Phosphopeptide Enrichment: An Antibody-based Immunoprecipitation Technique to Separate Specific Phosphorylated Peptides from Complex Peptide Mixtures

Overview

In this video, we demonstrate antibody-based immunoprecipitation of phosphopeptides from a peptide mixture. The enriched phosphopeptides are vacuum concentrated and stored for downstream analysis.

Protocol

1. Immunoprecipitation and Enrichment of pY Peptides

1. Resuspend the lyophilized powder of purified peptide digests with 0.5 mL of ice-cold immunoprecipitation (IP) binding buffer in each fraction. Pool the fractions by transferring the 0.5 mL resuspension volume from the second fraction to the first fraction and save the pipette tip. Vigorously vortex (instead of pipetting up and down) to ensure the sample is completely dissolved before transferring it to a 3.6 mL screw cap cryotube.
2. Rinse the lyophilization tubes with another 0.5 mL of IP binding buffer (Table 1) in each tube. Transfer the solution to the 3.6 mL screw-cap tube using the same pipette tip to minimize sample loss. Repeat the rinse 1x more, making the final resuspension volume 2 mL (for 5 mg of protein). Measure the sample pH to make sure it is approximately 7.4. If it is too acidic, iteratively add 10 µL of 1M Tris (untitrated, pH ~11). If it is too basic, iteratively add 10 µL of dilute HCl (1:25 or 1:100).
3. Pre-wash the pY beads (for 5 mg of starting lysate)
   1. 25 µg of 4G10 antibody and 12.5 µg of 27B10.4 antibody are needed per sample. After using a p200 pipette with a cut tip to transfer the antibodies into separate microcentrifuge tubes, wash the antibodies with 450 µL of ice-cold IP binding buffer 2x. Centrifuge them at 100 x g for 1 min at 4 °C and aspirate out the supernatant. 
   2. Resuspend the beads to a stock concentration of 0.5 mg/mL using IP binding buffer. (Do not vortex the beads.) After aliquoting the necessary slurry (50 µL of 4G10 antibody slurry and 25 µL 27B10.4 antibody slurry per sample) into a single tube, spin down the stock centrifuge tubes at 200 x g for 1 min at 4 °C. Wash the sidewalls with supernatant before returning the beads to storage in the refrigerator.
4. Add pre-washed pY beads to the resuspended sample solution in the screw cap cryotubes. Incubate them at 4 °C on an end-over-end rotator overnight.
5. Place the screw cap cryotubes in a 50 mL centrifuge tube lined with a delicate wipe. Spin down the beads at 100 x g for 1 min. Save the supernatant, which will be used to enrich pST peptides.
6. Resuspend the beads with 300 µL of IP binding buffer. Transfer them to a 2 mL microcentrifuge tube and spin them down at 100 x g for 1 min at 4 °C.
7. Rinse the incubation tube 3x with 200 µL of IP binding buffer. Transfer the contents to the same microcentrifuge tube each time. Then, spin them down.
8. Wash the beads in the microcentrifuge tube 3x with 500 µL of IP binding buffer and spin them down at 100 x g for 1 min. Then, wash the beads 4x with 450 µL of 25 mM NH₄HCO₃, pH 7.5, and spin them down at 100 x g for 1 min. Use a fresh 25 mM NH₄HCO₃ solution from powder every time.
9. Centrifuge the beads at 1,500 x g for 1 min. Use a gel-loading tip to remove the supernatant completely by dipping the tip of the gel-loading tip slightly below the beads’ surface.
10. Add 4x the bead volume of 0.1% TFA to the beads (i.e., add 300 µL of 0.1% TFA for 75 µg of pY bead slurry). Mix them well and incubate the mixture in a thermomixer at 1,000 rpm for 15 min at 37 °C.
11. Transfer the resuspension to a 0.2 µm spin filter. Quickly spin down the elution tube and transfer the residual volume to the same spin filter using a P10 pipette. Spin down the spin filter at 850 x g for 1 min. Transfer the elution to a low protein-binding microcentrifuge tube. Vacuum concentrate the eluate to dryness overnight at 40 °C and with a heat time of 300 min.
    NOTE: The experiment can be paused here. Freeze the samples at -80 °C and continue at a later date.

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
Lyophilizer	Labconco	7420020
CentriVap Benchtop Vacuum Concentrator	Labconco	7810010
Kimwipes	Fisher Scientific	06-666A
Millipore 0.2 µm spin filter	Millipore Sigma	UFC30GVNB
Low protein-binding Eppendorf tubes	Eppendorf	22431081
anti-Phosphotyrosine, Agarose, Clone: 4G10	Millipore Sigma	16101
27B10.4 antibody	Cytoskeleton	APY03-beads
Peptide assay kit	Thermo Scientific	23275
TopTip	PolyLC Inc	TT200TIO.96
SCX columns (PolySULFOETHYL A)	PolyLC Inc	SPESE1203
3 mL syringe	BD	309657
MilliQ water			Deionized water used to prepare all solutions and bufferes
Trifluoracetic Acid (TFA)	Fisher Scientific	PI-28904
Acetonitrile (ACN)	Fisher Scientific	A21-1
Potassium Chloride	Fisher Scientific	BP366-500
Lactic acid	Sigma-Aldrich	69785-250ML
Ammonium Hydroxide	Fisher Scientific	A669S-500
Tris Base	Fisher Scientific	BP152-5