Protein Extraction and Proteolysis: A Method to Obtain Proteins from Prostate Tumor Tissue Samples and their Enzymatic Digestion into Peptides
Published: April 30, 2023
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
- Prepare lysis buffer (Table 1). (The volume depends on the number of samples to be harvested.)
- Harvesting tissues
- 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.)
- 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.
- To reduce and alkylate, heat the homogenized samples at 95 °C for 5 min. Then, cool them on ice for 15 min. On ice, sonicate the lysate 3x (i.e., pulse for 30 s with 60 s pauses between pulses). The sample should not be viscous or clumpy at this point. Heat the lysate at 95 °C for 5 min.
- Centrifuge the lysate in the same sonication tube using a swing bucket rotor at 3,500 x g at 15 °C for 15 min. Collect the supernatant and discard the pellet.
- Determine the protein concentration by performing a Bradford assay. If necessary, dilute the lysate to 5 mg/mL with a lysis buffer. Store it at -20 °C.
NOTE: The experiment can be paused here. Freeze the samples at -80 °C and use it for further analysis.
2. Lysate Digestion
- Dilute the sample 12-fold by using 100 mM Tris (pH = 8.5) to reduce the amount of guanidinium. Dilute all samples to the same volume to minimize the effects of unequal digestion. Save 12.5 µg of the undigested lysate to confirm it on a Coomassie-stained gel.
- For 5 mg of protein, add 10 µg of Lysyl Endopeptidase (Lys-C) and incubate it at room temperature for 5-6 h. Adjust pH to 8.0 by adding 1 M untitrated Tris (pH ~11).
- Prepare 1 mg/mL of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin in 1 mM HCl (with 20 mM CaCl2). Add the trypsin at a 1:100 trypsin:protein ratio and incubate it at 37 °C for 3 h.
- Add the same amount of fresh trypsin as in step 2.3. Incubate it at 37 °C overnight.
- Save 12.5 µg of the digested lysate to confirm the complete digestion on a Coomassie-stained gel.
3. Reverse Phase Extraction
- Record the lysate volume. Filter the sample by using a 15 mL 10 kDa cutoff filter. Centrifuge the sample at 3,500 x g using the swing bucket rotor (or 3,500 x g in a fixed angle rotor) at 15 °C until the retentate volume is less than 250 µL (this takes approximately 45-60 min). Collect the flow-through and discard the retentate.
Table 1: Buffers and solutions. This table shows the compositions of the buffers and solutions used in this protocol.
| Buffer | Volume | Composition |
| 6 M guanidinium chloride lysis buffer | 50 mL | 6 M guanidinium chloride, 100 mM tris pH 8.5, 10 mM tris (2-carboxyethyl) phosphine, 40 mM chloroacetamide, 2 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 500 mg n-octyl-glycoside, ultra-pure water to volume |
| 100 mM sodium pyrophosphate | 50 mL | 2.23 g sodium pyrophosphate decahydrate, ultra-pure water to volume |
| 1M β-glycerophosphate | 50 mL | 15.31 g β-glycerophosphate, ultra-pure water to volume |
| 5% trifluoroacetic acid | 20 mL | Add 1 mL of 100% trifluoroacetic acid into 19 mL ultra-pure water |
| 0.1% trifluoroacetic acid | 250 mL | Add 5 mL 5% trifluoroacetic acid to 245 mL ultra-pure water |
| pY elution buffer | 250 mL | 0.1% trifluoroacetic acid, 40% acetonitrile, ultra-pure water to volume |
| pST elution buffer | 250 mL | 0.1% trifluoroacetic acid, 50% acetonitrile, ultra-pure water to volume |
| IP binding buffer | 200 mL | 50 mM tris pH 7.4, 50 mM sodium chloride, ultra-pure water to volume |
| 25 mM ammonium bicarbonate, pH 7.5 | 10 mL | Dissolve 19.7 mg into 10 mL sterile ultra-pure water, pH to 7.5 with 1 N hydrochloric acid (~10-15 µL/10 ml solution), make fresh |
| 1M phosphate buffer, pH 7 | 1,000 mL | 423 mL 1 M sodium dihydrogen phosphate, 577 mL 1 M sodium hydrogen phosphate |
| Equilibration buffer | 14 mL | 6.3 mL acetonitrile, 280 µL 5% trifluoroacetic acid, 1740 µL lactic acid, 5.68 mL ultra-pure water |
| Rinsing buffer | 20 mL | 9 mL acetonitrile, 400 µL 5% trifluoroacetic acid, 10.6 mL ultra-pure water |
| Mass spectrometry solution | 10 mL | 500 µL acetonitrile, 200 µL 5% trifluoroacetic acid, 9.3 mL ultra-pure water |
| Buffer A | 250 mL | 5 mM monopotassium phosphate (pH 2.65), 30% acetonitrile, 5 mM potassium chloride,ultra-pure water to volume |
| Buffer B | 250 mL | 5 mM monopotassium phosphate (pH 2.65), 30% acetonitrile, 350 mM potassium chloride, ultra-pure water to volume |
| 0.9% ammonium hydroxide | 10 mL | 300 μL 29.42% ammonium hydroxide, 9.7 mL ultra-pure water |
Divulgazioni
The authors have nothing to disclose.
Materials
| 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 bufferes | ||
| Sonic Dismembrator | Fisher Scientific | FB-120 | sonicator |
| Polytron System PT | Kinematica AG | PT 10-35 GT | homogenizer |
Citazione di questo articolo
Protein Extraction and Proteolysis: A Method to Obtain Proteins from Prostate Tumor Tissue Samples and their Enzymatic Digestion into Peptides. J. Vis. Exp. (Pending Publication), e20403, doi: (2023).