Unexpected Trypsin Cleavage at Ubiquitinated Lysines

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–48Ub–48Ub, linear Ub–63Ub–63Ub, and
the branched trimer [Ub]2–6,48Ub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.