Top‐down analysis of novel synthetic branched proteins

Abstract

A strategy for top‐down analysis of branched proteins has been reported earlier, which relies on electron transfer dissociation assisted by collisional activation, and software designed for graphic interpretation of tandem mass spectra and adapted for branched proteins. In the present study, the strategy is applied to identify unknown and novel products of reactions in which rationally mutated proteoforms of Rub1 are used to probe the selectivity of E1 and E2 enzymes normally active in ubiquitination. To test and demonstrate this application, components and attachment sites of three branched dimers are deduced and the mutations are confirmed.

Analysis of the topology of ubiquitin chains

“…These proteins were obtained by different synthetic approaches, such as using linkage-specific enzymes or a nonenzymatic chain assembly strategy. More information on the synthesis of the polyubiquitin chains, ubiquitinated conjugates and rubylated conjugates can be found elsewhere (Castañeda, Liu, Chaturvedi, et al, 2011;Castañeda, Liu, Kashyap, et al, 2011;Chen et al, 2018;Dixon, Castaneda, Kashyap, Wang, & Fushman, 2013;Gomes et al, 2019;Nakasone, Livnat-Levanon, Glickman, Cohen, & Fushman, 2013;Varadan et al, 2002Varadan et al, , 2004. In all cases protein purity was evaluated by SDS-PAGE.…”

“…The strategy below is focused on proteins modified by ubiquitin, however, it can also be applied to recognize proteins modified by other small protein modifiers such as SUMO or Rub1/Nedd8 as recently reported by Gomes et al (2019). 3.1.3.…”

“…A five step strategy was implemented to characterize monouniquitinated proteins (Chen et al, 2018). Additionally, Gomes et al (2019) recently applied this approach to characterize rubylated (Rub-1 modified) proteins. This strategy is summarized in Fig.…”

“…The ubiquitin conjugates studied were composed of ubiquitin carrying chemical modifications or mutations attached to the following anchor proteins: Rub1 (with T72R mutation), E2 enzyme UbcH5b (His-tagged) and a fluorescently labeled and chemically modified PTEN octadecapeptide (residues 5-21) (Chen et al, 2018). Additionally, the rubylated conjugates studied were Rub1 homodimers (with K4F and/or T72R mutations) and a rubylated ubiquitin (with Rub1 E63K and T72R mutation, and Ub lacking the C-terminal GG) (Gomes et al, 2019). The strategy described in this chapter was capable of fully characterizing these conjugates and of assigning chemical modifications that were introduced or inadvertently remained after synthesis.…”

FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer

“…Further purification of gel filtered fractions is needed to produce FITC-insulin conjugates at stoichiometric purity. Peaks 1 to 5 in absorbance chromatogram can be characterized by MALDI-TOF mass spectrometry and top-down analysis method for branched proteins by mass spectrometry [[12], [13], [14]].

Fig.

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