10.1021/acs.analchem.5b01960.s001 Meghan C. Burke Meghan C. Burke Yan Wang Yan Wang Amanda E. Lee Amanda E. Lee Emma Kimm Dixon Emma Kimm Dixon Carlos A. Castaneda Carlos A. Castaneda David Fushman David Fushman Catherine Fenselau Catherine Fenselau Unexpected Trypsin Cleavage at Ubiquitinated Lysines American Chemical Society 2015 K 48 residues trimer Lys 48. Trypsin R 74 ubiquitin peptide products tryptic conditions Ubiquitinated LysinesUnexpected tryptic cleavage Initial cleavage tryptic products Unexpected Trypsin Cleavage 2015-12-17 09:05:33 Journal contribution https://acs.figshare.com/articles/journal_contribution/Unexpected_Trypsin_Cleavage_at_Ubiquitinated_Lysines/2052732 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–<sup>48</sup>Ub–<sup>48</sup>Ub, linear Ub–<sup>63</sup>Ub–<sup>63</sup>Ub, and the branched trimer [Ub]<sub>2</sub>–<sup>6,48</sup>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.