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

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

1. 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.
2. Rinse the lyophilization tubes with another 0.5 mL of IP binding buffer (Table 1) in each tube. Transfer the solution to the 3.6 mL ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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