Quantitative proteomics and terminomics to elucidate the role of ubiquitination and proteolysis in adaptive immunity

Abstract

Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue 'Quantitative mass spectrometry'.

Keywords: LUBAC; MALT1; degradomics; linear ubiquitin; proteases; ubiquitin.

Figure 1.

Scheme displaying different homotypic polyubiquitin chains conjugated to protein substrates in vivo and biological outcome of ubiquitination. Ub, ubiquitin monomer; S, substrate. (Online version in colour.)

Figure 2.

Schematic overview of quantitative proteomics methods discussed in this paper. (a) Workflow of enrichment methods used in ubiquitin-profiling proteomics based on anti-ubiquitin (Ub) immunoprecipitation (left), ubiquitin pull down with selective ubiquitin-binding domain reagents (TUBEs, middle) and tryptic ubiquitin remnant peptide antibody immunoprecipitation (right). (b) Workflow of methods for enriching N-terminal peptides by chemical labelling of primary amines followed by removing non-labelled peptides via binding to an aldehyde-functionalized polymer (TAILS, left), chemical labelling of primary amines and sequential chromatographic fractionation to enrich for N-termini (COFRADIC), and enzymatic labelling of N-terminal α-amines and subsequent pull down of labelled peptides (right). (Online version in colour.)