Unexpected Trypsin Cleavage at Ubiquitinated Lysines

ABSTRACT: Unexpected tryptic cleavage has been characterized at modified K48 residues in polyubiquitins. In particular, the tryptic products of all seven of the lysine-linked dimers of ubiquitin and of three trimerslinear Ub−48Ub−48Ub, linear Ub−63Ub−63Ub, and the branched trimer [Ub]2−6,48Ubhave been analyzed. In addition to the peptide products expected under commonly used tryptic conditions, we observe that peptides are formed with an unexpected ε-glycinylglycinyl-Lys carboxyl terminus when the site of linkage is Lys48. Trypsin from three different commercial sources exhibited this aberration. Initial cleavage at R74 is proposed in a distal ubiquitin to produce a glycinylglycinyl-lysine residue which is bound by trypsin. Proteolysis by trypsin is commonly used in proteomicworkflows and other biochemical investigations. It is solidly established that the enzyme cleaves proteins and peptides on the carboxyl side of lysine and arginine residues1,