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...