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.
1. Extraction of Ubiquitinylated Peptides
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, and collected, and the flowthrough was subsequently transferred to the three remaining fresh beads fractions. The collected diGly peptides were analyzed by mass spectrometry on a Lumos Orbitrap mass spectrometer according to a two-tier scheme combining one cycle in which the most intense peaks were first selected for peptide fragmentation and the next cycle in which the least intense peaks were selected first. The complete set of nLC-MS/MS runs were then analyzed using MaxQuant. (B) One of the fractions should contain ubiquitin's own K48 modified tryptic diGly peptide LIFAGK(GG)QLEDGR (m/z 730.39). This is by far the most abundant peptide in the immunoprecipitated fraction and was characterized by the intense and broad peak in the LC chromatogram between 50–55 min on a 120 min gradient. If this benchmark peak is absent from the chromatogram the IP was most likely unsuccessful.
The authors have nothing to disclose.
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 |