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

JoVE Encyclopedia of Experiments

Kankeronderzoek

0 weergaven • 4:03 min • April 30th, 2023

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Begin with a prostate tumor tissue sample. Resuspend it in a chilled lysis buffer to initiate cell lysis. Use a homogenizer to break down the tissue structures further and release their intracellular components. Sonicate the sample on ice to ensure complete cell lysis and shear the DNA to prevent interference during peptide processing. Heat the lysate to disrupt the proteins' tertiary structure and unfold.

The reducing agents in the buffer cleave the disulfide bonds between cysteine residues. Subsequently, the alkylating agents in the buffer alkylate free thiol groups of reduced cysteines to prevent disulfide bond reformation. The additional denaturing agents in the buffer facilitate controlled denaturation and the further unfolding of proteins.

Centrifuge to pelletize the tissue debris and any contaminants. Collect the supernatant containing the protein mixture in a fresh tube. Add lysyl endopeptidase enzyme to cleave the peptide bonds in proteins at the C terminus of lysine residues, forming large peptides. Further, treat with the enzyme trypsin to cleave the peptide bonds at the C terminus of lysine and arginine residues and generate smaller peptides. Transfer the mixture to a centrifugal filter unit.

Centrifuge to allow the digested peptides to permeate through the pores of the filter membrane and collect as flow-through. Any larger fragments remain as the retentate.

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.

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Laatst bijgewerkt: 22 augustus 2026