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Post-translational modifications by the covalent attachment of Rub1 (NEDD8), ubiquitin, SUMO, and other small signaling proteins have profound impacts on the functions and fates of cellular proteins. Investigations of the relationship of these bioactive structures and their functions are limited by analytical methods that are scarce and tedious. A novel strategy is reported here for the analysis of branched proteins by top-down mass spectrometry and illustrated by application to four recombinant proteins and one synthetic peptide modified by covalent bonds with ubiquitin or Rub1. The approach allows an analyte to be recognized as a branched protein; the participating proteins to be identified; the site of conjugation to be defined; and other chemical, native, and recombinant modifications to be characterized. In addition to the high resolution and high accuracy provided by the mass spectrometer, success is based on …