Global strategies that use mass spectrometry (MS) to study ubiquitination. (a) Diagram of the lysine residues in ubiquitin; the carboxy-terminal Arg-Gly-Gly (RGG) motif is also indicated. (b) In ubiquitin profiling, 6×His-tagged ubiquitin expressed in cells is conjugated to substrate proteins, and this facilitates purification of ubiquitinated proteins under denaturing conditions by Ni-chelate chromatography, in which histidine-tagged proteins bind specifically to immobilized Ni2+ ions. Purified ubiquitinated proteins are digested with trypsin and the resulting peptides are analyzed by mass spectrometry to identify the proteins present in the sample. (c) Precise ubiquitination sites can be determined by mass spectrometry because of a characteristic mass shift caused by diglycine that is retained on ubiquitinated lysine residues within peptides after trypsin digestion. (d) A similar strategy allows differentiation between the various types of ubiquitin chain linkage that can lead to diverse ubiquitin-chain topologies. Depending on the lysine residue in ubiquitin that was used for the ubiquitin-ubiquitin linkage, different linkage-specific signature peptides with characteristic masses are produced by trypsin digestion. These signature peptides can be detected and distinguished by mass spectrometry.