About

The Bush Lab is a research group in the Department of Chemistry and the Biological Physics, Structure & Design Program at the University of Washington. Our research focuses on the development and application of mass spectrometry and ion mobility spectrometry techniques to elucidate the structures and assembly of protein complexes and subcellular machines.

Research topics

• Chemistry
• Biophysics
• Biochemistry
• Cell biology
• Biology
• Physics
• Computational chemistry
• Chemical physics
• Organic chemistry
• Atomic physics
• Nuclear physics

Selected publications

• Activation mechanism of small heat shock protein HSPB5 revealed by disease-associated mutants

  Proceedings of the National Academy of Sciences · 2025-05-16 · 9 citations

  articleOpen access

  Found from bacteria to humans, small heat shock proteins (sHSPs) are the least understood protein chaperones. HSPB5 (or αB-crystallin) is among the most widely expressed of the 10 human sHSPs, including in muscle, brain, and eye lens where it is constitutively present at high levels. A high content of disorder in HSPB5 has stymied efforts to uncover how its structure gives rise to function. To uncover its mechanisms of action, we compared human HSPB5 and two disease-associated mutants, R120G and D109H. Expecting to learn how the mutations lead to loss of function, we found instead that the mutants are constitutively activated chaperones while wild-type HSPB5 can transition reversibly between nonactivated (low activity) and activated (high activity) states in response to changing conditions. Techniques that provide information regarding interactions and accessibility of disordered regions revealed that the disordered N-terminal regions (NTR) that are required for chaperone activity exist in a complicated interaction network within HSPB5 oligomers and are sequestered from solvent in nonactivated states. Either mutation or an activating pH change causes rearrangements in the network that expose parts of the NTR, making them more available to bind an aggregating client. Although beneficial in the short-term, failure of the mutants to adopt a state with lower activity and lower NTR accessibility leads to increased coaggregation propensity and, presumably, early cataract. The results support a model where chaperone activity and solubility are modulated through the quasi-ordered NTR and its multiple competing interactions.

  PublisherDOI

• Disorder with consequence: Phosphorylation sites in HSPB5 yield distinct structural outcomes

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-28

  preprintOpen access

  HSPB5, a member of the small heat shock protein family, acts as a first responder to cellular stress. One proposed mechanism of stress activation is phosphorylation. HSPB5 is phosphorylated at three sites-serine residues at positions 19, 45, and 59-located within its disordered N-terminal region (NTR). The extent of phosphorylation of the different sites leads to different cellular outcomes. HSPB5 forms polydisperse oligomers, where the NTR regions can either be exposed to the solvent or buried within the oligomer, forming internal contacts. We assessed the effect of single and triple phospho-mimicry on HSPB5 oligomeric properties. Our findings indicate that single phosphorylation causes localized and subtle changes in oligomer size, subunit exchange, hydrogen-deuterium protection patterns, and ability to delay aggregation of a known eye lens client, γD-crystallin. In contrast, the triple phosphomimic shows substantial structural and functional alterations. We provide a rationale for the increased chaperone activity observed in the S45D phosphomimic. Taken together, our results offer structural insights into how different phosphorylation events lead to distinct cellular outcomes.

  PublisherOA PDFDOI

• SLIMPHONY: A SLIM-Based Instrument That Orchestrates Complex Ion Mobility–Mass Spectrometry Experiments

  Journal of the American Society for Mass Spectrometry · 2025-09-26

  articleOpen accessSenior authorCorresponding

  The inherent heterogeneity of biological macromolecules offers a unique challenge for analysis. The combination of ion mobility (IM) and mass spectrometry (MS) is sensitive to the size, shape, and dynamics of, for example, proteins and their complexes. Combining multiple dimensions of ion mobility and mass spectrometry (IM-IM-MS) while leveraging unique gas-phase manipulations between dimensions has great potential for increasing the information content for challenging analytes. Here, we introduce an instrument, SLIMPHONY, which was built using the Structures for Lossless Ion Manipulations (SLIM) architecture. SLIMPHONY is unique in that eight independently controlled traveling-wave regions work in concert to enable complex, multidimensional separations. Single-dimension IM-MS experiments were used to separate a mixture of protein and protein-complex ions and demonstrate that the peak-to-peak resolution increases roughly with the square root of the separation length for a pair of hexakis(fluoroalkoxy)phosphazine ions. Ion selection and trapping between dimensions was then used to probe the gas-phase unfolding of a subpopulation of ubiquitin ions. Finally, by varying the guard potential used to confine ions, we demonstrate tunable activation of ubiquitin subpopulations, which we analyzed using IM separations of various lengths. With the ability to select and activate ions in multiple regions, to vary the number of dimensions of IM, and to control the length of IM separation, SLIMPHONY is a flexible platform for characterizing protein ions.

  PublisherOA PDFDOI

• Selecting Reducing Agents for Native Mass Spectrometry

  ChemRxiv · 2024-02-01 · 1 citations

  preprintOpen accessSenior author

  In protein science, reducing agents are often added in cases where the protein, its cofactors, or its ligands are sensitive to oxidative stress. Although many native mass spectrometry (MS) workflows would benefit from maintaining reducing conditions throughout the analysis, there is a lack of consensus regarding the compatibility of reducing agents with that approach. This study systematically examines the effects of dithiothreitol (DTT), β-mercaptoethanol (βME), and tris(2-carboxyethyl)phosphine (TCEP) on the native mass spectra of protein standards. The selection and concentration of the reducing agents affected both the extent of nonspecific adduction and the charge-state distribution of the analyte. For a protein without disulfide bonds, increasing concentrations of DTT or βME resulted in shifts to higher charge states, whereas increasing concentrations of TCEP resulted in shifts to lower charge states. Based on these trends and additional properties of the reducing agents, we propose that DTT and βME are mild supercharging agents and that TCEP is a potent charge-reducing agent. The selection and concentration of the reducing agents, as well as the sample pH, also affected the extent of disulfide bond reduction, and for βME, the extent of covalent adduction by that molecule to cysteine. These results offer insights into the compatibility of reducing agents with the goal of obtaining high-quality native mass spectra. Based on our results, we present recommendations for the use of reducing agents in native MS experiments.

  PublisherOA PDFDOI

• Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism

  Molecular Pharmacology · 2024-04-05 · 8 citations

  articleOpen access

  Liver fatty acid binding protein (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data is available for human FABP1 (hFABP1). FABP1 has a large binding pocket and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (K_d,1=0.2 µM) and low affinity (K_d,2&gt;10 µM) binding sites. Nine drugs bound to hFABP1 with K_d values ranging from 1 to 20 µM. None of the tested drugs completely displaced DAUDA from hFABP1 and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The k_cat of diclofenac hydroxylation by CYP2C9 was decreased by ~50% (p&lt;0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. <b>Significance Statement</b> Many commonly prescribed drugs bind FABP1 forming ternary complexes with FABP1 and the fluorescent fatty acid DAUDA. These findings suggests that drugs will bind to apo-FABP1 and fatty acid bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1 may alter drug disposition processes <i>in vivo</i>.

  PublisherOA PDFDOI

• High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid

  Journal of Proteome Research · 2024-07-05 · 3 citations

  articleOpen accessSenior authorCorresponding

  In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

  PublisherOA PDFDOI

• Selecting Reducing Agents for Native Mass Spectrometry

  ChemRxiv · 2024-01-31 · 1 citations

  preprintOpen accessSenior author

  In protein science, reducing agents are often added in cases where the protein, its cofactors, or its ligands are sensitive to oxidative stress. Although many native mass spectrometry (MS) workflows would benefit from maintaining reducing conditions throughout the analysis, there is a lack of consensus regarding the compatibility of reducing agents with that approach. This study systematically examines the effects of dithiothreitol (DTT), β-mercaptoethanol (βME), and tris(2-carboxyethyl)phosphine (TCEP) on the native mass spectra of protein standards. The selection and concentration of the reducing agents affected both the extent of nonspecific adduction and the charge-state distribution of the analyte. For a protein without disulfide bonds, increasing concentrations of DTT or βME resulted in shifts to higher charge states, whereas increasing concentrations of TCEP resulted in shifts to lower charge states. Based on these trends and additional properties of the reducing agents, we propose that DTT and βME are mild supercharging agents and that TCEP is a potent charge-reducing agent. The selection and concentration of the reducing agents, as well as the sample pH, also affected the extent of disulfide bond reduction, and for βME, the extent of covalent adduction of cysteine by that molecule. These results offer insights into the compatibility of reducing agents with the goal of obtaining high-quality native mass spectra. Based on our results, we present recommendations for the use of reducing agents in native MS experiments.

  PublisherOA PDFDOI

• An artificial intelligence accelerated virtual screening platform for drug discovery

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-03-29 · 4 citations

  preprintOpen access

  Abstract Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we developed a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screened multi-billion compound libraries against two unrelated targets, a novel ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel Na V 1.7. On both targets, we discover hits, including seven novel hits (14% hit rate) to KLHDC2 and four novel hits (44% hit rate) to Na V 1.7 with single digit micromolar binding affinities. Screening in both cases was completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

  PublisherOA PDFDOI

• Metastability of Protein Solution Structures in the Absence of a Solvent: Rugged Energy Landscape and Glass-like Behavior

  Journal of the American Chemical Society · 2024-04-10 · 14 citations

  articleOpen access

  Native ion mobility/mass spectrometry is well-poised to structurally screen proteomes but characterizes protein structures in the absence of a solvent. This raises long-standing unanswered questions about the biological significance of protein structures identified through ion mobility/mass spectrometry. Using newly developed computational and experimental ion mobility/ion mobility/mass spectrometry methods, we investigate the unfolding of the protein ubiquitin in a solvent-free environment. Our data suggest that the folded, solvent-free ubiquitin observed by ion mobility/mass spectrometry exists in a largely native fold with an intact β-grasp motif and α-helix. The ensemble of folded, solvent-free ubiquitin ions can be partitioned into kinetically stable subpopulations that appear to correspond to the structural heterogeneity of ubiquitin in solution. Time-resolved ion mobility/ion mobility/mass spectrometry measurements show that folded, solvent-free ubiquitin exhibits a strongly stretched-exponential time dependence, which simulations trace to a rugged energy landscape with kinetic traps. Unfolding rate constants are estimated to be approximately 800 to 20,000 times smaller than in the presence of water, effectively quenching the unfolding process on the time scale of typical ion mobility/mass spectrometry measurements. Our proposed unfolding pathway of solvent-free ubiquitin shares substantial characteristics with that established for the presence of solvent, including a polarized transition state with significant native content in the N-terminal β-hairpin and α-helix. Our experimental and computational data suggest that (1) the energy landscape governing the motions of folded, solvent-free proteins is rugged in analogy to that of glassy systems; (2) large-scale protein motions may at least partially be determined by the amino acid sequence of a polypeptide chain; and (3) solvent facilitates, rather than controls, protein motions.

  PublisherOA PDFDOI

• An artificial intelligence accelerated virtual screening platform for drug discovery

  Nature Communications · 2024-09-05 · 176 citations

  articleOpen access

  Abstract Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel Na V 1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to Na V 1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

  PublisherOA PDFDOI

Recent grants

• Increasing the Selectivity of Hybrid Mass Spectrometry using Multidimensional Ion Mobility Spectrometry

  NIH · $1.3M · 2019–2025

• Bridging the Gap Between Observables from Ion Mobility Mass Spectrometry and the Structures of Native Proteins

  NSF · $412k · 2018–2022

• EAGER: Development of a Modular Ion Mobility Mass Spectrometry System for Structural Biology

  NSF · $146k · 2015–2017

• Advancing Native Mass Spectrometry for Probing Protein Equilibria and Dynamics

  NSF · $470k · 2022–2027

Frequent coauthors

• Evan R. Williams

  University of California, Berkeley

  49 shared

• Jos Oomens

  University of Amsterdam

  30 shared

• Richard J. Saykally

  25 shared

• Hugh I. Kim

  Korea University

  19 shared

• Valérie Gabelica

  University of Geneva

  19 shared

• Kevin Giles

  19 shared

• Rebecca A. Jockusch

  18 shared

• Nick C. Polfer

  University of Florida

  18 shared

Labs

• Bush LabPI

Education

• Ph. D., Department of Chemistry

  University of California Berkeley

  2008

• B.A.

  Carleton College

  2003

Awards & honors

• Arthur F. Findeis Award for Achievements by a Young Analytic…
• Sloan Research Fellowship, Alfred P. Sloan Foundation (2014)
• Young Investigator Award in Analytical Chemistry, Eli Lilly…
• Research Award, American Society for Mass Spectrometry (2013…
• Junior Research Fellowship, Jesus College, University of Oxf…