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MPI für Biochemie  

Proteomics and Signal Transduction
Matthias Mann

FFPE-FASP_protocol

 

FFPE-FASP_protocol as pdf

FFPE-FASP: Protocol for Processing of FFPE-samples for Mass Spectrometric Proteome Analysis

1. Materials

A. Solutions and Reagents

  1. Xylene
  2. Absolute ethanol
  3. Lysis solution (LS): 0.1 M Tris-HCl, pH 8.0, 0.1 M DTT
  4. 20% (w/v) solution of SDS in water
  5. Urea (UA) solution: 8 M urea (Sigma, U5128) in 0.1 M Tris/HCl pH 8.5
  6. IAA solution: 0.05 M iodoacetamide in UA
  7. Trypsin, stock 0.4 µg/µL
  8. Ammonium bicarbonate (ABC) solution: 0.05M NH4HCO3 in water

Note : LS, UA, and IAA solutions must be freshly prepared and used within a day.

B. Equipment

  1. Ultra-Turrax blender (T 10 basic Ultra, IKA, Staufen, Germany).
  2. Branson Sonifier (Heinemann, Schwäbisch Gmünd, Germany)
  3. Vivacon 500 (Sartorius Stedim Biotech)
  4. Refrigerated bench-top centrifuge, set to 18°C
  5. Incubator set to 37°C
  6. Wet chamber with a rack for Eppendorf tubes
  7. Heating block with agitation, set to 99°C
  8. Centrifugal vacuum-dryer (Speed-Vac)


2 Protocol

  1. Incubate FFPE tissues slices in 1 mL of xylene in an Eppendorf-type tube with gentle agitation at room temperature for 5 min.
  2. Remove the solution, add 1 mL of xylene and incubate as in (1).
  3. Remove the solution and repeat steps 1 and 2 in each using 1 mL of absolute ethanol.
  4. Remove ethanol and vacuum-dry the sample. 
  5. Mix the dried tissue with LS in a tissue to buffer ratio of 1:20 and homogenize on ice using the disperser for 3 min.  
  6. Sonicate the suspension on ice for 3 min. (output control 5; duty cycle 20%)
  7. Add 20% SDS to the suspension to a final concentration of 4%.
  8. Incubate in a heating block with agitation (600 rpm) at 99°C for 60 min.
  9. Remove the tube from the heating block and let it slowly chill to the room temperature.
  10. Centrifuge the extract at 16,000 × g for 10 min.  
  11.  ix up to 50 µL of the clarified lysate with 200 µL of UA in the filter unit and centrifuge at 14,000 × g for 30 min.
  12. Add 200 µL of UA to the filter unit and centrifuge at 14,000 × g for 20 min.
  13. Discard the flow-through form the collection tube.
  14. Add 100 µL IAA solution and mix at 600 rpm in a thermo-mixer for 1 min and incubate without mixing for 20 min.
  15. Centrifuge the filter units at 14,000 × g for 10 min.
  16. Add 100 µL of UA to the filter unit and centrifuge at 14,000 × g for 15 min. Repeat this step twice.
  17. Add 100 µL of ABC to the filter unit and centrifuge at 14,000 × g for 10 min. Repeat this step twice.
  18. Add 40 µL ABC with trypsin (enzyme to protein ratio 1:100) or another protease and mix at 600 rpm in thermo-mixer for 1 min.
  19. Incubate the units in a wet chamber at 37°C for 4 -18 h.
  20. Transfer the filter units to new collection tubes.
  21. Centrifuge the filter units at 14,000 × g for 10 min.
  22. Add 50 µL of ABC and centrifuge the filter units at 14,000 × g for 10 min.
  23. (optional) Measure the peptide yield in the filtrate using UV spectrophotometer.
 

 

References:

1. Ostasiewicz et al (2010) Proteome, N-Glycoproteome and Phosphoproteome are Quantitatively Preserved in Formalin-Fixed Paraffin-Embedded Tissue and Analyzable by High-Resolution Mass Spectrometry. J. Proteome Res. in press