Deciphering tissue-specific ubiquitylation by mass spectrometry

Abstract

Protein ubiquitylation is a highly conserved, central mechanism to regulate cellular events in all eukaryotes, such as proteasomal degradation, protein trafficking, DNA repair, synaptic plasticity, and immune response. The consequence of protein ubiquitylation is modulated by the structure of ubiquitin (Ub) moiety attached on the substrates, including ubiquitin monomer and diverse polyubiquitin chains with different linkages (N-terminus, K6, K11, K27, K29, K33, K48, and K63). The development of ubiquitin-enrichment strategies coupled with sensitive mass spectrometry enables direct analysis of ubiquitylated proteins in cells, providing an invaluable tool for ubiquitin research. In this chapter, we describe recent technology updates for analyzing tissue-specific ubiquitin conjugates in transgenic models, as well as targeted proteomics methods for quantifying different polyubiquitin chain linkages in any type of -samples, including human tissues.

Figure 1

The chemistry of the ubiquitin family. During the posttranslational modification by Ub and Ub like proteins, the C-terminal carboxyl group forms an isopeptide bond with the amine group on the side chain of Lys residues. Alternatively, N-terminal amine, Cys residues and even some lipid and other small molecules also serve as acceptors in this reaction.

Figure 2

The strategy for analyzing neuron-specific protein ubiquitination in the fly. The neuronal elavGAL4 driver allows the expression of BirA (negative control) or Ub₆-BirA in neurons using the GAL4/UAS system. Biotinylated Ub-conjugates are isolated and subjected to 1D SDS gel analysis followed by in-gel digestion and LC-MS/MS.

Figure 3

Measurement of the abundance of Ub linkages by mass spectrometry. (A) The synthesis of stable isotope-labeled internal standard as exemplified by K48 linkages. (B) and (C) Representative AQUA analysis of K11 and K48 linkages, respectively.