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

Overview

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 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.
5. 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.
6. 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

1. 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.
2. 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).
3. 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.
4. Add the same amount of fresh trypsin as in step 2.3. Incubate it at 37 °C overnight.
5. Save 12.5 µg of the digested lysate to confirm the complete digestion on a Coomassie-stained gel.

3. Reverse Phase Extraction

1. 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

Materials

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