Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top-down mass spectrometry.

The profound effects of ubiquitination on the movement and processing of cellular proteins depend exquisitely on the structures of monoubiquitin and polyubiquitin modifications. Unconjugated polyubiquitins also have a variety of intracellular functions. Structures and functions are not well correlated yet, because the structures of polyubiquitins and polyubiquitin modifications of proteins are difficult to decipher. We are moving towards a robust strategy to provide that structural information. In this report electron transfer dissociation mass spectra of six synthetic ubiquitin trimers (multiply branched proteins with molecular masses exceeding 25,600 Da) are examined using an Orbitrap Fusion Lumos instrument to determine how top-down mass spectrometry can characterize the chain topology and linkage sites in a single, facile workflow. The efficacy of this method relies on the formation, detection, and interpretation of extensive fragmentation.