Methods to measure ubiquitin chain length and linkage.

To understand the biological roles of different ubiquitin chains, it is important to determine the types of ubiquitin linkages, the lengths of the polymers, and the combinations of ubiquitin chains attached to substrates. In this chapter, we describe a mass spectrometry-based quantification method of ubiquitin chains, named Ub-AQUA/PRM (ubiquitin-absolute quantification/parallel reaction monitoring), for direct and highly sensitive measurement of the stoichiometry of all eight ubiquitin-ubiquitin linkage types simultaneously. We also show a method to quantify the K48/K63 branched ubiquitin chain, a recently identified ubiquitin signal with a complex topology. In addition, we describe a method to measure ubiquitin chain length of ubiquitylated substrates using a chain protector and limited digestion of ubiquitin chains, named Ub-ProT (ubiquitin chain protection from trypsinization). These strategies will contribute to our knowledge of polymeric ubiquitin signals and permit investigation of new mechanisms concerning the ubiquitin code.