Unexpected Trypsin Cleavage at Ubiquitinated Lysines

[Image: see text] 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 trimers—linear Ub–(48)Ub–(48)Ub, linear Ub–(63)Ub–(63)Ub, and the branched trimer [Ub](2)–(6,48)Ub—have 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.