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Methods Mol Biol. 2019;1934:191-221. doi: 10.1007/978-1-4939-9055-9_13.

Mass Spectrometric Determination of Protein Ubiquitination.

Author information

1
Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
2
Genome BC Proteomics Centre, University of Victoria, Victoria, BC, Canada.
3
Hagn Scientific, LLC, Efland, NC, USA.
4
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
5
American Cancer Society, Atlanta, GA, USA.
6
Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. christoph@proteincentre.com.
7
Genome BC Proteomics Centre, University of Victoria, Victoria, BC, Canada. christoph@proteincentre.com.
8
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada. christoph@proteincentre.com.
9
Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC, Canada. christoph@proteincentre.com.
10
Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, Canada. christoph@proteincentre.com.

Abstract

Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a portion of the ubiquitin side chain remains attached to the modified lysine. This "tag" can be used to distinguish a ubiquitinated peptide from the unmodified version, and can be incorporated into automated database searching. Several tags are discussed, the GGK and LRGGK tags, resulting from complete and incomplete tryptic digestion of the protein, and the STLHLVLRLRGG tag from a gluC-digested protein.A ubiquitinated peptide has two N-termini-one from the original peptide and the other from the ubiquitin side chain. Thus, it is possible to have two series of b ions and y ions, the additional series is the one that includes fragments containing portions of the ubiquitin side chain, and any diagnostic ions for the modification must include portions of this side chain. Fragment ions involving any part of the "normal" peptide will vary in mass according to the peptide being modified and will therefore not be of general diagnostic use. These diagnostic ions, found through examination of the MS/MS spectra of model ubiquitinated tryptic and gluC peptides, have not previously been reported. These ions can be used to trigger precursor ion scanning in automated MS/MS data acquisition scanning modes.

KEYWORDS:

Diagnostic ions; MS/MS; Mass spectrometry; Ubiquitination

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