Figure 4.

Detecting unique diglycine (–GG) signature peptides for each poly- ubiquitin chain linkage. Each poly-ubiquitin chain conformation can be detected by monitoring a unique signature peptide containing a –GG modified lysine residue, produced by trypsin cleavage. The full amino acid sequence of human ubiquitin is shown at the top. Ubiquitin-ubiquitin linkages correspond to isopeptide bonds formed between the C-terminal glycine (blue) of one ubiquitin and the ɛ-amino group of a lysine residue (red) within the second. These linkages can be formed through any of seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) (red). As an example, K48, K63, and K11 chains are demonstrated. Digestion of these linkages with trypsin produces peptides with distinct amino acid sequences (see insets). Trypsin cleaves at lysine and arginine residues within both primary and branched ubiquitin molecules (see arrows in insets), but cannot cut at lysines modified by isopeptide linked ubiquitin (see underlined in insets). The resulting tryptic peptides contain a –GG modified lysine, bearing an additional mass of 114.04 Da, denoting the original position of the modification. Database searching algorithms can utilize both the missed cleavage and –GG modification as search criteria when assigning precise sites of ubiquitination. In the case of a forked poly-ubiquitin chain, as demonstrated through K29 and K33, two –GG modified lysines are detected on the same peptide.