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