Titanium Dioxide-based Enrichment of Phosphopeptides: A Method for the Selective Isolation of Phosphopeptides from Peptide Library

Overview

This video demonstrates titanium dioxide-based enrichment of phosphopeptides extracted from prostate tumor tissue. These enriched phosphopeptides can be used for downstream applications such as protein characterization to understand cell signaling pathways involved in prostate cancer progression.

Protocol

1. Titanium Dioxide Enrichment of pY Peptides

1. Resuspend the dried down phosphopeptides in 200 µL of 50% ACN, 0.1% TFA. Vortex and centrifuge them at 10,000 x g for 30 s. Repeat this 1x to resuspend them well.
2. Preparing the TiO₂ beads contained in tips that have a capacity for 200 µL samples.
   1. Gently tap on the small tip-side of the tip to move the material to that end. Rinse the tip by adding 200 µL of 100% ACN, followed by inverting the tip and flicking the small end to move the liquid towards the cap.
   2. Using a razor blade, cut the small end of the tip and place it over a low protein-binding tube. (Avoid using polystyrene tubes as the TiO₂ will stick to the sides of the tube.) Remove the cap and insert a micropipette to plunge out the remaining ACN. Repeat the wash with 200 µL of 100% ACN. The TiO₂ beads are now located in the low protein-binding tube for the following steps.
   3. Precondition TiO₂ with 500 µL of 100% ACN 2x. Pipet it to mix the beads with the solvent. Centrifuge them at 100 x g for 1 min.
   4. Condition TiO₂ with 500 µL of 0.2 M sodium phosphate buffer (pH ~7) 2x. Wash the beads with 300 µL of equilibration buffer 3x. Because TiO₂ is very dense, the beads will settle quickly.
3. Add 400 µL of 50% ACN, 0.1% TFA into the low protein-binding tube, followed by adding 84 µL of lactic acid. Transfer the resuspended phosphopeptides into the low protein-binding tube and incubate them for 1 h at room temperature using an end-over-end rotator.
4. Centrifuge the beads at 100 x g for 1 min to pellet them. Wash them with 300 µL of equilibration buffer (Table 1) 2x and spin them down at 100 x g for 1 min.
5. Rinse the beads with 300 µL of rinsing buffer 2x. Transfer them to a 0.2 µm spin filter. Spin them at 1,500 x g for 1 min.
6. Transfer the filter unit to a clean 1.5 mL low protein-binding tube. Elute the contents 2x with 200 µL of 0.9% NH₃ in H₂O. Measure the pH with pH strips, which should be between 10 and 11. Vacuum concentrate the eluate to dry overnight to evaporate the ammonia.

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
Vacuum manifold	Restek	26080
Lyophilizer	Labconco	7420020
CentriVap Benchtop Vacuum Concentrator	Labconco	7810010
End-over-end rotator	ThermoFisher Scientific	415110Q
Razor blade	Fisher Scientific	620177
Amicon Ultra-15 Centrifugal Filter Units	Millipore Sigma	UFC901024
Nunc 15 mL conical tubes	ThermoFisher Scientific	12-565-268
Millipore 0.2 µm spin filter	Millipore Sigma	UFC30GVNB
Low protein-binding Eppendorf tubes	Eppendorf	22431081
anti-Phosphotyrosine, Agarose, Clone: 4G10	Millipore Sigma	16101
Peptide assay kit	Thermo Scientific	23275
Trifluoracetic Acid (TFA)	Fisher Scientific	PI-28904
Acetonitrile (ACN)	Fisher Scientific	A21-1
Lactic acid	Sigma-Aldrich	69785-250ML
Ammonium Hydroxide	Fisher Scientific	A669S-500
Potassium Phosphate Monobasic	Fisher Scientific	BP362-500
Potassium Chloride	Fisher Scientific	BP366-500
Calcium Chloride Dihydrate	Fisher Scientific	BP510-500
Tris Base	Fisher Scientific	BP152-5
MonoTip	PolyLC Inc	TT200TIO.96