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

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

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 l....

Materials

List of materials used in this article

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