Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions

Ubiquitination of proteins is characterized by the addition of ubiquitin - a regulatory mini-protein - via its di-glycine residues to the lysine residues of the substrate protein. This generates a lysine-glycine-glycine linkage between the substrate protein and ubiquitin.

To isolate ubiquitin-tagged peptides, begin by taking a peptide mix.

Treat these peptides with an endonuclease enzyme cocktail. During treatment, the enzymes Lys-C and trypsin efficiently cleave the ubiquitinylated peptides at the arginine residue, resulting in peptide fragments containing a signature di-glycine motif.

Add these peptide fragments to a slurry of antibody-conjugated hydrogel beads.

These antibodies contain a di-glycine recognition site, which binds exclusively to the ubiquitinylated peptides, forming a peptide-bead complex.

Centrifuge to pelletize ubiquitinylated peptide-bead complexes. Remove the unbound non-ubiquitinylated peptide-containing supernatant.

Load the complexes onto a filter assembly. Centrifuge to remove the buffer and trap peptide-conjugated beads.

Add an acidified reagent that lowers the pH, causing disengagement of peptides from the beads.

Recover the free peptides from the filtrate. Load them onto a fresh filter with a hydrophobic matrix. The peptides retain in the matrix while acidic salts get washed away.

Using a suitable solvent, detach the peptides.

Transfer the ubiquitinylated peptides to a fresh tube and dry them to remove any traces of solvent.