Preparing to read the ubiquitin code: top-down analysis of unanchored ubiquitin tetramers

Abstract

The characterization of polyubiquitin chains has been an analytical challenge for several decades. It has been shown that anchored and unanchored polyubiquitin chains with different isopeptide linkages and lengths exhibit a wide range of profoundly different cellular functions. However, structure function studies have been hindered by the difficulty of characterizing these complex chain structures. This report presents a broadly applicable workflow to characterize ubiquitin tetramers without the need for genetic mutations or reiterative immunoprecipitations. We use a top-down proteomic strategy that exploits ETciD activation on an orbitrap Fusion Lumos, and manual interpretation aided by graphical interpretation of mass shifts to facilitate characterization of chain topography and lysine linkage sites. Our workflow differentiates all topological features of the numerous isomers of tetraubiquitin, which have molecular masses in excess of 34,000 Da, and identifies linkage sites in these branched proteins.

Top-Down Analysis of Branched Proteins Using Mass Spectrometry

“…In foundation maps the fragment ions in the complete spectrum are mapped to the sequences of both unmodified proteins. 13 14. Only a series of b/c ions can be mapped onto the modifier-protein whose C-terminal is conjugated, while series of both b/c and y/z ions are usually recognized in the anchor protein.…”

“…Top-down MS/MS spectra of homo polymers of ubiquitin, 12 isomeric ubiquitin trimers 13 and isomeric ubiquitin tetramers 14 have been reported recently, in which UVPD or CID-assisted ETD was used to activate the heavy ions. However, examples of top-down analysis of branched proteins, proteins modified by ubiquitin or Rub1/NEDD8 have not been reported.…”

Top‐down analysis of novel synthetic branched proteins

“…Ubiquitin is cleaved at R74 to leave GG tags on the modified lysines in peptides from the substrate proteins . This bottom‐up proteomic strategy has the drawback that the GG tag does not allow the distinction of ubiquitination from rubylation (because both ubiquitin and Rub1 have the same C‐terminal LRGG sequence) and does not provide information on the length and connectivity of polymeric modifiers . It also requires that the GG‐tagged peptide be located within a complex mixture of tryptic peptide products.…”

“…Top‐down mass spectrometry is an attractive alternative technique, providing molecular mass and structural information on intact proteins . It has been successfully demonstrated for analyses of a series of intact polyubiquitins and a set of ubiquitinated polypeptide substrates . Top‐down analysis of these branched proteins is facilitated by the high resolution, high mass accuracy, and extensive activation provided by contemporary tandem instrumentation.…”

“…However, these large complex spectra are not easily interpreted manually. We have developed a quasi‐interpretative approach supported by a novel use of graphic interpretation programs to recognize branched proteins, distinguish anchor proteins and modifying proteins, locate mutations and modifications, and assign lysine attachment sites . This strategy is modified (Scheme ) and further tested in the present study, whose objective is to characterize primary structures and branching sites of novel Rub1‐containing branched proteins formed by conjugation catalyzed by with ubiquitin cognate E1 and E2 enzymes in which Rub1 is the target protein or both the target and the modifier.…”