UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites

Vyacheslav Akimov; Inigo Barrio-Hernandez; Sten V.F. Hansen; Philip Hallenborg; Anna-Kathrine Pedersen; Dorte B. Bekker-Jensen; Michele Puglia; Stine D K Christensen; Jens T. Vanselow; Mogens M. Nielsen; Irina Kratchmarova; Christian D. Kelstrup; Jesper V. Olsen; Blagoy Blagoev

doi:10.1038/s41594-018-0084-y

UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites

Vyacheslav Akimov, Inigo Barrio-Hernandez, Sten V.F. Hansen, Philip Hallenborg, Anna-Kathrine Pedersen, Dorte B. Bekker-Jensen, Michele Puglia, Stine D K Christensen, Jens T. Vanselow, Mogens M. Nielsen, Irina Kratchmarova, Christian D. Kelstrup, Jesper V. Olsen, Blagoy Blagoev

104 Citations (Scopus)

Abstract

Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

Original language	English
Journal	Nature Structural & Molecular Biology
Volume	25
Pages (from-to)	631-640
Number of pages	10
ISSN	1545-9993
DOIs	https://doi.org/10.1038/s41594-018-0084-y
Publication status	Published - 2 Jul 2018

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Akimov, V., Barrio-Hernandez, I., Hansen, S. V. F., Hallenborg, P., Pedersen, A.-K., Bekker-Jensen, D. B., Puglia, M., Christensen, S. D. K., Vanselow, J. T., Nielsen, M. M., Kratchmarova, I., Kelstrup, C. D., Olsen, J. V., & Blagoev, B. (2018). UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites. Nature Structural & Molecular Biology, 25, 631-640. https://doi.org/10.1038/s41594-018-0084-y

Akimov, V, Barrio-Hernandez, I, Hansen, SVF, Hallenborg, P, Pedersen, A-K, Bekker-Jensen, DB, Puglia, M, Christensen, SDK, Vanselow, JT, Nielsen, MM, Kratchmarova, I, Kelstrup, CD, Olsen, JV & Blagoev, B 2018, 'UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites', Nature Structural & Molecular Biology, vol. 25, pp. 631-640. https://doi.org/10.1038/s41594-018-0084-y

@article{8b6c3aa15ba24dc087f48a17c284de68,

title = "UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites",

abstract = "Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.",

author = "Vyacheslav Akimov and Inigo Barrio-Hernandez and Hansen, {Sten V.F.} and Philip Hallenborg and Anna-Kathrine Pedersen and Bekker-Jensen, {Dorte B.} and Michele Puglia and Christensen, {Stine D K} and Vanselow, {Jens T.} and Nielsen, {Mogens M.} and Irina Kratchmarova and Kelstrup, {Christian D.} and Olsen, {Jesper V.} and Blagoy Blagoev",

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doi = "10.1038/s41594-018-0084-y",

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T1 - UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites

AU - Akimov, Vyacheslav

AU - Barrio-Hernandez, Inigo

AU - Hansen, Sten V.F.

AU - Hallenborg, Philip

AU - Pedersen, Anna-Kathrine

AU - Bekker-Jensen, Dorte B.

AU - Puglia, Michele

AU - Christensen, Stine D K

AU - Vanselow, Jens T.

AU - Nielsen, Mogens M.

AU - Kratchmarova, Irina

AU - Kelstrup, Christian D.

AU - Olsen, Jesper V.

AU - Blagoev, Blagoy

PY - 2018/7/2

Y1 - 2018/7/2

N2 - Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

AB - Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

U2 - 10.1038/s41594-018-0084-y

DO - 10.1038/s41594-018-0084-y

M3 - Journal article

C2 - 29967540

SN - 1545-9993

VL - 25

SP - 631

EP - 640

JO - Nature Structural & Molecular Biology

JF - Nature Structural & Molecular Biology

ER -

UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites

Abstract

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