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

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).

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 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. <li…

開示

The authors have nothing to disclose.

Materials

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

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記事を引用
Phosphopeptide Enrichment: An Antibody-based Immunoprecipitation Technique to Separate Specific Phosphorylated Peptides from Complex Peptide Mixtures. J. Vis. Exp. (Pending Publication), e20405, doi: (2023).

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