TY - JOUR
T1 - Unexpected Trypsin Cleavage at Ubiquitinated Lysines
AU - Burke, Meghan C.
AU - Wang, Yan
AU - Lee, Amanda E.
AU - Dixon, Emma Kimm
AU - Castaneda, Carlos A.
AU - Fushman, David
AU - Fenselau, Catherine
N1 - Publisher Copyright:
© 2015 American Chemical Society.
PY - 2015/8/18
Y1 - 2015/8/18
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.analchem.5b01960
DO - 10.1021/acs.analchem.5b01960
M3 - Article
C2 - 26182167
AN - SCOPUS:84939857323
SN - 0003-2700
VL - 87
SP - 8144
EP - 8148
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 16
ER -