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…

開示

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

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記事を引用
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).

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