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

Abstract

Source: Cheng, L. C. et al. Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer. J. Vis. Exp. (2018)

This video describes the technique of protein extraction and digestion to obtain peptides from prostate tumor tissue. These peptides on further analysis can help identify novel targets for oncotherapy.

Protocol

1. Protein Extraction

1. Prepare lysis buffer (Table 1). (The volume depends on the number of samples to be harvested.)
2. Harvesting tissues
   1. Weigh the tumor and add 2 mL of ice-cold lysis buffer for every 100 mg of tissue in a culture test tube. (Typically, 50 to 150 mg of tissue wet weight is needed.)
3. Homogenize the lysate using a hand-held or benchtop homogenizer (pulse 2x for 15 s). Clean the homogenizer before the first sample and between samples by using 10% bleach, 70% ethanol, and deionized water in succession.
4. To reduce and alkylate, heat the homogenized samples at....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ultra-Low Temperature Freezer	Panasonic	MDF-U76V
Freezer -20 °C	VWR	scpmf-2020
Swing rotor bucket	ThermoFisher Scientific	75004377
End-over-end rotator	ThermoFisher Scientific	415110Q
Low protein-binding Eppendorf tubes	Eppendorf	22431081
Trypsin, TPCK Treated	Worthington Biochemicals	LS003740
Lysyl Endopeptidase	Wako Pure Chemical Industries, Ltd.	125-05061
MilliQ water			Deionized water used to prepare all solutions and buffers
Sonic Dismembrator	Fisher Scientific	FB-120	Sonicator
Polytron System PT	Kinematica AG	PT 10-35 GT	Homogenizer