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…
Divulgaciones
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 |
Citar este artículo
Phosphopeptide Enrichment: An Antibody-based Immunoprecipitation Technique to Separate Specific Phosphorylated Peptides from Complex Peptide Mixtures. J. Vis. Exp. (Pending Publication), e20405, doi: (2023).