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

Abstract

The profound effects of ubiquitination on the movement and processing of cellular proteins depend exquisitely on the structures of mono 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.

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.…”