Protein Extraction and Proteolysis: A Method to Obtain Proteins from Prostate Tumor Tissue Samples and their Enzymatic Digestion into Peptides

To harvest tumor tissue, weigh the tumor, and in a culture test tube, add 2 milliliters of ice-cold lysis buffer for every 100 milligrams of tissue. Then, use a hand-held or benchtop homogenizer to homogenize the lysate.

To reduce and alkylate the sample, heat the tube at 95 degrees Celsius for 5 minutes, then cool the sample on ice for 15 minutes. While still on ice, sonicate the lysate three times. Then, heat the sample at 95 degrees Celsius for 5 minutes.

Place this sonication tube in a swinging bucket rotor and centrifuge the lysate at 3,500 x g and 15 degrees Celsius for 15 minutes. Then, collect the supernatant and discard the pellet. To digest the lysate, use 100 millimolar Tris to dilute the sample 12-fold to reduce the amount of guanidinium. For 5 milligrams of protein, add 10 micrograms of Lysyl Endopeptidase, or Lys-C, and incubate the sample at room temperature for 5 to 6 hours.

Prepare 1 milligram per milliliter of TPCK-treated trypsin in 1 millimolar HCl with 20 millimolar calcium chloride and trypsin added at a 1 to 100 trypsin to protein ratio. Incubate the sample at 37 degrees Celsius for 3 hours. Then, add an additional aliquot of trypsin to the tube and incubate the sample at 37 degrees Celsius overnight.

Add the sample to a 15-milliliter, 10 kDa cutoff filter. Centrifuge the sample in a swinging bucket or fixed angle rotor at 3,500 x g and 15 degrees Celsius until the retentate volume is less than 250 microliters. Then, collect the flow-through and discard the retentate.