-loading process using EvoTips to prepare the samples for LC-MS/MS proteomics analysis.

Expected Outcome/Data:
Cell samples lysed, digested, and desalted for analysis on MS instrument.  Samples to be analyzed within one or two days of desalting.

[GUIDELINES]
Definitions:
1. ACN is Acetonitrile
2. BCA is Bicinchoninic Acid Assay
3. IAA is Iodoacetamide
4. MeOH is Methyl Alcohol/Methanol
5. TCEP is Tris(2-carboxyethyl)phosphine
6. TFA is Trifluoroacetic Acid
7. TFE is Tetrafluoroethylene

[STEPS]
SECTION: Tissue Lysis
2. Place tubes in dry ice for 5 min
SECTION: Tissue Lysis
3. Defrost tubes on wet ice for5 min and then vortex briefly.

SECTION: Tissue Lysis
4. Add ice to water in the sonicator to make an icy slurry.
SECTION: Tissue Lysis
5. Sonicate samples in ice bath for 10 min and vortex. 

SECTION: Tissue Lysis
6. Spin tubes in microcentrifuge for 5 min at 14000 rpm.


SECTION: Tissue Lysis
7. Pipet supernatant into new labeled Eppendorf tube.  Discard pelleted tissue.
SECTION: Tissue Lysis
8. Determine protein concentration of samples via Pierce BCA Protein Assay kit:
Prepare BSA standard curve with lysis buffer following BCA kit instructions.
Pipette 25 µL of standards into the “curve” wells in a clear flat bottom plate.
Pipette 20 µL of lysis buffer into the sample wells.
Pipette 5 µL of sample into each sample well and mix 5x.
Prepare working reagent as instructed in BCA protocol.
Add 200 µL working reagent to each curve/sample well.
Incubate samples for 30 min at 37 °C.
Add template to Softmax Pro during 30 min 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.







SECTION: Acetone Precipitation
9. Add 100 µg of protein sample to a 1.5mL labeled eppendorf tube.

SECTION: Acetone Precipitation
10. Add lysis buffer to the sample to equal 100 µL.

SECTION: Acetone Precipitation
11. Add 300 µL ice cold 75:25 acetone:methanol to the sample.

SECTION: Acetone Precipitation
12. Vortex sample and incubate for 120 min at -80 °C.  
Alternatively, incubate overnight at -20 °C.  

Place tube rotor in cold centrifuge at 4 °C.

SECTION: Acetone Precipitation
13. Remove tubes from freezer and centrifuge samples for 15 min at 4000 RPM.  When removing from centrifuge, place on ice or cold block to prevent pellet from dislodging.

SECTION: Acetone Precipitation
14. Carefully remove and discard supernatent.
SECTION: Acetone Precipitation
15. Add 300 µL of ice cold acetone to all samples and spin for 15 min at 4000 RPM.


SECTION: Acetone Precipitation
16. 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.
SECTION: Protein Digestion
19. Prepare 0.5M IAA:
Dilute 1 vial of pre-weighed IAA with 100 µL of Rapid Trypsin Digestion Buffer.
Keep IAA in a drawer (light sensitive).

SECTION: Protein Digestion
17. Resuspend the 100 µg pellet in 10 µL of neat TFE and 10 µL of 100mM Tris (pH 8.0).  Vortex to dissolve pellet.
SECTION: Protein Digestion
22. Prepare Promega Rapid Trypsin:
Add 100 µL of Promega Resuspension Buffer to 1 bottle of rapid trypsin.

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

SECTION: Protein Digestion
24. Gently vortex each sample.
Incubate plate at 55 °C for 45 min .


SECTION: Protein Digestion
25. Remove samples from incubator and pulse in centrifuge to to push any condensation out of the cap.

SECTION: Protein Digestion
26. Add 5 µL 60% Formic Acid to each well sample to stop the digestion.

SECTION: Protein Digestion
27. Place samples on ice to prepare for desalting or place them in -80 °C for future use.

SECTION: Desalting Samples and Preparing for LCMS
28. Prepare tip soaking plate by placing  100 µL 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.
SECTION: Desalting Samples and Preparing for LCMS
29. Wash
Add  20 µL of Solvent B to each tip.
Place the Evotips box in a centrifuge with appropriate counter balance.
Centrifuge at 700g for 700 x g  for 1 min.  
SECTION: Desalting Samples and Preparing for LCMS
30. Condition
Place Evotip adaptor rack on top of the MTP plate with 1-propanol.
Soak for 1 min.  
Inspect the tips to make sure that all tips are pale white.  If not, soak longer.
SECTION: Desalting Samples and Preparing for LCMS
31. Equilibrate
Add  20 µL of Solvent A to each tip.
Place the Evotips box in a centrifuge with appropriate counter balance.
Centrifuge at 700g for 700 x g  for 1 min.  
32. Sample Load
Add of sample or control to each tip, for a total of  20 µL .
    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 700 x g  for 1 min.  
SECTION: Protein Digestion
18. Reduce samples with  1 µL premade TCEP (0.5M) at room temperature for 30 min .
SECTION: Protein Digestion
20. Alkylate samples with by adding 2 µL of 0.5M IAA to each sample.
Incubate in a dark drawer at room temperature for  30 min .
SECTION: Protein Digestion
21. Add 73 µL of Rapid Trypsin Buffer to each sample.
33. Wash
Add  20 µL of Solvent A to each tip.
Place the Evotips box in a centrifuge with appropriate counter balance.
Centrifuge at 700g for 700 x g  for 1 min.  
34. Preservation
Add  100 µL of Solvent A to each tip.
Place the Evotips box in a centrifuge with appropriate counter balance.
Pulse the centrifuge at 700g for 700 x g  for 10 s.  
This will keep the tips wet.
35. Samples are now ready to for LCMS analysis.
SECTION: Tissue Lysis
1. Begin with tissue cryosections in eppendorf tubes. Add 100µL-500µL lysis buffer to tissue.