Identification of Sites of Ubiquitination in Proteins:  A Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Approach

• Published in: Analytical chemistry (Washington) 2004-12, Vol.76 (23), p.6982-6988
• Main Authors: Cooper, Helen J, Heath, John K, Jaffray, Ellis, Hay, Ronald T, Lam, TuKiet T, Marshall, Alan G
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical chemistry; Chemistry; Exact sciences and technology; Fourier Analysis; Fourier transforms; Humans; Infrared Rays; Lasers; Magnetics; Mass Spectrometry - instrumentation; Mass Spectrometry - methods; Molecular Sequence Data; Peptides; Proteins; Proteins - chemistry; Scientific imaging; Sensitivity and Specificity; Spectrometric and optical methods; Static Electricity; Ubiquitins - chemistry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0401063

Structural elucidation of posttranslationally modified peptides and proteins is of key importance in the understanding of an array of biological processes. Ubiquitination is a reversible modification that regulates many cellular functions. Consequences of ubiquitination depend on whether a single ubiquitin or polyubiquitin chain is added to the tagged protein. The lysine residue through which the polyubiquitin chain is formed is also critical for biological activity. Robust methods are therefore required to identify sites of ubiquitination modification, both in the target protein and in ubiquitin. Here, we demonstrate the suitability of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, in conjunction with activated ion electron capture dissociation (AI ECD) or infrared multiphoton dissociation (IRMPD), for the analysis of ubiquitinated proteins. Polyubiquitinated substrate protein GST-Ubc5 was generated in vitro. Tryptic digests of polyubiquitinated species contain modified peptides in which the ubiquitin C-terminal Gly-Gly residues are retained on the modified lysine residues. Direct infusion microelectrospray FT-ICR of the digest and comparison with an in silico digest enables identification of modified peptides and therefore sites of ubiquitination. Fifteen sites of ubiquitination were identified in GST-Ubc5 and four sites in ubiquitin. Assignments were confirmed by AI ECD or IRMPD. The Gly-Gly modification is stable and both tandem mass spectrometric techniques are suitable, providing extensive sequence coverage and retention of the modification on backbone fragments.