Category: Uncategorized

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.

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 Rae Eaton, who successfully defended her thesis to a packed audience! We all wish you great success in your future endeavors!

Congratulations to Daniele Canzani, who was recently awarded a Graduate Student Merit Fellowship. This award recognizes his accomplishments in research and coursework. We all thank the support of Mickey and Karen Schurr, who have endowed this award.

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, 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!

The Department of Chemistry congratulates Assistant Professor Matthew Bush on his promotion to associate professor with tenure, effective September 16, 2019. For more information, see the announcement on the Department of Chemistry website.

Principles of Ion Selection, Alignment, and Focusing in Tandem Ion Mobility Implemented Using Structures for Lossless Ion Manipulations (SLIM).
Rachel M. Eaton, Samuel J. Allen, Matthew F. Bush. J. Am. Soc. Mass Spectrom. 2019, DOI: 10.1007/s13361-019-02170-1. (Link)

Tandem ion mobility (IM) enables the characterization of subpopulations of ions from larger ensembles, including differences that cannot be resolved in a single dimension of IM. Tandem IM consists of at least two IM regions that are each separated by an ion selection region. In many implementations of tandem IM, ions eluting from a dimension of separation are filtered and immediately transferred to the subsequent dimension of separation (selection-only experiments). We recently reported a mode of operation in which ions eluting from a dimension are trapped prior to the subsequent dimension (selection-trapping experiments), which was implemented on an instrument constructed using the structures for lossless ion manipulations (SLIM) architecture. Here, we use a combination of experiments and trajectory simulations to characterize aspects of the selection, trapping, and separation processes underlying these modes of operation. For example, the actual temporal profile of filtered ions can be very similar to the width of the waveforms used for selection, but depending on experimental parameters, can differ by up to ± 500 μs. Experiments and simulations indicate that ions in selection-trapping experiments can be spatially focused between dimensions, which removes the broadening that occurred during the preceding dimension. During focusing, individual ions are thermalized, which aligns and establishes common initial conditions for the subsequent dimension. Therefore, selection-trapping experiments appear to offer significant advantages relative to selection-only experiments, which we anticipate will become more pronounced in future experiments that make use of longer IM separations, additional dimensions of analysis, and the outcomes of this study.

The Bush Lab was just awarded a grant from the National Institutes of Health for a project titled “Increasing the Selectivity of Hybrid Mass Spectrometry Using Multidimensional Ion Mobility Spectrometry” (R01 GM130708). We are excited to pursue this research and are grateful for this financial support from the NIH.