New Publication: Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase

Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase Using an Integrative Mass Spectrometry Strategy. Daniele Canzani, Domnita̧ -Valeria Rusnac, Ning Zheng, and Matthew F. Bush. Anal. Chem. 2019, DOI: 10.1021/acs.analchem.9b02331. (Link)

Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with KLHDC2, a human E3 that recognizes the extreme C-terminus of substrates. Using a combination of native MS, native top-down MS, MS of destabilized samples, and liquid chromatography MS, we identified and quantified a near complete fraction of the KLHDC2-binding peptidome in E. coli cells. This degronome includes peptides that originate from a variety of proteins. Although all identified protein fragments are terminated by diglycine or glycylalanine, the preceding amino acids are diverse. These results significantly expand our understanding of the sequences that can be recognized by KLHDC2, which provides insight into the potential substrates of this E3 in humans. We anticipate that this integrative MS strategy could be leveraged more broadly to characterize the degronomes of other E3 ligase substrate receptors, including those that adhere to the more common N-end rule for substrate recognition. Therefore, this work advances “degronomics,” i.e., identifying, quantifying, and validating functional E3:peptide interactions in order to determine the individual roles of each E3.

New Publication: Collision-Induced Unfolding Is Sensitive to Polarity

Collision-Induced Unfolding is Sensitive to the Polarity of Proteins and Protein Complexes. Seoyeon Hong, Matthew F. Bush. J. Am. Soc. Mass Spectrom. 2019, in press. (Link)

Collision-induced unfolding (CIU) uses ion mobility to probe the structures of ions of proteins and noncovalent complexes as a function of the extent of gas-phase activation prior to analysis. CIU can be sensitive to domain structures, isoform identities, and binding partners, which makes it appealing for many applications. Almost all previous applications of CIU have probed cations. Here, we evaluate the application of CIU to anions and compare the results for anions with those for cations. Towards that end, we developed a “similarity score” that we used to quantify the differences between the results of different CIU experiments and evaluate the significance of those differences relative to the variance of the underlying measurements. Many of the differences between anions and cations that were identified can be attributed to the lower absolute charge states of anions. For example, the extents of the increase in collision cross section over the full range of energies depended strongly on absolute charge state. However, over intermediate energies, there are significant difference between anions and cations with the same absolute charge state. Therefore, CIU is sensitive to the polarity of protein ions. Based on these results, we propose that the utility of CIU to differentiate similar proteins or noncovalent complexes may also depend on polarity. More generally, these results indicate that the relationship between the structures and dynamics of native-like cations and anions deserve further attention and that future studies may benefit from integrating results from ions of both polarities.

Congratulations to Anna Bakhtina!

Congratulations to Anna Bakhtina, who has accepted an offer to enter the Ph.D. program in Genome Sciences at the University of Washington. As an undergraduate student researcher in the Bush Lab, Anna investigated the effects of drift gas selection on the ion mobility of petroleum and biomolecular ions. We are excited to follow her research over in Genome Sciences!

Congratulations to Evan Hubbard!

Congratulations to Evan Hubbard, who has accepted an offer to enter the Ph.D. program in Chemistry at the University of California, Riverside. As an undergraduate student researcher in the Bush Lab, Evan investigated fundamental aspects of electrospray ionization in the context of native mass spectrometry. We are excited to follow his research at UCR!