This article discusses the process of ubiquitination, where ubiquitin is attached to substrate proteins, and outlines a method for isolating ubiquitin-tagged peptides. The protocol involves enzymatic cleavage, antibody binding, and purification steps to recover the peptides for analysis.
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.
One batch of ubiquitin remnant motif antibodies conjugated to protein A agarose bead slurry is split into six equal fractions. Dissolve the three peptide fractions according to the manuscript directions and spin down the debris.
Add the supernatants of the three fractions to the bead slurry while keeping the other three fractions on ice and incubate them for 2 hours at 4 degrees Celsius on a rotator unit.
Then, spin down the beads. Transfer the supernatant to a fresh batch of beads and repeat the incubation with the remaining three bead slurry fractions.
Store the supernatants for subsequent global proteome analysis and transfer the beads to 200-microliter pipette tips equipped with a GF/F filter plug.
Put the tips into 1.5-milliliter tubes equipped with a centrifuge tip adapter and wash the beads three times with 200 microlitres of ice-cold IAP buffer, followed by three washes with ice-cold purified water.
Spin down the columns at 200 x g for 2 minutes between washes, making sure not to let the column run dry. After the final wash, elute the peptides with two cycles of 50 microlitres of 0.15% TFA. Desalt the peptides with a C18 stage tip and dry them with vacuum centrifugation.
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