Characterizing Ubiquitination Sites by Peptide-based Immunoaffinity Enrichment

Abstract

Advances in high resolution tandem mass spectrometry and peptide enrichment technologies have transformed the field of protein biochemistry by enabling analysis of end points that have traditionally been inaccessible to molecular and biochemical techniques. One field benefitting from this research has been the study of ubiquitin, a 76-amino acid protein that functions as a covalent modifier of other proteins. Seminal work performed decades ago revealed that trypsin digestion of a branched protein structure known as A24 yielded an enigmatic diglycine signature bound to a lysine residue in histone 2A. With the onset of mass spectrometry proteomics, identification of K-GG-modified peptides has emerged as an effective way to map the position of ubiquitin modifications on a protein of interest and to quantify the extent of substrate ubiquitination. The initial identification of K-GG peptides by mass spectrometry initiated a flurry of work aimed at enriching these post-translationally modified peptides for identification and quantification en masse. Recently, immunoaffinity reagents have been reported that are capable of capturing K-GG peptides from ubiquitin and its thousands of cellular substrates. Here we focus on the history of K-GG peptides, their identification by mass spectrometry, and the utility of immunoaffinity reagents for studying the mechanisms of cellular regulation by ubiquitin. Molecular & Cellular Proteomics 11: 10.1074/mcp.R112.019117, 1529 -1540, 2012.Post-translational modification by ubiquitin and ubiquitinlike proteins represents a major regulatory system in eukaryotic organisms (1, 2) and certain pathogenic bacteria (3). A conserved enzymatic cascade couples the C terminus of ubiquitin to the epsilon amino group of lysine residues on substrate proteins (4). Evidence has also emerged implicating ubiquitination via cysteine, serine, and threonine, as well as N-terminal residues (5-8). Depending on signaling context and the enzymes involved, protein substrates can be monoubiquitinated, multiubiquitinated, or polyubiquitinated. Ubiquitin-dependent processes are commonly modulated via formation of polyubiquitin chains, whereby the C terminus of a chain extending ubiquitin becomes linked to the N terminus or one of seven lysine residues (Lys-6, Lys-11, Lys-27, Lys-29, Lys-33, Lys-48, and Lys-63) within a substrate-bound ubiquitin molecule (9, 10). The functions of these structurally diverse modifications have been extensively studied through in vitro biochemistry (11-13), ubiquitin replacement in cellular models (14, 15), linkage specific antibodies (16 -19), and mass spectrometry (20 -26). A growing body of evidence indicates that polyubiquitin chains regulate biological processes not only by eliciting proteasomal degradation but also by altering subcellular localization, modulating enzymatic activity, and facilitating protein-protein interactions of ubiquitinated substrates (9, 24). Although in depth characterization of model substrates has led to significant advances in our understanding of bioche...

N- and O-Glycosylation in the Murine Synaptosome

“…Numerous large-scale studies have been conducted on intracellular, regulatory modifications, such as phosphorylation (2), GlcNAcylation (3), ubiquitination (4), and acetylation (5). Although ER/Golgi-derived glycosylation is one of the most frequently occurring classes of PTMs (6), these extracellular modifications continue to be a major challenge because of technical difficulties both in their structural characterization and in deciphering their biological roles.…”

Using the Ubiquitin-modified Proteome to Monitor Protein Homeostasis Function

“…Protein-based Ubiquitin Proteomics-The need for the development of sensitive and reliable methods for monitoring spatial and temporal patterns of ubiquitylation in vivo has driven innovation in the field of mass spectrometry (MS)-based proteomics (3)(4)(5). MS is an ideal platform for studying protein modifications such as ubiquitylation because it allows for the simultaneous identification of endogenous ubiquitylation substrates and the precise lysine residue that is modified (6).…”

Proteomic techniques to probe the ubiquitin landscape

“…The modification has different chemical properties and can be resolved chromatographically as a peak doublet [57]; it is also not well-recognized by the K-GG peptide antibody [58,59]. The detailed analysis of MS/MS spectra of these chemical artifacts shows a higher tendency for a -57 Da or a -144 Da neutral loss, which can be used as an additional quality criterion [58]. IAA alkylation of lysine is temperature-dependent with modification only occuring at higher incubation temperatures [56], and so can be largely avoided.…”