Dissecting the ubiquitin pathway by mass spectrometry

Abstract

Protein modification by ubiquitin is a central regulatory mechanism in eukaryotic cells. Recent proteomics developments in mass spectrometry enable systematic analysis of cellular components in the ubiquitin pathway. Here, we review the advances in analyzing ubiquitinated substrates, determining modified lysine residues, quantifying polyubiquitin chain topologies, as well as profiling deubiquitinating enzymes based on the activity. Moreover, proteomic approaches have been developed for probing the interactome of proteasome and for identifying proteins with ubiquitin-binding domains. Similar strategies have been applied on the studies of the modification by ubiquitin-like proteins as well. These strategies are discussed with respect to their advantages, limitations and potential improvements. While the utilization of current methodologies has rapidly expanded the scope of protein modification by the ubiquitin family, a more active role is anticipated in the functional studies with the emergence of quantitative mass spectrometry.

Fig. 1

The key steps in the ubiquitin pathway including protein conjugation, recognition and function.

Fig. 2

(A) The strategy for identifying Ub modification sites in protein targets and for analyzing polyUb chain topologies. All tryptic sites in ubiquitin are shown. (B) Measurement of the abundance of Ub linkages by the AQUA strategy using mass spectrometry. The synthesis of stable isotope labeled internal standard as exemplified by K48 linkage. It is also shown to use liquid chromatography coupled with multiple reaction monitoring (MRM) to quantify K48 and K63 linkages.