Unexpected Trypsin Cleavage at Ubiquitinated Lysines

Meghan C. Burke; Yan Wang; Amanda E. Lee; Emma Kimm Dixon; Carlos A. Castaneda; David Fushman; Catherine Fenselau

doi:10.1021/acs.analchem.5b01960

Unexpected Trypsin Cleavage at Ubiquitinated Lysines

Meghan C. Burke, Yan Wang, Amanda E. Lee, Emma Kimm Dixon, Carlos A. Castaneda, David Fushman, Catherine Fenselau

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

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 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.

Original language	English (US)
Pages (from-to)	8144-8148
Number of pages	5
Journal	Analytical Chemistry
Volume	87
Issue number	16
DOIs	https://doi.org/10.1021/acs.analchem.5b01960
State	Published - Aug 18 2015
Externally published	Yes

ASJC Scopus subject areas

Analytical Chemistry

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title = "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 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.",

author = "Burke, {Meghan C.} and Yan Wang and Lee, {Amanda E.} and Dixon, {Emma Kimm} and Castaneda, {Carlos A.} and David Fushman and Catherine Fenselau",

note = "Publisher Copyright: {\textcopyright} 2015 American Chemical Society.",

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doi = "10.1021/acs.analchem.5b01960",

language = "English (US)",

volume = "87",

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journal = "Analytical Chemistry",

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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

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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JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 16

ER -

Unexpected Trypsin Cleavage at Ubiquitinated Lysines

Abstract

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