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J Biomol Tech. 2007 February; 18(1): 26.
PMCID: PMC2292063

P76-S Targeted Mass Spectrometric Strategies for Global Mapping of Ubiquitination on Proteins


Protein modification by ubiquitin is an important event that represents a highly versatile means for regulation of protein function. Ubiquitination acts at multiple levels to regulate gene expression, including targeting for degradation, proteolytic activation, and intracellular localization. Mapping of protein ubiquitination sites is important because it can facilitate the elucidation of mechanisms of this modification, yielding insights into cell regulation. Recently, mass spectrometry has been employed for determination of ubiquitination sites; however, this protein analysis approach is incomplete and not saturated for any given protein. Existing targeted mass spectrometric approaches can monitor only a few predicted ubiquitinated peptides at any given time, thus requiring multiple analysis and samples. Additionally, proteins are typically enzymatically digested with trypsin. However, ubiquitinated peptides are modified on their lysine residues with the addition of GG motifs, thus generating large tryptic peptides that are often multiply charged. Hence, MS/MS spectra can be complicated and challenging to interpret. Here we employ a unique approach to target and sequence sites of ubiquitination on proteins and their relevant peptides.

Ubiquitinated proteins were purified and digested with various enzymes to obtain varying lengths of peptides. Sample analysis was performed using reverse-phase nano-HPLC interfaced to a hybrid quadrupole linear ion trap mass spectrometer. MRM-initiated detection and sequencing workflow was used to monitor 70–100 predicted ubiquitinated peptide masses and their fragment ions in one analysis for simultaneous detection, identification, and relative quantification of the peptides. A number of ubiquitinated peptides were identified by this analysis, and the results will be discussed. This targeted approach has been shown to provide a high-throughput analysis in addition to better specificity and sensitivity for ubiquitination site mapping.

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