Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions
Published: April 30, 2023
Abstract
Source: Bezstarosti, K. et al. Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry. J. Vis. Exp. (2020)
This video describes a method to extract and purify ubiquitinylated peptides containing remnant di-glycine peptides from a complex peptide mixture. The presented method may help in identifying original ubiquitination sites in the protein.
Protocol
1. Extraction of Ubiquitinylated Peptides Use high pH reverse-phase (RP) C18 chromatography with polymeric stationary phase material (300 Å, 50 µM; see Table of Materials) loaded into an empty column cartridge to fractionate the tryptic peptides. The stationary phase bed size must be adjusted to the amount of protein digest to be fractionated. Prepare an empty 6 mL column cartridge (see Table of Materials) filled with 0.5 g of stationary phase material for ~10 mg of protein digest. …
Representative Results
Figure 1: Experimental overview. (A) Overview of the experimental approach. Samples were prepared, trypsinized, and fractionated into three fractions using reverse-phase chromatography with high pH elution. One batch of commercial α-diGly peptide antibody beads was split into six equal fractions and the three peptide fractions were then loaded on three of the bead fractions. The diGly peptides were immunopurified, eluted, …
Disclosures
The authors have nothing to disclose.
Materials
| Lysyl Endopeptidase(LysC) | Wako Pure Chemicals | 129-02541 | |
| NanoLC oven | MPI design, MS Wil GmbH | ||
| N-Lauroylsarcosine sodium salt | Sigma-Aldrich | L-5125 | |
| Orbitrap Fusion Lumos mass spectrometer |
ThermoFisher | ||
| PLRP-S (300 Å, 50 µm) polymeric reversed phase particles |
Agilent Technologies | PL1412-2K01 | |
| PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit |
Cell Signaling Technologies | 5562 | |
| Sep-Pak tC18 6 cc Vac Cartridge | Waters | WAT036790 | Remove the tC18 material from the cartridge before filling the cartridge with PLRP-S |
| Sodium deoxycholate | Sigma-Aldrich | 30970 | |
| Tris-base | Sigma-Aldrich | T6066 | |
| Tris-HCl | Sigma-Aldrich | T5941 | |
| Trypsin, TPCK Treated | ThermoFisher | 20233 |
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Cite This Article
Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions. J. Vis. Exp. (Pending Publication), e20622, doi: (2023).