Springer Protocols: Abstract: Mass Spectrometric Determination of Protein Ubiquitination

Mass Spectrometric Determination of Protein Ubiquitination

By: Carol E. Parker³, Maria R. E. Warren³, Viorel Mocanu³, Susanna F. Greer⁴, Christoph H. Borchers³

Abstract

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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. The ubiquitinated peptide thus has two N-termini – one from the original peptide and one 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. 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.

Affiliation(s): (3) Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
(4) Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Book Title: Post-translational Modifi cations of Proteins: Tools for Functional Proteomics

Series: Methods in Molecular Biology | Volume: 446 | Pub. Date: Apr-04-2008 | Page Range: 109-130 | DOI: 10.1007/978-1-60327-084-7_8

Subject: Protein Science

Key Words: Mass spectrometry - ubiquitination - diagnostic ions - MS/MS

References

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