Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides.

Ubiquitin, a 76 amino acid protein, is a key component that contributes to cellular protein homeostasis. The specificity of this modification is due to a series of enzymes: ligases, attaching the ubiquitin to a lysine, and deubiquitinases, which remove it. More than a hundred of such proteins are implicated in the regulation of protein turnover.

Microwave-mediated reduction of disulfide bridges with supported (tris(2-carboxyethyl)phosphine) as resin-bound reducing agent.

We report on the synthesis and use of a new supported reagent consisting in tris(2-carboxyethyl)phosphine (TCEP) immobilized on hydrophilic PEG based resin beads. Used in conjunction with a 5 min microwave (MW) irradiation, "supported TCEP" reduced disulfide bridges in free thiols in peptides having two or more cysteine residues. Separation of reaction products from reducing agent was easily performed by simple filtration.

Supported oligomethionine sulfoxide and Ellman's reagent for cysteine bridges formation.

A large number of bioactive peptides are cyclized through a disulfide bridge. This structural feature is very important for both bioactivity and stability. The oxidation of cysteine side chains is challenging not only to avoid intermolecular reaction leading to oligomers and oxidation of other residues but also to remove solvents and oxidant such as dimethyl sulfoxide.