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, …

開示

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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記事を引用
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).

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