Titanium Dioxide-based Enrichment of Phosphopeptides: A Method for the Selective Isolation of Phosphopeptides from Peptide Library
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 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
- 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.
- Preparing the TiO2 beads contained in tips that have a capacity for 200 µL samples.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 |
Offenlegungen
The authors have nothing to disclose.
Materials
| 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 |
Diesen Artikel zitieren
Titanium Dioxide-based Enrichment of Phosphopeptides: A Method for the Selective Isolation of Phosphopeptides from Peptide Library. J. Vis. Exp. (Pending Publication), e20406, doi: (2023).