Improvement of ubiquitylation site detection by Orbitrap mass spectrometry.

Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-ε-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome.