| Abstract
| - Ubiquitin (Ub) is a 76 amino acid polypeptide thatmodifies a wide range of proteins in the types of monomeror polymers, and functional consequence of ubiquitinationis modulated by the length and topologies of polyUbchains. Whereas polyUb chains are usually analyzed byfully trypsin digestion and mass spectrometry (MS), wepresent here a middle-down strategy to characterize thestructure of polyUb chains by high-resolution mass spectrometry (MS). Under optimized condition, native foldedpolyUb is partially trypsinized exclusively at the R74residue, generating a large Ub fragment (1−74 residuestermed UbR74) and its ubiquitinated form with a diglycinetag (UbR74-GG). The molar ratio between UbR74 andUbR74-GG reflects the length of homogeneous polyUbchains (i.e., 1:1 for the dimer, 1:2 for the trimer, 1:3 forthe tetramer, and so on). Moreover, lysine residues inubiquitin used for chain linkages are detectable by MS/MS and MS/MS/MS of large GG-tagged Ub fragments. Thestrategy was validated using a number of ubiquitin polymers, including K48-linked human di-Ub, K63-linkedhuman tetra-Ub, as well as His-tagged polyUb chainspurified from yeast under native condition. The potentialof this strategy to analyze polyUb chains with mixedlinkages (e.g., forked chains) is also discussed. Together,this middle-down MS strategy provides a novel complementary method for studying the length and linkages ofcomplex polyUb chain structures.
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