Michael T Marty
- Associate Professor, Chemistry and Biochemistry-Sci
- Assistant Professor, Chemistry and Biochemistry - Med
- Member of the Graduate Faculty
- Associate Professor, BIO5 Institute
- (520) 621-1501
- Carl S. Marvel Labs of Chem., Rm. 542
- Tucson, AZ 85721
- mtmarty@arizona.edu
Biography
My research program focuses on the characterization of the structure, biophysics, and interactions of membrane proteins and peptides by mass spectrometry and builds upon my background in biochemistry and analytical chemistry. As a graduate student, I worked with Prof. Stephen Sligar at the University of Illinois Urbana-Champaign to interface nanodiscs, self-assembled nanoscale lipoprotein complexes, with various analytical techniques including photonic microring resonator biosensors and mass spectrometry (MS). I also developed new methods for forming and characterizing nanodiscs using heterogeneous populations of membrane proteins. Through a collaboration with Prof. Michael Gross (Washington University, St. Louis) to perform native MS on nanodiscs, I was the first to show that nanodiscs could be maintained intact in the gas phase for native MS.
As a postdoc with Prof. Dame Carol Robinson at the University of Oxford, I continued this vein of research but shifted my focus to native MS of membrane protein oligomers in nanodiscs. Challenging data motivated me to develop new MS data analysis algorithms and software that have since been widely adopted in the field. Combining new data analysis approaches with advances in high-mass Orbitrap instrumentation, I discovered that membrane protein oligomers ejected from nanodiscs retain a large number of bound lipids. I found that the stoichiometry of lipids bound to the membrane protein agrees with simulations for various shells of lipid interactions, including the lipid annular belt. Building on these results, I led a study to characterize nanodiscs with heterogeneous lipid populations by native MS, which has been further expanded to a wide range of lipids by my independent research group.
I began my independent research laboratory in the Department of Chemistry and Biochemistry at the University of Arizona in 2016. To advance nanodiscs as a platform for native MS, my research group engineered a series of mutant membrane scaffold proteins that facilitate the interpretation of mass spectra from membrane protein nanodiscs. I have also continued development of UniDec software, adding new features for high-throughput deconvolution of native MS data and improvements to the deconvolution algorithm to reduce artifacts. Combining these techniques, we discovered that charge manipulation reagents provide a powerful tool for manipulating the gas-phase stability of nanodiscs. We were the first to measure the mass of an intact nanodisc with a membrane protein inside, which revealed the oligomeric state of the membrane protein without disrupting the membrane. We then applied native MS of intact nanodiscs to characterize interactions of antimicrobial peptides with nanodiscs containing different lipids. Our novel approach revealed a wide range of different types of peptide-peptide and peptide-lipid interactions between different antimicrobial peptides.
Degrees
- Ph.D. Chemistry
- University of Illinois, Urbana, Illinois, United States
- Analysis of Membrane Systems Using Lipoprotein Nanodiscs
- B.A. Chemistry and Mathematics
- St. Olaf College, Northfield, Minnesota, United States
- Single and Double Displacement of Ethylene Bridges in a Molybdenum Dithiolate Dimer with Nitrile-Activated Transition Metal Carbonyl Complexes
Work Experience
- University of Oxford (2013 - 2016)
Awards
- Galileo Circle Curie Award
- University of Arizona College of Science, Fall 2020
- Outstanding Reviewer Award
- Journal of the American Society for Mass Spectrometry, Summer 2019
- NSF Career Award
- National Science Foundation, Spring 2019
- American Society for Mass Spectrometry Research Award
- American Society for Mass Spectrometry, Summer 2018
- Bisgrove Scholar Award
- Science Foundation Arizona, Spring 2017
Interests
Research
Membrane proteins play a number of critical biochemical roles and make up the majority of drug targets. Despite their importance, membrane proteins remain challenging systems for analysis due to their amphipathic nature and low expression levels. Moreover, the lipid bilayer can play an important but largely unexplored role in regulating membrane protein structure and function. New analytical and biochemical methods are necessary to better understand and design drugs to target membrane proteins.Research in the Marty lab is focused on developing mass spectrometry methods to study the structure and biophysics of membrane proteins. Working at the interface of Analytical Chemistry and Biochemistry, we utilize lipoprotein Nanodiscs to solubilize membrane proteins in a lipid bilayer with a defined composition. Nanodiscs are nanoscale discoidal lipid bilayers encircled by two amphipathic membrane scaffold proteins.By using nondenaturing nano-electrospray ionization, we can keep the Nanodisc complex intact inside the mass spectrometer and gradually release the membrane protein with collisional activation. This approach, known as noncovalent or native mass spectrometry, allows us to measure the stoichiometry and lipid composition in large protein-lipid complexes to better understand protein-lipid interactions in membrane protein receptors and transporters.Following electrospray ionization, Nanodiscs are activated by gas-phase collision induced dissociation (CID) to release the membrane protein bound to dozens of lipids for high-resolution Orbitrap mass analysis, which results in a highly complex spectral pattern.Because mass spectra of Nanodiscs are highly complex, we are interested in development of new computational approaches and software for analysis of native MS data. This work builds on UniDec software we have developed to rapidly and robustly deconvolve mass and ion mobility spectra. We are also interested in developing cross-linking and covalent labeling approaches to use bottom-up proteomics to study the structure of membrane proteins in their native membrane environment.
Teaching
My teaching interests focus on teaching analytical approaches to study biochemical systems. I have taught graduate Mass Spectrometry, which focused on characterization of therapeutic antibodies through mass spectrometry techniques. I have also taught undergraduate Analytical Chemistry and have restructured the curriculum to focus on the analytical methods and teach statistical methods through cloud-based Python coding.
Courses
2024-25 Courses
-
Analytical Chemistry
CHEM 325 (Fall 2024) -
Analytical Chemistry Lab
CHEM 326 (Fall 2024) -
Directed Research
BIOC 392 (Fall 2024) -
Dissertation
CHEM 920 (Fall 2024) -
Exchange Chemical Info
CHEM 695B (Fall 2024) -
Honors Directed Research
BIOC 492H (Fall 2024) -
Introduction to Research
BIOC 792 (Fall 2024) -
Research
CHEM 900 (Fall 2024)
2023-24 Courses
-
Dissertation
BIOC 920 (Spring 2024) -
Dissertation
CHEM 920 (Spring 2024) -
Exchange Chemical Info
CHEM 695B (Spring 2024) -
Introduction to Research
BIOC 792 (Spring 2024) -
Mass Spectrometry
CHEM 525A (Spring 2024) -
Professional Devlpmnt Part II
CHEM 595B (Spring 2024) -
Research
BIOC 900 (Spring 2024) -
Research
CHEM 900 (Spring 2024) -
Analytical Chemistry
CHEM 325 (Fall 2023) -
Analytical Chemistry
CHEM 696A (Fall 2023) -
Analytical Chemistry Lab
CHEM 326 (Fall 2023) -
Directed Research
PSIO 492 (Fall 2023) -
Dissertation
CHEM 920 (Fall 2023) -
Exchange Chemical Info
CHEM 695B (Fall 2023) -
Honors Thesis
CHEM 498H (Fall 2023) -
Introduction to Research
BIOC 792 (Fall 2023) -
Professional Devlpmnt Part 1
CHEM 595A (Fall 2023) -
Research
CHEM 900 (Fall 2023)
2022-23 Courses
-
Directed Research
CHEM 392 (Spring 2023) -
Dissertation
BIOC 920 (Spring 2023) -
Dissertation
CHEM 920 (Spring 2023) -
Exchange Chemical Info
CHEM 695B (Spring 2023) -
Independent Study
PSIO 399 (Spring 2023) -
Research
BIOC 900 (Spring 2023) -
Research
CHEM 900 (Spring 2023) -
Directed Research
CHEM 392 (Fall 2022) -
Dissertation
CHEM 920 (Fall 2022) -
Exchange Chemical Info
CHEM 695B (Fall 2022) -
Research
CHEM 900 (Fall 2022)
2021-22 Courses
-
Analytical Chemistry
CHEM 696A (Spring 2022) -
Directed Research
CHEM 492 (Spring 2022) -
Dissertation
BIOC 920 (Spring 2022) -
Dissertation
CHEM 920 (Spring 2022) -
Exchange Chemical Info
CHEM 695B (Spring 2022) -
Honors Thesis
MCB 498H (Spring 2022) -
Mass Spectrometry
CHEM 525A (Spring 2022) -
Research
CHEM 900 (Spring 2022) -
Analytical Chemistry
CHEM 325 (Fall 2021) -
Directed Research
MCB 792 (Fall 2021) -
Dissertation
CHEM 920 (Fall 2021) -
Exchange Chemical Info
CHEM 695B (Fall 2021) -
Honors Thesis
MCB 498H (Fall 2021) -
Introduction to Research
BIOC 792 (Fall 2021) -
Research
CHEM 900 (Fall 2021)
2020-21 Courses
-
Analytical Chemistry
CHEM 325 (Spring 2021) -
Analytical Chemistry Lab
CHEM 326 (Spring 2021) -
Dissertation
BIOC 920 (Spring 2021) -
Dissertation
CHEM 920 (Spring 2021) -
Exchange Chemical Info
CHEM 695B (Spring 2021) -
Honors Independent Study
MCB 399H (Spring 2021) -
Honors Thesis
CHEM 498H (Spring 2021) -
Research
BIOC 900 (Spring 2021) -
Research
CHEM 900 (Spring 2021) -
Analytical Chemistry
CHEM 325 (Fall 2020) -
Directed Research
MCB 792 (Fall 2020) -
Dissertation
CHEM 920 (Fall 2020) -
Exchange Chemical Info
CHEM 695B (Fall 2020) -
Honors Independent Study
MCB 399H (Fall 2020) -
Honors Thesis
CHEM 498H (Fall 2020) -
Journal Club
BIOC 595B (Fall 2020) -
Research
CHEM 900 (Fall 2020)
2019-20 Courses
-
Directed Research
CHEM 492 (Spring 2020) -
Dissertation
CHEM 920 (Spring 2020) -
Exchange Chemical Info
CHEM 695B (Spring 2020) -
Honors Directed Research
BIOC 392H (Spring 2020) -
Honors Thesis
PSIO 498H (Spring 2020) -
Introduction to Research
BIOC 795A (Spring 2020) -
Journal Club
BIOC 595B (Spring 2020) -
Research
CHEM 900 (Spring 2020) -
Analytical Chemistry
CHEM 325 (Fall 2019) -
Dissertation
CHEM 920 (Fall 2019) -
Exchange Chemical Info
CHEM 695B (Fall 2019) -
Honors Thesis
PSIO 498H (Fall 2019) -
Journal Club
BIOC 595B (Fall 2019) -
Research
CHEM 900 (Fall 2019)
2018-19 Courses
-
Directed Research
CHEM 392 (Spring 2019) -
Dissertation
CHEM 920 (Spring 2019) -
Exchange Chemical Info
CHEM 695B (Spring 2019) -
Honors Independent Study
BIOC 499H (Spring 2019) -
Introduction to Research
BIOC 795A (Spring 2019) -
Introduction to Research
MCB 795A (Spring 2019) -
Journal Club
BIOC 595B (Spring 2019) -
Research
BIOC 900 (Spring 2019) -
Research
CHEM 900 (Spring 2019) -
Research
MCB 900 (Spring 2019) -
Senior Capstone
BIOC 498 (Spring 2019) -
Thesis
MCB 910 (Spring 2019) -
Exchange Chemical Info
CHEM 695B (Fall 2018) -
Honors Independent Study
BIOC 399H (Fall 2018) -
Journal Club
BIOC 595B (Fall 2018) -
Research
CHEM 900 (Fall 2018) -
Senior Capstone
BIOC 498 (Fall 2018) -
Thesis
MCB 910 (Fall 2018)
2017-18 Courses
-
Directed Research
BIOC 392 (Spring 2018) -
Exchange Chemical Info
CHEM 695B (Spring 2018) -
Honors Independent Study
BIOC 399H (Spring 2018) -
Honors Thesis
BIOC 498H (Spring 2018) -
Journal Club
BIOC 595B (Spring 2018) -
Research
BIOC 900 (Spring 2018) -
Research
CHEM 900 (Spring 2018) -
Senior Capstone
BIOC 498 (Spring 2018) -
Exchange Chemical Info
CHEM 695B (Fall 2017) -
Honors Independent Study
BIOC 399H (Fall 2017) -
Honors Thesis
BIOC 498H (Fall 2017) -
Introduction to Research
MCB 795A (Fall 2017) -
Journal Club
BIOC 595B (Fall 2017) -
Mass Spectrometry
CHEM 525A (Fall 2017) -
Research
CHEM 900 (Fall 2017) -
Senior Capstone
BIOC 498 (Fall 2017)
2016-17 Courses
-
Exchange Chemical Info
CHEM 695B (Spring 2017) -
Introduction to Research
BIOC 795A (Spring 2017) -
Introduction to Research
MCB 795A (Spring 2017) -
Research
CHEM 900 (Spring 2017) -
Senior Capstone
MCB 498 (Spring 2017) -
Honors Independent Study
BIOC 499H (Fall 2016) -
Mass Spectrometry
CHEM 525A (Fall 2016) -
Senior Capstone
MCB 498 (Fall 2016)
Scholarly Contributions
Journals/Publications
- Hale, O. J., Cooper, H. J., & Marty, M. T. (2023). High-Throughput Deconvolution of Native Protein Mass Spectrometry Imaging Data Sets for Mass Domain Analysis. Analytical chemistry, 95(37), 14009-14015.More infoProtein mass spectrometry imaging (MSI) with electrospray-based ambient ionization techniques, such as nanospray desorption electrospray ionization (nano-DESI), generates data sets in which each pixel corresponds to a mass spectrum populated by peaks corresponding to multiply charged protein ions. Importantly, the signal associated with each protein is split among multiple charge states. These peaks can be transformed into the mass domain by spectral deconvolution. When proteins are imaged under native/non-denaturing conditions to retain non-covalent interactions, deconvolution is particularly valuable in helping interpret the data. To improve the acquisition speed, signal-to-noise ratio, and sensitivity, native MSI is usually performed using mass resolving powers that do not provide isotopic resolution, and conventional algorithms for deconvolution of lower-resolution data are not suitable for these large data sets. UniDec was originally developed to enable rapid deconvolution of complex protein mass spectra. Here, we developed an updated feature set harnessing the high-throughput module, MetaUniDec, to deconvolve each pixel of native MSI data sets and transform /-domain image files to the mass domain. New tools enable the reading, processing, and output of open format .imzML files for downstream analysis. Transformation of data into the mass domain also provides greater accessibility, with mass information readily interpretable by users of established protein biology tools such as sodium dodecyl sulfate polyacrylamide gel electrophoresis.
- Jayasekera, H. S., Mohona, F. A., Ewbank, M., & Marty, M. T. (2023). SIMULTANEOUS NATIVE MASS SPECTROMETRY ANALYSIS OF SINGLE AND DOUBLE MUTANTS TO PROBE LIPID BINDING TO MEMBRANE PROTEINS. bioRxiv : the preprint server for biology.More infoLipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that the W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into lipid binding to specific sites on membrane proteins.
- Larson, E. J., Pergande, M. R., Moss, M. E., Rossler, K. J., Wenger, R. K., Krichel, B., Josyer, H., Melby, J. A., Roberts, D. S., Pike, K., Shi, Z., Chan, H. J., Knight, B., Rogers, H. T., Brown, K. A., Ong, I. M., Jeong, K., Marty, M. T., McIlwain, S. J., & Ge, Y. (2023). MASH Native: a unified solution for native top-down proteomics data processing. Bioinformatics (Oxford, England), 39(6).More infoNative top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking.
- Liu, W., Jayasekera, H. S., Sanders, J. D., Zhang, G., Viner, R., & Marty, M. T. (2023). Online Buffer Exchange Enables Automated Membrane Protein Analysis by Native Mass Spectrometry. Analytical chemistry, 95(47), 17212-17219.More infoMembrane proteins represent the majority of clinical drug targets and are actively involved in a range of cellular processes. However, the complexity of membrane mimetics for membrane protein solubilization poses challenges for native mass spectrometry (MS) analyses. The most common approach for native MS analyses of membrane proteins remains offline buffer exchange into native MS-compatible buffers prior to manual sample loading into static nano-ESI emitters. This laborious process requires relatively high sample consumption and optimization for the individual proteins. Here, we developed online buffer exchange coupled to native mass spectrometry (OBE-nMS) for analyzing membrane proteins in different membrane mimetics, including detergent micelles and nanodiscs. Detergent screening for OBE-nMS reveals that mobile phases containing ammonium acetate with lauryl-dimethylamine oxide are most universal for characterizing both bacterial and mammalian membrane proteins in detergent. Membrane proteins in nanodiscs simply require ammonium acetate as the mobile phase. To preserve the intact nanodiscs, a novel switching electrospray approach was used to capture the high-flow separation on the column with a low-flow injection to MS. Rapid OBE-nMS completes each membrane protein measurement within minutes and thus enables higher-throughput assessment of membrane protein integrity prior to its structural elucidation.
- Lyu, J., Zhang, T., Marty, M. T., Clemmer, D., Russell, D. H., & Laganowsky, A. (2023). Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions. bioRxiv : the preprint server for biology.More infoStructural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D--oct-2-ulosonic acid (Kdo)-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5'-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.
- Odenkirk, M. T., Zhang, G., & Marty, M. T. (2023). Do Nanodisc Assembly Conditions Affect Natural Lipid Uptake?. Journal of the American Society for Mass Spectrometry, 34(9), 2006-2015.More infoLipids play critical roles in modulating membrane protein structure, interactions, and activity. Nanodiscs provide a tunable membrane mimetic that can model these endogenous protein-lipid interactions in a nanoscale lipid bilayer. However, most studies of membrane proteins with nanodiscs use simple synthetic lipids that lack the headgroup and fatty acyl diversity of natural extracts. Prior research has successfully used natural lipid extracts in nanodiscs that more accurately mimic natural environments, but it is not clear how nanodisc assembly may bias the incorporated lipid profiles. Here, we applied lipidomics to investigate how nanodisc assembly conditions affect the profile of natural lipids in nanodiscs. Specifically, we tested the effects of assembly temperature, nanodisc size, and lipidome extract complexity. Globally, our analysis demonstrates that the lipids profiles are largely unaffected by nanodisc assembly conditions. However, a few notable changes emerged within individual lipids and lipid classes, such as a differential incorporation of cardiolipin and phosphatidylglycerol lipids from the polar lipid extract at different temperatures. Conversely, some classes of brain lipids were affected by nanodisc size at higher temperatures. Collectively, these data enable the application of nanodiscs to study protein-lipid interactions in complex lipid environments.
- Phung, W., Bakalarski, C. E., Hinkle, T. B., Sandoval, W., & Marty, M. T. (2023). UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data. Analytical chemistry, 95(30), 11491-11498.More infoRecent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec processing pipeline (UPP) for the analysis of batched biotherapeutic intact MS data. UPP is built into the UniDec software package, which provides fast processing, deconvolution, and peak detection. The user and programming interfaces for UPP read a spreadsheet that contains the data file names, deconvolution parameters, and quantitation settings. After iterating through the spreadsheet and analyzing each file, it returns a spreadsheet of results and HTML reports. We demonstrate the use of UPP to measure the correct pairing percentage on a set of bispecific antibody data and to measure drug-to-antibody ratios from antibody-drug conjugates. Moreover, because the software is free and open-source, users can easily build on this platform to create customized workflows and calculations. Thus, UPP provides a flexible workflow that can be deployed in diverse settings and for a wide range of biotherapeutic applications.
- Reid, D. J., Dash, T., Wang, Z., Aspinwall, C. A., & Marty, M. T. (2023). Investigating Daptomycin-Membrane Interactions Using Native MS and Fast Photochemical Oxidation of Peptides in Nanodiscs. Analytical chemistry, 95(11), 4984-4991.More infoDaptomycin is a cyclic lipopeptide antibiotic that targets the lipid membrane of Gram-positive bacteria. Membrane fluidity and charge can affect daptomycin activity, but its mechanisms are poorly understood because it is challenging to study daptomycin interactions within lipid bilayers. Here, we combined native mass spectrometry (MS) and fast photochemical oxidation of peptides (FPOP) to study daptomycin-membrane interactions with different lipid bilayer nanodiscs. Native MS suggests that daptomycin incorporates randomly and does not prefer any specific oligomeric states when integrated into bilayers. FPOP reveals significant protection in most bilayer environments. Combining the native MS and FPOP results, we observed that stronger membrane interactions are formed with more rigid membranes, and pore formation may occur in more fluid membranes to expose daptomycin to FPOP oxidation. Electrophysiology measurements further supported the observation of polydisperse pore complexes from the MS data. Together, these results demonstrate the complementarity of native MS, FPOP, and membrane conductance experiments to shed light on how antibiotic peptides interact with and within lipid membranes.
- Ryan, J. P., Kostelic, M. M., Hsieh, C. C., Powers, J., Aspinwall, C., Dodds, J. N., Schiel, J. E., Marty, M. T., & Baker, E. S. (2023). Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods. Journal of the American Society for Mass Spectrometry, 34(12), 2811-2821.More infoAdeno-associated virus (AAV) capsids are among the leading gene delivery platforms used to treat a vast array of human diseases and conditions. AAVs exist in a variety of serotypes due to differences in viral protein (VP) sequences with distinct serotypes targeting specific cells and tissues. As the utility of AAVs in gene therapy increases, ensuring their specific composition is imperative for the correct targeting and gene delivery. From a quality control perspective, current analytical tools are limited in their selectivity for viral protein (VP) subunits due to their sequence similarities, instrumental difficulties in assessing the large molecular weights of intact capsids, and the uncertainty in distinguishing empty and filled capsids. To address these challenges, we combined two distinct analytical workflows that assess the intact capsids and VP subunits separately. First, a selective temporal overview of resonant ion (STORI)-based charge detection-mass spectrometry (CD-MS) was applied for characterization of the intact capsids. Liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) separations were then used for the capsid denaturing measurements. This multimethod combination was applied to three AAV serotypes (AAV2, AAV6, and AAV8) to evaluate their intact empty and filled capsid ratios and then examine the distinct VP sequences and modifications present.
- Sanders, H. M., Chalyavi, F., Fields, C. R., Kostelic, M. M., Li, M. H., Raleigh, D. P., Zanni, M. T., & Marty, M. T. (2023). Interspecies Variation Affects Islet Amyloid Polypeptide Membrane Binding. Journal of the American Society for Mass Spectrometry, 34(6), 986-990.More infoThe aggregation of islet amyloid polypeptide (IAPP) is associated with β-cell dysfunction in type 2 diabetes (T2D) in humans. One possible mechanism of toxicity is the interaction of IAPP oligomers with lipid membranes to disrupt the bilayer integrity and/or homeostasis of the cell. Amino acid sequence variations of IAPPs between species can greatly decrease their propensity for aggregation. For example, human IAPP is toxic to β-cells, but rat and pig IAPP are not. However, it is not clear how these differences affect membrane association. Using native mass spectrometry with lipid nanodiscs, we explored the differences in the association of human, rat, and pig IAPP with lipid bilayers. We discovered that human and rat IAPP bound nanodiscs with anionic dipalmitoyl-phosphatidylglycerol (DPPG) lipids, but pig IAPP did not. Furthermore, human and rat IAPP interacted differently with the membrane. Human IAPP show potential tetramer complexes, but rat IAPP associated with the membrane sequentially. Thus, overall IAPP-bilayer interactions are not necessarily related to disease, but small differences in oligomeric behavior at the membrane may instead play a role.
- Townsend, J. A., Fapohunda, O., Wang, Z., Pham, H., Taylor, M. T., Kloss, B., Park, S. H., Opella, S., Aspinwall, C. A., & Marty, M. T. (2023). Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu. bioRxiv : the preprint server for biology.More infoViroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry (MS) in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for mechanisms of their biological functions as well as their potential as therapeutic targets.
- Zhang, G., Odenkirk, M. T., Janczak, C. M., Lee, R., Richardson, K., Wang, Z., Aspinwall, C. A., & Marty, M. T. (2023). Identifying Membrane Protein-Lipid Interactions with Lipidomic Lipid Exchange-Mass Spectrometry. Journal of the American Chemical Society, 145(38), 20859-20867.More infoLipids can play important roles in modulating membrane protein structure and function. However, it is challenging to identify natural lipids bound to membrane proteins in complex bilayers. Here, we developed lipidomic lipid exchange-mass spectrometry (LX-MS) to study the lipid affinity for membrane proteins on a lipidomic scale. We first mix membrane protein nanodiscs with empty nanodiscs that have no embedded membrane proteins. After allowing lipids to passively exchange between the two populations, we separate the two types of nanodiscs and perform lipidomic analysis on each with liquid chromatography and MS. Enrichment of lipids in the membrane protein nanodiscs reveals the affinity of individual lipids for binding the target membrane protein. We apply this approach to study three membrane proteins. With the ammonium transporter AmtB and aquaporin AqpZ in nanodiscs with polar lipid extracts, we detected binding of cardiolipin and phosphatidyl-glycerol lipids to the proteins. With the acetylcholine receptor in nanodiscs with brain polar lipid extracts, we discovered a complex set of lipid interactions that depended on the head group and tail composition. Overall, lipidomic LX-MS provides a detailed understanding of the lipid-binding affinity and thermodynamics for membrane proteins in complex bilayers and provides a unique perspective on the chemical environment surrounding membrane proteins.
- Zhu, Y., Odenkirk, M. T., Qiao, P., Zhang, T., Schrecke, S., Zhou, M., Marty, M. T., Baker, E. S., & Laganowsky, A. (2023). Combining native mass spectrometry and lipidomics to uncover specific membrane protein-lipid interactions from natural lipid sources. Chemical science, 14(32), 8570-8582.More infoWhile it is known that lipids play an essential role in regulating membrane protein structure and function, it remains challenging to identify specific protein-lipid interactions. Here, we present an innovative approach that combines native mass spectrometry (MS) and lipidomics to identify lipids retained by membrane proteins from natural lipid extracts. Our results reveal that the bacterial ammonia channel (AmtB) enriches specific cardiolipin (CDL) and phosphatidylethanolamine (PE) from natural headgroup extracts. When the two extracts are mixed, AmtB retains more species, wherein selectivity is tuned to bias headgroup selection. Using a series of natural headgroup extracts, we show TRAAK, a two-pore domain K channel (K2P), retains specific acyl chains that is independent of the headgroup. A brain polar lipid extract was then combined with the K2Ps, TRAAK and TREK2, to understand lipid specificity. More than a hundred lipids demonstrated affinity for each protein, and both channels were found to retain specific fatty acids and lysophospholipids known to stimulate channel activity, even after several column washes. Natural lipid extracts provide the unique opportunity to not only present natural lipid diversity to purified membrane proteins but also identify lipids that may be important for membrane protein structure and function.
- Keener, J. E., Jayasekera, H. S., & Marty, M. T. (2022). Investigating the Lipid Selectivity of Membrane Proteins in Heterogeneous Nanodiscs. Analytical chemistry, 94(23), 8497-8505.More infoThe structure and function of membrane proteins can be significantly impacted by the surrounding lipid environment, but membrane protein-lipid interactions in lipid bilayers are often difficult to study due to their transient and polydisperse nature. Here, we used two native mass spectrometry (MS) approaches to investigate how the ammonium transporter trimer (AmtB) and aquaporin Z (AqpZ) selectively remodel their local lipid environment in heterogeneous lipoprotein nanodiscs. First, we used gas-phase ejection to isolate the membrane protein with bound lipids from heterogeneous nanodiscs with different combinations of lipids. Second, we used solution-phase detergent extraction as an orthogonal approach to study membrane protein remodeling of lipids in the nanodisc with native MS. Our results showed that Triton X-100 and lauryldimethylamine oxide retain lipid selectivity that agrees with gas-phase ejection, but C8E4 distorts some preferential lipid interactions. Both approaches reveal that AmtB has a few selective binding sites for phosphatidylcholine (PC) lipids, is selective for binding phosphatidylglycerols (PG) overall, and is nonselective for phosphatidylethanolamines (PE). In contrast, AqpZ prefers either PC or PG over PE and prefers PC over PG. Overall, these experiments provide a picture of how membrane proteins bind different lipid head groups in the context of mixed lipid bilayers.
- Kostelic, M. M., & Marty, M. T. (2022). Deconvolving Native and Intact Protein Mass Spectra with UniDec. Methods in molecular biology (Clifton, N.J.), 2500, 159-180.More infoIntact protein, top-down, and native mass spectrometry (MS) generally requires the deconvolution of electrospray ionization (ESI) mass spectra to assign the mass of components from their charge state distribution. For small, well-resolved proteins, the charge can usually be assigned based on the isotope distribution. However, it can be challenging to determine charge states with larger proteins that lack isotopic resolution, in complex mass spectra with overlapping charge states, and in native spectra that show adduction. To overcome these challenges, UniDec uses Bayesian deconvolution to assign charge states and to create a zero-charge mass distribution. UniDec is fast, user-friendly, and includes a range of advanced tools to assist in intact protein, top-down, and native MS data analysis. This chapter provides a step-by-step protocol and an in-depth explanation of the UniDec algorithm, and highlights the parameters that affect the deconvolution. It also covers advanced data analysis tools, such as macromolecular mass defect analysis and tools for assigning potential PTMs and bound ligands. Overall, this chapter provides users with a deeper understanding of UniDec, which will enhance the quality of deconvolutions and allow for more intricate MS experiments.
- Kostelic, M. M., Hsieh, C. C., Sanders, H. M., Zak, C. K., Ryan, J. P., Baker, E. S., Aspinwall, C. A., & Marty, M. T. (2022). Surface Modified Nano-Electrospray Needles Improve Sensitivity for Native Mass Spectrometry. Journal of the American Society for Mass Spectrometry, 33(6), 1031-1037.More infoNative mass spectrometry (MS) and charge detection-mass spectrometry (CD-MS) have become versatile tools for characterizing a wide range of proteins and macromolecular complexes. Both commonly use nanoelectrospray ionization (nESI) from pulled borosilicate needles, but some analytes are known to nonspecifically adsorb to the glass, which may lower sensitivity and limit the quality of the data. To improve the sensitivity of native MS and CD-MS, we modified the surface of nESI needles with inert surface modifiers, including polyethylene-glycol. We found that the surface modification improved the signal intensity for native MS of proteins and for CD-MS of adeno-associated viral capsids. Based on mechanistic comparisons, we hypothesize that the improvement is more likely due to an increased flow rate with coated ESI needles rather than less nonspecific adsorption. In any case, these surface-modified needles provide a simple and inexpensive method for improving the sensitivity of challenging analytes.
- Kostelic, M. M., Ryan, J. P., Brown, L. S., Jackson, T. W., Hsieh, C. C., Zak, C. K., Sanders, H. M., Liu, Y., Chen, V. S., Byrne, M., Aspinwall, C. A., Baker, E. S., & Marty, M. T. (2022). Stability and Dissociation of Adeno-Associated Viral Capsids by Variable Temperature-Charge Detection-Mass Spectrometry. Analytical chemistry, 94(34), 11723-11727.More infoAdeno-associated viral (AAV) vectors have emerged as gene therapy and vaccine delivery systems. Differential scanning fluorimetry or differential scanning calorimetry is commonly used to measure the thermal stability of AAVs, but these global methods are unable to distinguish the stabilities of different AAV subpopulations in the same sample. To address this challenge, we combined charge detection-mass spectrometry (CD-MS) with a variable temperature (VT) electrospray source that controls the temperature of the solution prior to electrospray. Using VT-CD-MS, we measured the thermal stabilities of empty and filled capsids. We found that filled AAVs ejected their cargo first and formed intermediate empty capsids before completely dissociating. Finally, we observed that pH stress caused a major decrease in thermal stability. This new approach better characterizes the thermal dissociation of AAVs, providing the simultaneous measurement of the stabilities and dissociation pathways of different subpopulations.
- Marty, M. T. (2022). Fundamentals: How Do We Calculate Mass, Error, and Uncertainty in Native Mass Spectrometry?. Journal of the American Society for Mass Spectrometry, 33(10), 1807-1812.More infoMass spectrometry (MS) is uniquely powerful for measuring the mass of intact proteins and other biomolecules. New applications have expanded intact protein analysis into biopharmaceuticals, native MS, and top-down proteomics, all of which have driven the need for more automated data-processing pipelines. However, key metrics in the field are often not precisely defined. For example, there are different views on how to calculate uncertainty from spectra. This Critical Insight will explore the different definitions of mass, error, and uncertainty. It will discuss situations where different definitions may be more suitable and provide recommendations for best practices. Targeting both beginners and experts, the goal of the discussion is to provide a common foundation of terminology, enhance statistical rigor, and improve automation of data analysis.
- Marty, M. T. (2022). Guest Editorial: Bias in Mass Spectrometry Technical Support. Journal of the American Society for Mass Spectrometry, 33(11), 2013-2014.
- Reid, D. J., Rohrbough, J. G., Kostelic, M. M., & Marty, M. T. (2022). Investigating Antimicrobial Peptide-Membrane Interactions Using Fast Photochemical Oxidation of Peptides in Nanodiscs. Journal of the American Society for Mass Spectrometry, 33(1), 62-67.More infoAntimicrobial peptides (AMPs) are an important part of the innate immune system and demonstrate promising applications in the fight against antibiotic-resistant infections due to their unique mechanism of targeting bacterial membranes. However, it is challenging to study the interactions of these peptides within lipid bilayers, making it difficult to understand their mechanisms of toxicity and selectivity. Here, we used fast photochemical oxidation of peptides, an irreversible footprinting technique that labels solvent accessible residues, and native charge detection-mass spectrometry to study AMP-lipid interactions with different lipid bilayer nanodiscs. We observed differences in the oxidation of two peptides, indolicidin and LL-37, in three distinct lipid environments, which reveal their affinity for lipid bilayers. Our findings suggest that indolicidin interacts with lipid head groups via a simple charge-driven mechanism, but LL-37 is more specific for nanodiscs. These results provide complementary information on the potential modes of action and lipid selectivity of AMPs.
- Sacco, M. D., Wang, S., Adapa, S. R., Zhang, X., Lewandowski, E. M., Gongora, M. V., Keramisanou, D., Atlas, Z. D., Townsend, J. A., Gatdula, J. R., Morgan, R. T., Hammond, L. R., Marty, M. T., Wang, J., Eswara, P. J., Gelis, I., Jiang, R. H., Sun, X., & Chen, Y. (2022). A unique class of Zn-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile. Nature communications, 13(1), 4370.More infoTreatment with β-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to β-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for β-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with β-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn-binding motif that influences β-lactam binding and protein stability. The Zn-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.
- Sanders, H. M., Jovcevski, B., Marty, M. T., & Pukala, T. L. (2022). Structural and mechanistic insights into amyloid-β and α-synuclein fibril formation and polyphenol inhibitor efficacy in phospholipid bilayers. The FEBS journal, 289(1), 215-230.More infoUnder certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid-β 40 and α-synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well-known polyphenol fibril inhibitors, (-)-epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off-pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development.
- Sanders, H. M., Kostelic, M. M., Zak, C. K., & Marty, M. T. (2022). Lipids and EGCG Affect α-Synuclein Association and Disruption of Nanodiscs. Biochemistry, 61(11), 1014-1021.More infoLipid membranes have recently been implicated in protein misfolding and disease etiology, including for α-synuclein and Parkinson's disease. However, studying the intersection of protein complex formation, membrane interactions, and bilayer disruption simultaneously is challenging. In particular, the efficacies of small molecule inhibitors for toxic protein aggregation are not well understood. Here, we used native mass spectrometry in combination with lipid nanodiscs to study α-synuclein-membrane interactions. α-Synuclein did not interact with zwitterionic 1,2-dimyristoyl--glycero-3-phosphocholine lipids but interacted strongly with anionic 1,2-dimyristoyl--glycero-3-phospho(1'--glycerol) lipids, eventually leading to membrane disruption. Unsaturated 1-palmitoyl-2-oleoyl--glycero-3-phospho(1'--glycerol) (POPG) lipid nanodiscs were also prone to bilayer disruption, releasing α-synuclein:POPG complexes. Interestingly, the fibril inhibitor, (-)-epigallocatechin gallate (EGCG), prevented membrane disruption but did not prevent the incorporation of α-synuclein into nanodisc complexes. Thus, although EGCG inhibits fibrillization, it does not inhibit α-synuclein from associating with the membrane.
- Sivinski, J., Ngo, D., Zerio, C. J., Ambrose, A. J., Watson, E. R., Kaneko, L. K., Kostelic, M. M., Stevens, M., Ray, A. M., Park, Y., Wu, C., Marty, M. T., Hoang, Q. Q., Zhang, D. D., Lander, G. C., Johnson, S. M., & Chapman, E. (2022). Allosteric differences dictate GroEL complementation of E. coli. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(3), e22198.More infoGroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.
- Swansiger, A. K., Marty, M. T., & Prell, J. S. (2022). Fourier-Transform Approach for Reconstructing Macromolecular Mass Defect Profiles. Journal of the American Society for Mass Spectrometry, 33(1), 172-180.More infoState-of-the-art native mass spectrometry (MS) methods have been developed for analysis of highly heterogeneous intact complexes and have provided much insight into the structure and properties of noncovalent assemblies that can be difficult to study using denatured proteins. These native MS methods can often be used to study even highly polydisperse membrane proteins embedded in detergent micelles, nanodiscs, and other membrane mimics. However, characterizing highly polydisperse native complexes which are also heterogeneous presents additional challenges for native MS. Macromolecular mass defect (MMD) analysis aims to characterize heterogeneous ion populations obfuscated by adduct polydispersity and reveal the distribution of "base" masses, and was recently implemented in the Bayesian analysis software UniDec. Here, we illustrate an alternative, orthogonal MMD analysis method implemented in the deconvolution program iFAMS, which takes advantage of Fourier transform (FT) to deconvolve low-resolution data with few user-input parameters and which can provide high quality results even for mass spectra with a signal-to-noise ratio of ∼5:1. Agreement between this method, which is based on frequency-domain data, and the mass-domain algorithm of UniDec provides strong evidence that both methods can accurately characterize highly polydisperse and heterogeneous ion populations. The FT algorithm is expected to be very useful in characterizing many types of analytes ranging from membrane proteins to polymer-conjugated proteins, branched polymers, and other large analytes, as well as for reconstructing isotope profiles for highly complex but still isotope-resolved mass spectra.
- Walker, L. R., & Marty, M. T. (2022). Lipid tails modulate antimicrobial peptide membrane incorporation and activity. Biochimica et biophysica acta. Biomembranes, 1864(4), 183870.More infoMembrane disrupting antimicrobial peptides (AMPs) are often amphipathic peptides that interact directly with lipid bilayers. AMPs are generally thought to interact mostly with lipid head groups, but it is less clear how the lipid alkyl chain length and saturation modulate interactions with membranes. Here, we used native mass spectrometry to measure the stoichiometry of three different AMPs-LL-37, indolicidin, and magainin-2-in lipid nanodiscs. We also measured the activity of these AMPs in unilamellar vesicle leakage assays. We found that LL-37 formed specific hexamer complexes but with different intermediates and affinities that depended on the bilayer thickness. LL-37 was also most active in lipid bilayers containing longer, unsaturated lipids. In contrast, indolicidin incorporated to a higher degree into more fluid lipid bilayers but was more active with bilayers with thinner, less fluid lipids. Finally, magainin-2 incorporated to a higher degree into bilayers with longer, unsaturated alkyl chains and showed more activity in these same conditions. Together, these data show that higher amounts of peptide incorporation generally led to higher activity and that AMPs tend to incorporate more into longer unsaturated lipid bilayers. However, the activity of AMPs was not always directly related to amount of peptide incorporated.
- Wijetunge, A. N., Davis, G. J., Shadmehr, M., Townsend, J. A., Guzmán, L. E., Marty, M. T., & Jewett, J. C. (2022). Correction to Copper-Free Click Enabled Triazabutadiene for Bioorthogonal Protein Functionalization. Bioconjugate chemistry, 33(3), 541.
- Cater, R. J., Chua, G. L., Erramilli, S. K., Keener, J. E., Choy, B. C., Tokarz, P., Chin, C. F., Quek, D. Q., Kloss, B., Pepe, J. G., Parisi, G., Wong, B. H., Clarke, O. B., Marty, M. T., Kossiakoff, A. A., Khelashvili, G., Silver, D. L., & Mancia, F. (2021). Structural basis of omega-3 fatty acid transport across the blood-brain barrier. Nature, 595(7866), 315-319.More infoDocosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources. This nutrient is transported across the blood-brain and blood-retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na-dependent manner. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents.
- Davis, G. J., Townsend, J. A., Morrow, M. G., Hamie, M., Shepard, A. J., Hsieh, C. C., Marty, M. T., & Jewett, J. C. (2021). Protein Modification via Mild Photochemical Isomerization of Triazenes to Release Aryl Diazonium Ions. Bioconjugate chemistry, 32(11), 2432-2438.More infoThis work describes the development of phenyl diazenyl piperidine triazene derivatives that can be activated to release aryl diazonium ions for labeling of proteins using light. These probes show marked bench stability at room temperature and can be photoisomerized via low-intensity UVA irradiation at physiological pH. Upon isomerization, the triazenes are rendered more basic and readily protonate to release reactive aryl diazonium ions. It was discovered that the intensity and duration of the UV light was essential to the observed diazonium ion reactivity in competition with the traditionally observed photolytic radical pathways. The combination of their synthetic efficiency coupled with their overall stability makes triazenes an attractive candidate for use in bioconjugation applications. Bioorthogonal handles on the triazenes are used to demonstrate the ease by which proteins can be modified.
- Keener, J. E., Zhang, G., & Marty, M. T. (2021). Native Mass Spectrometry of Membrane Proteins. Analytical chemistry, 93(1), 583-597.
- Kitamura, N., Sacco, M. D., Ma, C., Hu, Y., Townsend, J. A., Meng, X., Zhang, F., Zhang, X., Ba, M., Szeto, T., Kukuljac, A., Marty, M. T., Schultz, D., Cherry, S., Xiang, Y., Chen, Y., & Wang, J. (2021). Expedited Approach toward the Rational Design of Noncovalent SARS-CoV-2 Main Protease Inhibitors. Journal of medicinal chemistry.More infoThe main protease (M) of SARS-CoV-2 is a validated antiviral drug target. Several M inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including , , , and , with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, () that is structurally distinct from the canonical M inhibitor . Significantly, is highly selective compared with covalent inhibitors such as , especially toward host proteases. The cocrystal structure of SARS-CoV-2 M with revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered , one of the most potent and selective noncovalent SARS-CoV-2 M inhibitors reported to date, and a novel binding pocket in M that can be explored for inhibitor design.
- Kostelic, M. M., Zak, C. K., Jayasekera, H. S., & Marty, M. T. (2021). Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry. Analytical chemistry, 93(14), 5972-5979.More infoNative mass spectrometry (MS) with nanodiscs is a promising technique for characterizing membrane protein and peptide interactions in lipid bilayers. However, prior studies have used nanodiscs made of only one or two lipids, which lack the complexity of a natural lipid bilayer. To better model specific biological membranes, we developed model mammalian, bacterial, and mitochondrial nanodiscs with up to four different phospholipids. Careful selection of lipids with similar masses that balance the fluidity and curvature enabled these complex nanodiscs to be assembled and resolved with native MS. We then applied this approach to characterize the specificity and incorporation of LL-37, a human antimicrobial peptide, in single-lipid nanodiscs versus model bacterial nanodiscs. Overall, development of these model membrane nanodiscs reveals new insights into the assembly of complex nanodiscs and provides a useful toolkit for studying membrane protein, peptide, and lipid interactions in model biological membranes.
- Kostelic, M. M., Zak, C. K., Liu, Y., Chen, V. S., Wu, Z., Sivinski, J., Chapman, E., & Marty, M. T. (2021). UniDecCD: Deconvolution of Charge Detection-Mass Spectrometry Data. Analytical chemistry, 93(44), 14722-14729.More infoNative mass spectrometry (MS) has become a versatile tool for characterizing high-mass complexes and measuring biomolecular interactions. Native MS usually requires the resolution of different charge states produced by electrospray ionization to measure the mass, which is difficult for highly heterogeneous samples that have overlapping and unresolvable charge states. Charge detection-mass spectrometry (CD-MS) seeks to address this challenge by simultaneously measuring the charge and / for isolated ions. However, CD-MS often shows uncertainty in the charge measurement that limits the resolution. To overcome this charge state uncertainty, we developed UniDecCD (UCD) software for computational deconvolution of CD-MS data, which significantly improves the resolution of CD-MS data. Here, we describe the UCD algorithm and demonstrate its ability to improve the CD-MS resolution of proteins, megadalton viral capsids, and heterogeneous nanodiscs made from natural lipid extracts. UCD provides a user-friendly interface that will increase the accessibility of CD-MS technology and provide a valuable new computational tool for CD-MS data analysis.
- Ma, C., Sacco, M. D., Xia, Z., Lambrinidis, G., Townsend, J. A., Hu, Y., Meng, X., Szeto, T., Ba, M., Zhang, X., Gongora, M., Zhang, F., Marty, M. T., Xiang, Y., Kolocouris, A., Chen, Y., & Wang, J. (2021). Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay. ACS central science, 7(7), 1245-1260.More infoThe papain-like protease (PL) of SARS-CoV-2 is a validated antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PL inhibitors including and with improved enzymatic inhibition and antiviral activity compared to , which was reported as a SARS-CoV PL inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PL inhibitors in the BSL-2 setting. X-ray crystal structure of PL in complex with showed that binding of to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that and engaged in more extensive interactions than . Overall, the PL inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PL assay is a suitable surrogate for screening PL inhibitors in the BSL-2 setting.
- Ma, C., Xia, Z., Sacco, M. D., Hu, Y., Townsend, J. A., Meng, X., Choza, J., Tan, H., Jang, J., Gongora, M. V., Zhang, X., Zhang, F., Xiang, Y., Marty, M. T., Chen, Y., & Wang, J. (2021). Discovery of Di- and Trihaloacetamides as Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity. Journal of the American Chemical Society, 143(49), 20697-20709.More infoThe main protease (M) is a validated antiviral drug target of SARS-CoV-2. A number of M inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 M inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates (dichloroacetamide) and (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to , these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent M inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 M with and reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.
- Marty, M. T. (2021). Illuminating Individual Membrane Protein Complexes with Mass Photometry. Chem, 7(1), 16-17. doi:10.1016/j.chempr.2020.12.012More infoMembrane proteins play critical roles but have been challenging for structural biology. In this issue of Chem, Olerinyova et al. apply an emerging technology, mass photometry, to characterize membrane proteins in different solubilization vehicles. Mass photometry streamlines characterization of membrane proteins in different membrane mimetics, enabling rapid analysis of homogeneity.
- Norris, C. E., Keener, J. E., Perera, S. M., Weerasinghe, N., Fried, S. D., Resager, W. C., Rohrbough, J. G., Brown, M. F., & Marty, M. T. (2021). Native Mass Spectrometry Reveals the Simultaneous Binding of Lipids and Zinc to Rhodopsin. International journal of mass spectrometry, 460.More infoRhodopsin, a prototypical G-protein-coupled receptor, is responsible for scoptic vision at low-light levels. Although rhodopsin's photoactivation cascade is well understood, it remains unclear how lipid and zinc binding to the receptor are coupled. Using native mass spectrometry, we developed a novel data analysis strategy to deconvolve zinc and lipid bound to the proteoforms of rhodopsin and investigated the allosteric interaction between lipids and zinc binding. We discovered that phosphatidylcholine bound to rhodopsin with a greater affinity than phosphatidylserine or phosphatidylethanolamine, and that binding of all lipids was influenced by zinc but with different effects. In contrast, zinc binding was relatively unperturbed by lipids. Overall, these data reveal that lipid binding can be strongly and differentially influenced by metal ions.
- Petras, D., Phelan, V. V., Acharya, D., Allen, A. E., Aron, A. T., Bandeira, N., Bowen, B. P., Belle-Oudry, D., Boecker, S., Cummings, D. A., Deutsch, J. M., Fahy, E., Garg, N., Gregor, R., Handelsman, J., Navarro-Hoyos, M., Jarmusch, A. K., Jarmusch, S. A., Louie, K., , Maloney, K. N., et al. (2021). GNPS Dashboard: collaborative exploration of mass spectrometry data in the web browser. Nature methods.
- Shepard, A. J., Townsend, J. A., Foley, C., Hulme, C., Marty, M. T., & Jewett, J. C. (2021). Suzuki Coupling of Protected Aryl Diazonium Ions: Expanding the Knowledge of Triazabutadiene Compatible Reactions. Organic letters, 23(5), 1851-1855.More infoAryl diazonium ions are important in synthesis and chemical biology, and the acid-labile triazabutadiene can protect this handle for future use. We report a Suzuki coupling strategy that is compatible with the triazabutadiene scaffold, expanding the scope of synthetically available triazabutadienes. Shown herein, the triazabutadiene scaffold remains intact and reactive after coupling, as demonstrated by releasing the aryl diazonium ion to label a tyrosine-rich model protein.
- Sung, Y. S., Wu, W., Ewbank, M. A., Utterback, R. D., Marty, M. T., & Tomat, E. (2021). Albumin Conjugates of Thiosemicarbazone and Imidazole-2-thione Prochelators: Iron Coordination and Antiproliferative Activity. ChemMedChem, 16(18), 2764-2768.More infoThe central role of iron in tumor progression and metastasis motivates the development of iron-binding approaches in cancer chemotherapy. Disulfide-based prochelators are reductively activated upon cellular uptake to liberate thiol chelators responsible for iron sequestration. Herein, a trimethyl thiosemicarbazone moiety and the imidazole-2-thione heterocycle are incorporated in this prochelator design. Iron binding of the corresponding tridentate chelators leads to the stabilization of a low-spin ferric center in 2 : 1 ligand-to-metal complexes. Native mass spectrometry experiments show that the prochelators form stable disulfide conjugates with bovine serum albumin, thus affording novel bioconjugate prochelator systems. Antiproliferative activities at sub-micromolar levels are recorded in a panel of breast, ovarian and colorectal cancer cells, along with significantly lower activity in normal fibroblasts.
- Townsend, J. A., Sanders, H. M., Rolland, A. D., Park, C. K., Horton, N. C., Prell, J. S., Wang, J., & Marty, M. T. (2021). Influenza AM2 Channel Oligomerization Is Sensitive to Its Chemical Environment. Analytical chemistry, 93(48), 16273-16281.More infoViroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best-characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry and other techniques to characterize the oligomeric state of both the full-length and transmembrane (TM) domain of AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by the local chemical environment. Native mass spectrometry of AM2 in nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled the measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 can form a wider range of oligomeric states than previously thought possible, which may provide new potential mechanisms of influenza pathology and pharmacology.
- Wijetunge, A. N., Davis, G. J., Shadmehr, M., Townsend, J. A., Marty, M. T., & Jewett, J. C. (2021). Copper-Free Click Enabled Triazabutadiene for Bioorthogonal Protein Functionalization. Bioconjugate chemistry.More infoAryl diazonium ions have long been used in bioconjugation due to their reactivity toward electron-rich aryl residues, such as tyrosine. However, their utility in biological systems has been restricted due to the requirement of harsh conditions for their generation , as well as limited hydrolytic stability. Previous work describing a scaffold known as triazabutadiene (TBD) has shown the ability to protect aryl diazonium ions allowing for increased synthetic utility, as well as triggered release under biologically relevant conditions. Herein, we describe the synthesis and application of a novel TBD, capable of installation of a cyclooctyne on protein surfaces for later use of copper-free click reactions involving functional azides. The probe shows efficient protein labeling across a wide pH range that can be accomplished in a convenient and timely manner. Orthogonality of the cyclooctyne modification was showcased by labeling a model protein in the presence of hen egg proteins, using an azide-containing fluorophore. We further confirmed that the azobenzene modification can be cleaved using sodium dithionite treatment.
- Xia, Z., Sacco, M. D., Ma, C., Townsend, J. A., Kitamura, N., Hu, Y., Ba, M., Szeto, T., Zhang, X., Meng, X., Zhang, F., Xiang, Y., Marty, M. T., Chen, Y., & Wang, J. (2021). Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay. bioRxiv : the preprint server for biology.More infoThe papain-like protease (PL ) of SARS-CoV-2 is a validated antiviral drug target. PL is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PL inhibitors with IC values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC values ranging from 0.56 to 0.90 µM. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PL inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PL assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC values of 8.89 and 8.32 µM, respectively, which were 3-fold more potent than GRL0617 (EC = 25.1 µM). The X-ray crystal structures of PL in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PL inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PL assay might be a suitable surrogate for screening PL inhibitors in the BSL-2 setting.
- Harvey, S. R., VanAernum, Z. L., Kostelic, M. M., Marty, M. T., & Wysocki, V. H. (2020). Probing the structure of nanodiscs using surface-induced dissociation mass spectrometry. Chemical communications (Cambridge, England), 56(100), 15651-15654.More infoIn the study of membrane proteins and antimicrobial peptides, nanodiscs have emerged as a valuable membrane mimetic to solubilze these molecules in a lipid bilayer. We present the structural characterization of nanodiscs using native mass spectrometry and surface-induced dissociation, which are powerful tools in structural biology.
- Keener, J. E., Mowad, M., Marty, M. T., & Townsend, J. A. (2020). Measuring Membrane Protein-Lipid Interactions in Nanodiscs with Native Mass Spectrometry. Biophysical Journal, 118(3), 240a. doi:10.1016/j.bpj.2019.11.1413
- Ma, C., Hu, Y., Townsend, J. A., Lagarias, P. I., Marty, M. T., Kolocouris, A., & Wang, J. (2020). Ebselen, Disulfiram, Carmofur, PX-12, Tideglusib, and Shikonin Are Nonspecific Promiscuous SARS-CoV-2 Main Protease Inhibitors. ACS pharmacology & translational science, 3(6), 1265-1277.More infoAmong the drug targets being investigated for SARS-CoV-2, the viral main protease (M) is one of the most extensively studied. M is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites. It is highly conserved and has a unique substrate preference for glutamine in the P1 position. Therefore, M inhibitors are expected to have broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as M inhibitors. In this study, we investigated the mechanism of action of six previously reported M inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12, using a consortium of techniques including FRET-based enzymatic assay, thermal shift assay, native mass spectrometry, cellular antiviral assays, and molecular dynamics simulations. Collectively, the results showed that the inhibition of M by these six compounds is nonspecific and that the inhibition is abolished or greatly reduced with the addition of reducing reagent 1,4-dithiothreitol (DTT). Without DTT, these six compounds inhibit not only M but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease and 2A and 3C from enterovirus A71 (EV-A71) and EV-D68. However, none of the compounds inhibits the viral replication of EV-A71 or EV-D68, suggesting that the enzymatic inhibition potency IC values obtained in the absence of DTT cannot be used to faithfully predict their cellular antiviral activity. Overall, we provide compelling evidence suggesting that these six compounds are nonspecific SARS-CoV-2 M inhibitors and urge the scientific community to be stringent with hit validation.
- Ma, C., Hu, Y., Townsend, J. A., Lagarias, P. I., Marty, M. T., Kolocouris, A., & Wang, J. (2020). Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors. bioRxiv : the preprint server for biology.More infoThere is an urgent need for vaccines and antiviral drugs to combat the COVID-19 pandemic. Encouraging progress has been made in developing antivirals targeting SARS-CoV-2, the etiological agent of COVID-19. Among the drug targets being investigated, the viral main protease (M ) is one of the most extensively studied drug targets. M is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites and it is highly conserved among coronaviruses. In addition, M has a unique substrate preference for glutamine in the P1 position. Taken together, it appears that M inhibitors can achieve both broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as M inhibitors, with several of which also showed antiviral activity in cell culture. In this study, we investigated the mechanism of action of six previously reported M inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 using a consortium of techniques including FRET-based enzymatic assay, thermal shift assay, native mass spectrometry, cellular antiviral assays, and molecular dynamics simulations. Collectively, the results showed that the inhibition of M by these six compounds is non-specific and the inhibition is abolished or greatly reduced with the addition of reducing reagent DTT. In the absence of DTT, these six compounds not only inhibit M , but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease, the 2A and 3C from enterovirus A71 (EV-A71) and EV-D68. However, none of the compounds inhibits the viral replication of EV-A71 or EV-D68, suggesting that the enzymatic inhibition potency IC values obtained in the absence of DTT cannot be used to faithfully predict their cellular antiviral activity. Overall, we provide compelling evidence suggesting that ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 are non-specific SARS-CoV-2 M inhibitors, and urge the scientific community to be stringent with hit validation.
- Ma, C., Sacco, M. D., Hurst, B., Townsend, J. A., Hu, Y., Szeto, T., Zhang, X., Tarbet, B., Marty, M. T., Chen, Y., & Wang, J. (2020). Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease. Cell research, 30(8), 678-692.More infoA new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic M inhibitors. A complex crystal structure of SARS-CoV-2 M with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.
- Ma, C., Sacco, M. D., Hurst, B., Townsend, J. A., Hu, Y., Szeto, T., Zhang, X., Tarbet, B., Marty, M. T., Chen, Y., & Wang, J. (2020). Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease. bioRxiv : the preprint server for biology.More infoA novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC values ranging from 0.49 to 3.37 μM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known M inhibitors. A complex crystal structure of SARS-CoV-2 M with GC-376, determined at 2.15 Å resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.
- Marty, M. T. (2020). A Universal Score for Deconvolution of Intact Protein and Native Electrospray Mass Spectra. Analytical chemistry, 92(6), 4395-4401.More infoThe growing use of intact protein mass analysis, top-down proteomics, and native mass spectrometry have created a need for improved data analysis pipelines for deconvolution of electrospray (ESI) mass spectra containing multiple charge states and potentially without isotopic resolution. Although there are multiple deconvolution algorithms, there is no consensus for how to judge the quality of the deconvolution, and many scoring schemes are not published. Here, an intuitive universal score (UniScore) for ESI deconvolution is presented. The UniScore is the weighted average of deconvolution scores (DScores) for each peak multiplied by the of the fit to the data. Each DScore is composed of separate components to score (1) the uniqueness and fit of the deconvolution to the data, (2) the consistency of the peak shape across different charge states, (3) the smoothness of the charge state distribution, and (4) symmetry and separation of the peak. Example scores are provided for a range of experimental and simulated data. By providing a means of judging the quality of the overall deconvolution as well as individual mass peaks, the UniScore scheme provides a foundation for standardizing ESI data analysis of larger molecules and enabling the use of ESI deconvolution in automated data analysis pipelines.
- Marty, M. T. (2020). Nanodiscs and Mass Spectrometry: Making Membranes Fly. International journal of mass spectrometry, 458.More infoCells are surrounded by a protective lipid bilayer membrane, and membrane proteins in the bilayer control the flow of chemicals, information, and energy across this barrier. Many therapeutics target membrane proteins, and some directly target the lipid membrane itself. However, interactions within biological membranes are challenging to study due to their heterogeneity and insolubility. Mass spectrometry (MS) has become a powerful technique for studying membrane proteins, especially how membrane proteins interact with their surrounding lipid environment. Although detergent micelles are the most common membrane mimetic, nanodiscs are emerging as a promising platform for MS. Nanodiscs, nanoscale lipid bilayers encircled by two scaffold proteins, provide a controllable lipid bilayer for solubilizing membrane proteins. This Young Scientist Perspective focuses on native MS of intact nanodiscs and highlights the unique experiments enabled by making membranes fly, including studying membrane protein-lipid interactions and exploring the specificity of fragile transmembrane peptide complexes. It will also explore current challenges and future perspectives for interfacing nanodiscs with MS.
- Marty, M. T. (2020). Nanodiscs and Mass Spectrometry: Making Membranes Fly.. International journal of mass spectrometry, 458, 116436. doi:10.1016/j.ijms.2020.116436More infoCells are surrounded by a protective lipid bilayer membrane, and membrane proteins in the bilayer control the flow of chemicals, information, and energy across this barrier. Many therapeutics target membrane proteins, and some directly target the lipid membrane itself. However, interactions within biological membranes are challenging to study due to their heterogeneity and insolubility. Mass spectrometry (MS) has become a powerful technique for studying membrane proteins, especially how membrane proteins interact with their surrounding lipid environment. Although detergent micelles are the most common membrane mimetic, nanodiscs are emerging as a promising platform for MS. Nanodiscs, nanoscale lipid bilayers encircled by two scaffold proteins, provide a controllable lipid bilayer for solubilizing membrane proteins. This Young Scientist Perspective focuses on native MS of intact nanodiscs and highlights the unique experiments enabled by making membranes fly, including studying membrane protein-lipid interactions and exploring the specificity of fragile transmembrane peptide complexes. It will also explore current challenges and future perspectives for interfacing nanodiscs with MS.
- Marty, M. T., Kostelic, M. M., Zak, C. K., Kostelic, M. M., & Jurkowitz, D. B. (2020). Modeling Natural Bilayers with Mixed Lipid Nanodiscs for Native MS. Biophysical Journal, 118(3), 343a. doi:10.1016/j.bpj.2019.11.1984
- Norris, C. E., Keener, J. E., Weerasinghe, N., Weerasinghe, N., Brown, M. F., Brown, M. F., & Marty, M. T. (2020). Investigating the Influences of Lipid Binding on Rhodopsin Activation using Native Mass Spectrometry. Biophysical Journal, 118(3), 17a-18a. doi:10.1016/j.bpj.2019.11.279
- Sacco, M. D., Ma, C., Lagarias, P., Gao, A., Townsend, J. A., Meng, X., Dube, P., Zhang, X., Hu, Y., Kitamura, N., Hurst, B., Tarbet, B., Marty, M. T., Kolocouris, A., Xiang, Y., Chen, Y., & Wang, J. (2020). Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M and cathepsin L. Science advances, 6(50).More infoThe main protease (M) of SARS-CoV-2 is a key antiviral drug target. While most M inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently found that several M inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host protease that is important for viral entry. In this study, we solved x-ray crystal structures of M in complex with calpain inhibitors II and XII and three analogs of The structure of M with calpain inhibitor II confirmed that the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. The structure of calpain inhibitor XII revealed an unexpected, inverted binding pose. Together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of dual inhibitors as SARS-CoV-2 antivirals.
- Sacco, M. D., Ma, C., Lagarias, P., Gao, A., Townsend, J. A., Meng, X., Dube, P., Zhang, X., Hu, Y., Kitamura, N., Hurst, B., Tarbet, B., Marty, M. T., Kolocouris, A., Xiang, Y., Chen, Y., & Wang, J. (2020). Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against M and cathepsin L. bioRxiv : the preprint server for biology.More infoThe main protease (M) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 M inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently discovered several M inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of M in complex with calpain inhibitors II and XII, and three analogues of , one of the most potent M inhibitors . The structure of M with calpain inhibitor II confirmed the S1 pocket of M can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1' pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1' pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three analogues , , and provide insight to the sidechain preference of the S1', S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the α-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of M inhibitors as SARS-CoV-2 antivirals.
- Sahin, C., Reid, D. J., Marty, M. T., & Landreh, M. (2020). Scratching the surface: native mass spectrometry of peripheral membrane protein complexes. Biochemical Society transactions, 48(2), 547-558.More infoA growing number of integral membrane proteins have been shown to tune their activity by selectively interacting with specific lipids. The ability to regulate biological functions via lipid interactions extends to the diverse group of proteins that associate only peripherally with the lipid bilayer. However, the structural basis of these interactions remains challenging to study due to their transient and promiscuous nature. Recently, native mass spectrometry has come into focus as a new tool to investigate lipid interactions in membrane proteins. Here, we outline how the native MS strategies developed for integral membrane proteins can be applied to generate insights into the structure and function of peripheral membrane proteins. Specifically, native MS studies of proteins in complex with detergent-solubilized lipids, bound to lipid nanodiscs, and released from native-like lipid vesicles all shed new light on the role of lipid interactions. The unique ability of native MS to capture and interrogate protein-protein, protein-ligand, and protein-lipid interactions opens exciting new avenues for the study of peripheral membrane protein biology.
- Tan, Y. Z., Rodrigues, J., Keener, J. E., Zheng, R. B., Brunton, R., Kloss, B., Giacometti, S. I., Rosário, A. L., Zhang, L., Niederweis, M., Clarke, O. B., Lowary, T. L., Marty, M. T., Archer, M., Potter, C. S., Carragher, B., & Mancia, F. (2020). Cryo-EM structure of arabinosyltransferase EmbB from Mycobacterium smegmatis. Nature communications, 11(1), 3396.More infoArabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding.
- Walker, L. R., & Marty, M. T. (2020). Revealing the Specificity of a Range of Antimicrobial Peptides in Lipid Nanodiscs by Native Mass Spectrometry. Biochemistry, 59(23), 2135-2142.More infoAntimicrobial peptides (AMPs) interact directly with lipid membranes of pathogens and may have the potential to combat antibiotic resistance. Although many AMPs are thought to form toxic oligomeric pores, their interactions within lipid membranes are not well understood. Here, we used native mass spectrometry to measure the incorporation of a range of different AMPs in lipoprotein nanodiscs. We found that the truncation of human LL37 increases the lipid specificity but decreases the specificity of complex formation. We also saw that the reduction of disulfide bonds can have a dramatic effect on the ability of AMPs to interact with lipid bilayers. Finally, by examining a wider range of peptides we discovered that AMPs tend to interact specifically with anionic lipids but form nonspecific complexes with wide oligomeric state distributions. Overall, these data reveal that each AMP has unique behaviors but some common trends apply to many AMPs.
- Zhang, G., Keener, J. E., & Marty, M. T. (2020). Measuring Remodeling of the Lipid Environment Surrounding Membrane Proteins with Lipid Exchange and Native Mass Spectrometry. Analytical chemistry, 92(8), 5666-5669.More infoDue to their crucial biochemical roles, membrane proteins are important drug targets. Although it is clear that lipids can influence membrane protein function, the chemistry of lipid binding remains difficult to study because protein-lipid interactions are polydisperse, competitive, and transient. Furthermore, detergents, which are often used to solubilize membrane proteins in micelles, may disrupt lipid interactions that occur in bilayers. Here, we present two new approaches to quantify protein-lipid interactions in bilayers and understand how membrane proteins remodel their surrounding lipid environment. First, we used mass spectrometry (MS) to measure the exchange of lipids between lipoprotein nanodiscs with and without an embedded membrane protein. Shifts in the lipid distribution toward the membrane protein nanodiscs revealed lipid binding, and titrations allowed measurement of the optimal lipid composition for the membrane protein. Second, we used native or nondenaturing MS to ionize membrane protein nanodiscs with heterogeneous lipids. Ejecting the membrane protein complex with bound lipids in the mass spectrometer revealed enrichment of specific lipids around the membrane protein. Both new approaches showed that the ammonium transporter AmtB prefers phosphatidylglycerol lipids overall but has a minor affinity for phosphatidylcholine lipids.
- Kostelic, M. M., Ryan, A. M., Reid, D. J., Noun, J. M., & Marty, M. T. (2019). Expanding the Types of Lipids Amenable to Native Mass Spectrometry of Lipoprotein Complexes. Journal of the American Society for Mass Spectrometry, 30(8), 1416-1425.More infoNative mass spectrometry (MS) has become an important tool for the analysis of membrane proteins. Although detergent micelles are the most commonly used method for solubilizing membrane proteins for native MS, nanoscale lipoprotein complexes such as nanodiscs are emerging as a promising complementary approach because they solubilize membrane proteins in a lipid bilayer environment. However, prior native MS studies of intact nanodiscs have employed only a limited set of phospholipids that are similar in mass. Here, we extend the range of lipids that are amenable to native MS of nanodiscs by combining lipids with masses that are simple integer multiples of each other. Although these lipid combinations create complex distributions, overlap between resonant peak series allows interpretation of nanodisc spectra containing glycolipids, sterols, and cardiolipin. We also investigate the gas-phase stability of nanodiscs with these new lipids towards collisional activation. We observe that negative ionization mode or charge reduction stabilizes nanodiscs and is essential to preserving labile lipids such as sterols. These new approaches to native MS of nanodiscs will enable future studies of membrane proteins embedded in model membranes that more accurately mimic natural bilayers. Graphical Abstract.
- Marty, M. T. (2019). Eliminating Artifacts in Electrospray Deconvolution with a SoftMax Function. Journal of the American Society for Mass Spectrometry, 30(10), 2174-2177.More infoUniDec provides a rapid and robust approach to deconvolving electrospray mass spectra into their corresponding mass and charge components. However, the UniDec algorithm can produce artifacts depending on the quality and complexity of the data. Here, a SoftMax function is applied to the charge state distribution of each data point, which pushes the algorithm towards assigning each data point to one primary charge state. As shown for several data sets of increasing complexity, the SoftMax function significantly reduces deconvolution artifacts, even for data with overlapping charge states.
- Marty, M. T., Kostelic, M. M., Walker, L. R., Marzluff, E. M., & Kostelic, M. M. (2019). Measuring the Stoichiometry of Antimicrobial Peptides in Nanodiscs with Native Mass Spectrometry. Biophysical Journal, 116(3), 85a-86a. doi:10.1016/j.bpj.2018.11.504
- Marty, M. T., Prell, J., Pemberton, J. E., Deodhar, B. S., Reid, D. J., Zak, C. K., Zhang, G., Zambrano, D. E., & Keener, J. E. (2019). Chemical Additives Enable Native Mass Spectrometry Measurement of Membrane Protein Oligomeric State within Intact Nanodiscs. J. Amer. Chem. Soc., 141, 1054-1061. doi:10.1021/jacs.8b11529
- Musharrafieh, R., Ma, C., Zhang, J., Hu, Y., Diesing, J. M., Marty, M. T., & Wang, J. (2019). Validating Enterovirus D68-2A as an Antiviral Drug Target and the Discovery of Telaprevir as a Potent D68-2A Inhibitor. Journal of virology.More infoEnterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2A) and characterization of its enzymatic activity. Furthermore, we discovered telaprevir, an FDA-approved drug used for the treatment of Hepatitis C virus infections, as a potent antiviral against EV-D68 by targeting the 2A enzyme. Using FRET-based substrate cleavage assay, we showed that the purified EV-D68 2A has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2A through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar to low micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2A was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the enzymatic activity of EV-D68 2A and the identification of telaprevir as a potent EV-D68 2A inhibitor. These findings implicate EV-D68 2A as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection. A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2A, and identify telaprevir-an FDA approved drug used to treat hepatitis C virus (HCV) infections-as a potent antiviral with a novel mechanism towards 2A 2A functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. Binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2A inhibition. This is the first inhibitor to selectively target the 2A from EV-D68 and can be used as a starting point for the development of selective EV-D68 therapeutics.
- Reid, D. J., Marty, M. T., Wales, J. A., Reid, D. J., Perry, S. M., Montfort, W. R., Miller, M. A., Marty, M. T., & Diesing, J. M. (2019). MetaUniDec: High-Throughput Deconvolution of Native Mass Spectra.. Journal of the American Society for Mass Spectrometry, 30(1), 118-127. doi:10.1007/s13361-018-1951-9More infoThe expansion of native mass spectrometry (MS) methods for both academic and industrial applications has created a substantial need for analysis of large native MS datasets. Existing software tools are poorly suited for high-throughput deconvolution of native electrospray mass spectra from intact proteins and protein complexes. The UniDec Bayesian deconvolution algorithm is uniquely well suited for high-throughput analysis due to its speed and robustness but was previously tailored towards individual spectra. Here, we optimized UniDec for deconvolution, analysis, and visualization of large data sets. This new module, MetaUniDec, centers around a hierarchical data format 5 (HDF5) format for storing datasets that significantly improves speed, portability, and file size. It also includes code optimizations to improve speed and a new graphical user interface for visualization, interaction, and analysis of data. To demonstrate the utility of MetaUniDec, we applied the software to analyze automated collision voltage ramps with a small bacterial heme protein and large lipoprotein nanodiscs. Upon increasing collisional activation, bacterial heme-nitric oxide/oxygen binding (H-NOX) protein shows a discrete loss of bound heme, and nanodiscs show a continuous loss of lipids and charge. By using MetaUniDec to track changes in peak area or mass as a function of collision voltage, we explore the energetic profile of collisional activation in an ultra-high mass range Orbitrap mass spectrometer. Graphical abstract ᅟ.
- Townsend, J. A., Keener, J. E., Miller, Z. M., Prell, J. S., & Marty, M. T. (2019). Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry. Analytical chemistry, 91(22), 14765-14772.More infoNoncovalent interactions between biomolecules are critical to their activity. Native mass spectrometry (MS) has enabled characterization of these interactions by preserving noncovalent assemblies for mass analysis, including protein-ligand and protein-protein complexes for a wide range of soluble and membrane proteins. Recent advances in native MS of lipoprotein nanodiscs have also allowed characterization of antimicrobial peptides and membrane proteins embedded in intact lipid bilayers. However, conventional native electrospray ionization (ESI) can disrupt labile interactions. To stabilize macromolecular complexes for native MS, charge reducing reagents can be added to the solution prior to ESI, such as triethylamine, trimethylamine oxide, and imidazole. Lowering the charge acquired during ESI reduces Coulombic repulsion that leads to dissociation, and charge reduction reagents may also lower the internal energy of the ions through evaporative cooling. Here, we tested a range of imidazole derivatives to discover improved charge reducing reagents and to determine how their chemical properties influence charge reduction efficacy. We measured their effects on a soluble protein complex, a membrane protein complex in detergent, and lipoprotein nanodiscs with and without embedded peptides, and used computational chemistry to understand the observed charge-reduction behavior. Together, our data revealed that hydrophobic substituents at the 2 position on imidazole can significantly improve both charge reduction and gas-phase stability over existing reagents. These new imidazole derivatives will be immediately beneficial for a range of native MS applications and provide chemical principles to guide development of novel charge reducing reagents.
- Walker, L. R., Marzluff, E. M., Townsend, J. A., Resager, W. C., & Marty, M. T. (2019). Native Mass Spectrometry of Antimicrobial Peptides in Lipid Nanodiscs Elucidates Complex Assembly. Analytical chemistry, 91(14), 9284-9291.More infoAntimicrobial peptides (AMPs) are generally cationic and amphipathic peptides that show potential applications to combat the growing threat of antibiotic resistant infections. AMPs are known to interact with bacterial membranes, but their mechanisms of toxicity and selectivity are poorly understood, in part because it is challenging to characterize AMP oligomeric complexes within lipid bilayers. Here, we used native mass spectrometry to measure the stoichiometry of AMPs inserted into lipoprotein nanodiscs with different lipid components. Titrations of increasing peptide concentration and collisional activation experiments reveal that AMPs can exhibit a range of behaviors from nonspecific incorporation into the nanodisc to formation of specific complexes. This new approach to characterizing formation of AMP complexes within lipid membranes will provide unique insights into AMP mechanisms.
- Fried, S. D., Resager, W. C., Perera, S. C., Perera, S. C., Brown, M. F., Brown, M. F., & Marty, M. T. (2018). Investigation of Photoinduced Oligomerization of Rhodopsin by Native Mass Spectrometry. Biophysical Journal, 114(3), 268a. doi:10.1016/j.bpj.2017.11.1550
- Hochberg, G. K., Shepherd, D. A., Marklund, E. G., Santhanagoplan, I., Degiacomi, M. T., Laganowsky, A., Allison, T. M., Basha, E., Marty, M. T., Galpin, M. R., Struwe, W. B., Baldwin, A. J., Vierling, E., & Benesch, J. L. (2018). Structural principles that enable oligomeric small heat-shock protein paralogs to evolve distinct functions. Science (New York, N.Y.), 359(6378), 930-935.More infoOligomeric proteins assemble with exceptional selectivity, even in the presence of closely related proteins, to perform their cellular roles. We show that most proteins related by gene duplication of an oligomeric ancestor have evolved to avoid hetero-oligomerization and that this correlates with their acquisition of distinct functions. We report how coassembly is avoided by two oligomeric small heat-shock protein paralogs. A hierarchy of assembly, involving intermediates that are populated only fleetingly at equilibrium, ensures selective oligomerization. Conformational flexibility at noninterfacial regions in the monomers prevents coassembly, allowing interfaces to remain largely conserved. Homomeric oligomers must overcome the entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer subunits than homomers that have no paralogs.
- Keener, J. E., Reid, D. J., Reid, D. J., Marty, M. T., Zambrano, D. E., & Zak, C. K. (2018). Characterizing the Lipid Annulus Surrounding Membrane Proteins with Native Mass Spectrometry of Nanodiscs. Biophysical Journal, 114(3), 457a-458a. doi:10.1016/j.bpj.2017.11.2527
- Keener, J. E., Zambrano, D. E., Zhang, G., Zak, C. K., Reid, D. J., Deodhar, B. S., Pemberton, J. E., Prell, J. S., & Marty, M. T. (2018). Chemical additives enable native mass spectrometry measurement of membrane protein oligomeric state within intact nanodiscs. Journal of the American Chemical Society.More infoMembrane proteins play critical biochemical roles but remain challenging to study. Recently, native or nondenaturing mass spectrometry (MS) has made great strides in characterizing membrane protein interactions. However, conventional native MS relies on detergent micelles, which may disrupt natural interactions. Lipoprotein nanodiscs provide a platform to present membrane proteins for native MS within a lipid bilayer environment, but prior native MS of membrane proteins in nanodiscs has been limited by the intermediate stability of nanodiscs. It is difficult to eject membrane proteins from nanodiscs for native MS but also difficult to retain intact nanodisc complexes with membrane proteins inside. Here, we employed chemical reagents that modulate the charge acquired during electrospray ionization (ESI). By modulating ESI conditions, we could either eject the membrane protein complex with few bound lipids or capture the intact membrane protein nanodisc complex-allowing measurement of membrane protein oligomeric state within an intact lipid bilayer environment. The dramatic differences in the stability of nanodiscs under different ESI conditions opens new applications for native MS of nanodiscs.
- Lippens, J. L., Egea, P. F., Spahr, C., Vaish, A., Keener, J. E., Marty, M. T., Loo, J. A., & Campuzano, I. D. (2018). Rapid LC-MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins. Analytical chemistry, 90(22), 13616-13623.More infoTherapeutic target characterization involves many components, including accurate molecular weight (MW) determination. Knowledge of the accurate MW allows one to detect the presence of post-translational modifications, proteolytic cleavages, and importantly, if the correct construct has been generated and purified. Denaturing liquid chromatography-mass spectrometry (LC-MS) can be an attractive method for obtaining this information. However, membrane protein LC-MS methodology has remained relatively under-explored and under-incorporated in comparison to methods for soluble proteins. Here, systematic investigation of multiple gradients and column chemistries has led to the development of a 5 min denaturing LC-MS method for acquiring membrane protein accurate MW measurements. Conditions were interrogated with membrane proteins, such as GPCRs and ion channels, as well as bispecific antibody constructs of variable sizes with the aim to provide the community with rapid LC-MS methods necessary to obtain chromatographic and accurate MW measurements in a medium- to high-throughput manner. The 5 min method detailed has successfully produced MW measurements for hydrophobic proteins with a wide MW range (17.5 to 105.3 kDa) and provided evidence that some constructs indeed contain unexpected modifications or sequence clipping. This rapid LC-MS method is also capable of baseline separating formylated and nonformylated aquaporinZ membrane protein.
- Reid, D. J., Diesing, J. M., Miller, M. A., Perry, S. M., Wales, J. A., Montfort, W. R., & Marty, M. T. (2018). MetaUniDec: High-Throughput Deconvolution of Native Mass Spectra. Journal of the American Society for Mass Spectrometry.More infoThe expansion of native mass spectrometry (MS) methods for both academic and industrial applications has created a substantial need for analysis of large native MS datasets. Existing software tools are poorly suited for high-throughput deconvolution of native electrospray mass spectra from intact proteins and protein complexes. The UniDec Bayesian deconvolution algorithm is uniquely well suited for high-throughput analysis due to its speed and robustness but was previously tailored towards individual spectra. Here, we optimized UniDec for deconvolution, analysis, and visualization of large data sets. This new module, MetaUniDec, centers around a hierarchical data format 5 (HDF5) format for storing datasets that significantly improves speed, portability, and file size. It also includes code optimizations to improve speed and a new graphical user interface for visualization, interaction, and analysis of data. To demonstrate the utility of MetaUniDec, we applied the software to analyze automated collision voltage ramps with a small bacterial heme protein and large lipoprotein nanodiscs. Upon increasing collisional activation, bacterial heme-nitric oxide/oxygen binding (H-NOX) protein shows a discrete loss of bound heme, and nanodiscs show a continuous loss of lipids and charge. By using MetaUniDec to track changes in peak area or mass as a function of collision voltage, we explore the energetic profile of collisional activation in an ultra-high mass range Orbitrap mass spectrometer. Graphical abstract ᅟ.
- Moutal, A., Wang, Y., Yang, X., Ji, Y., Luo, S., Dorame, A., Bellampalli, S. S., Chew, L. A., Cai, S., Dustrude, E. T., Keener, J. E., Marty, M. T., Vanderah, T. W., & Khanna, R. (2017). Dissecting the role of the CRMP2-neurofibromin complex on pain behaviors. Pain, 158(11), 2203-2221.More infoNeurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.
- Reid, D. J., Keener, J. E., Wheeler, A. P., Zambrano, D. E., Diesing, J. M., Reinhardt-Szyba, M., Makarov, A., & Marty, M. T. (2017). Engineering Nanodisc Scaffold Proteins for Native Mass Spectrometry. Analytical Chemistry, 89(21), 11189-11192.More infoLipoprotein nanodiscs are ideally suited for native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane proteins into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. To simplify interpretation of nanodisc spectra, we engineered a series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Finally, we demonstrate the use of mixed belt nanodiscs with embedded membrane proteins to confirm the dissociation of MSP prior to desolvation.
- Gault, J., Donlan, J. A., Liko, I., Hopper, J. T., Gupta, K., Housden, N. G., Struwe, W. B., Marty, M. T., Mize, T., Bechara, C., Zhu, Y., Wu, B., Kleanthous, C., Belov, M., Damoc, E., Makarov, A., & Robinson, C. V. (2016). High-resolution mass spectrometry of small molecules bound to membrane proteins. Nature methods, 13(4), 333-6.More infoSmall molecules are known to stabilize membrane proteins and to modulate their function and oligomeric state, but such interactions are often hard to precisely define. Here we develop and apply a high-resolution, Orbitrap mass spectrometry-based method for analyzing intact membrane protein-ligand complexes. Using this platform, we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.
- Hoi, K. K., Robinson, C. V., & Marty, M. T. (2016). Unraveling the Composition and Behavior of Heterogeneous Lipid Nanodiscs by Mass Spectrometry. Analytical chemistry, 88(12), 6199-204.More infoMass spectrometry (MS) has emerged as a powerful tool to study membrane protein complexes and protein-lipid interactions. Because they provide a precisely defined lipid bilayer environment, lipoprotein Nanodiscs offer a promising cassette for membrane protein MS analysis. However, heterogeneous lipids create several potential challenges for native MS: additional spectral complexity, ambiguous assignments, and differing gas-phase behaviors. Here, we present strategies to address these challenges and streamline analysis of heterogeneous-lipid Nanodiscs. We show that using two lipids of similar mass limits the complexity of the spectra in heterogeneous Nanodiscs and that the lipid composition can be determined by using a dual Fourier transform approach to obtain the average lipid mass. Further, the relationship between gas-phase behavior, lipid composition, and instrumental polarity was investigated to determine the effects of lipid headgroup chemistry on Nanodisc dissociation mechanisms. These results provide unique mechanistic and methodological insights into characterization of complex and heterogeneous systems by mass spectrometry.
- Landreh, M., Marty, M. T., Gault, J., & Robinson, C. V. (2016). A sliding selectivity scale for lipid binding to membrane proteins. Current opinion in structural biology, 39, 54-60.More infoBiological membranes form barriers that are essential for cellular integrity and compartmentalisation. Proteins in the membrane have co-evolved with their hydrophobic lipid environment, which serves as a solvent for proteins with very diverse requirements. As a result, their interactions range from non-selective to highly discriminating. Mass spectrometry enables us to monitor how lipids interact with membrane proteins and assess their effects on structure and dynamics. Recent studies illustrate the ability to differentiate specific lipid binding, preferential interactions with lipid subsets, and nonselective annular contacts. Here, we consider the biological implications of different lipid-binding scenarios and propose that binding occurs on a sliding selectivity scale, in line with the view of biological membranes as facilitators of dynamic protein and lipid organization.
- Marty, M. T., Hoi, K. K., & Robinson, C. V. (2016). Interfacing Membrane Mimetics with Mass Spectrometry. Accounts of chemical research, 49(11), 2459-2467.More infoMembrane proteins play critical physiological roles and make up the majority of drug targets. Due to their generally low expression levels and amphipathic nature, membrane proteins represent challenging molecular entities for biophysical study. Mass spectrometry offers several sensitive approaches to study the biophysics of membrane proteins. By preserving noncovalent interactions in the gas phase and using collisional activation to remove solubilization agents inside the mass spectrometer, native mass spectrometry (MS) is capable of studying isolated assemblies that would be insoluble in aqueous solution, such as membrane protein oligomers and protein-lipid complexes. Conventional methods use detergent to solubilize the protein prior to electrospray ionization. Gas-phase activation inside the mass spectrometer removes the detergent to yield the isolated proteins with bound ligands. This approach has proven highly successful for ionizing membrane proteins. With the appropriate choice of detergents, membrane proteins with bound lipid species can be observed, which allows characterization of protein-lipid interactions. However, detergents have several limitations. They do not necessarily replicate the native lipid bilayer environment, and only a small number of protein-lipid interactions can be resolved. In this Account, we summarize the development of different membrane mimetics as cassettes for MS analysis of membrane proteins. Examples include amphipols, bicelles, and picodiscs with a special emphasis on lipoprotein nanodiscs. Polydispersity and heterogeneity of the membrane mimetic cassette is a critical issue for study by MS. Ever more complex data sets consisting of overlapping protein charge states and multiple lipid-bound entities have required development of new computational, theoretical, and experimental approaches to interpret both mass and ion mobility spectra. We will present the rationale and limitations of these approaches. Starting with the early work on empty nanodiscs, we chart developments that culminate in recent high-resolution studies of membrane protein-lipid complexes ejected from nanodiscs. For the latter, increasing collision energies allowed progressive removal of nanodisc components, beginning with the scaffold proteins and continuing through successive shells of lipids, allowing direct characterization of the stoichiometry of the annular lipid belt that surrounds the membrane protein. We consider future directions for the study of membrane proteins in membrane mimetics, including the development of mixed lipid systems and native bilayer environments. Unambiguous assignment of these heterogeneous systems will rely heavily upon further enhancements in both data analysis protocols and instrumental resolution. We anticipate that these developments will provide new insights into the factors that control dynamic protein-lipid interactions in a variety of tailored and natural lipid environments.
- Marty, M. T., Hoi, K. K., Gault, J., & Robinson, C. V. (2016). Probing the Lipid Annular Belt by Gas-Phase Dissociation of Membrane Proteins in Nanodiscs. Angewandte Chemie (International ed. in English), 55(2), 550-4.More infoInteractions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas-phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein-lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.
- Marty, M. T., Baldwin, A. J., Marklund, E. G., Hochberg, G. K., Benesch, J. L., & Robinson, C. V. (2015). Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Analytical chemistry, 87(8), 4370-6.More infoInterpretation of mass spectra is challenging because they report a ratio of two physical quantities, mass and charge, which may each have multiple components that overlap in m/z. Previous approaches to disentangling the two have focused on peak assignment or fitting. However, the former struggle with complex spectra, and the latter are generally computationally intensive and may require substantial manual intervention. We propose a new data analysis approach that employs a Bayesian framework to separate the mass and charge dimensions. On the basis of this approach, we developed UniDec (Universal Deconvolution), software that provides a rapid, robust, and flexible deconvolution of mass spectra and ion mobility-mass spectra with minimal user intervention. Incorporation of the charge-state distribution in the Bayesian prior probabilities provides separation of the m/z spectrum into its physical mass and charge components. We have evaluated our approach using systems of increasing complexity, enabling us to deduce lipid binding to membrane proteins, to probe the dynamics of subunit exchange reactions, and to characterize polydispersity in both protein assemblies and lipoprotein Nanodiscs. The general utility of our approach will greatly facilitate analysis of ion mobility and mass spectra.
- Reading, E., Walton, T. A., Liko, I., Marty, M. T., Laganowsky, A., Rees, D. C., & Robinson, C. V. (2015). The Effect of Detergent, Temperature, and Lipid on the Oligomeric State of MscL Constructs: Insights from Mass Spectrometry. Chemistry & biology, 22(5), 593-603.More infoThe mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology.
- Shepherd, D. A., Marty, M. T., Giles, K., Baldwin, A. J., & Benesch, J. L. (2015). Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins. International Journal of Mass Spectrometry, 377, 663-671. doi:10.1016/j.ijms.2014.09.007More infoPolydispersity presents a considerable challenge for the detailed molecular characterisation of many proteins. This is because in most biophysical and structural biology approaches the molecules in solution are ensemble-averaged, obscuring differences between individual proteins or conformational states. Mass spectrometry is however inherently dispersive, allowing the specific interrogation of molecules with distinct mass-to-charge ratios. Here, we exploit this intrinsic benefit to develop a means for determining directly the stoichiometries and sizes of oligomers comprising a polydisperse protein ensemble. Our method exploits the quadrupole-(ion-mobility)-(time-of-flight) geometry by submitting selected mass-to-charge ranges for ion mobility separation followed by collision-induced dissociation. In this sequential experiment the ion mobility information of the precursors is reported by the arrival times of the fragments, which are highly separated in mass-to-charge by virtue of the dissociation process. We observe small differences in the measured arrival time between fragments arising due to ion transit conditions after the ion mobility cell. To accommodate these systematic deviations, we develop a mass-to-charge dependent correction, leading to a reduction in the error of the collision cross-section measurement to around 0.5%. We characterise our method using HSP16.9, a small heat-shock protein that undergoes a mono- to polydisperse transition upon lowering pH, and reveal that the oligomers it forms have collisional cross-sections consistent with the polyhedral and double-ring architectures exhibited by other members of the protein family.
- Wilcox, K. C., Marunde, M. R., Das, A., Velasco, P. T., Kuhns, B. D., Marty, M. T., Jiang, H., Luan, C. H., Sligar, S. G., & Klein, W. L. (2015). Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers. PloS one, 10(4), e0125263.More infoDespite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery for membrane protein targets.
- Marty, M. T., Zhang, H., Cui, W., Gross, M. L., & Sligar, S. G. (2014). Interpretation and deconvolution of nanodisc native mass spectra. Journal of the American Society for Mass Spectrometry, 25(2), 269-77.More infoNanodiscs are a promising system for studying gas-phase and solution complexes of membrane proteins and lipids. We previously demonstrated that native electrospray ionization allows mass spectral analysis of intact Nanodisc complexes at single lipid resolution. This report details an improved theoretical framework for interpreting and deconvoluting native mass spectra of Nanodisc lipoprotein complexes. In addition to the intrinsic lipid count and charge distributions, Nanodisc mass spectra are significantly shaped by constructive overlap of adjacent charge states at integer multiples of the lipid mass. We describe the mathematical basis for this effect and develop a probability-based algorithm to deconvolute the underlying mass and charge distributions. The probability-based deconvolution algorithm is applied to a series of dimyristoylphosphatidylcholine Nanodisc native mass spectra and used to provide a quantitative picture of the lipid loss in gas-phase fragmentation.
- Marty, M. T., & Beussman, D. J. (2013). Simulating a Time-of-Flight Mass Spectrometer: A LabView Exercise. Journal of Chemical Education, 90(2), 239-243. doi:10.1021/ed200158qMore infoAn in-depth understanding of all parameters that affect an instrumental analysis method, allowing students to explore how these instruments work so that they are not just a “black box”, is key to being able to optimize the technique and obtain the best possible results. It is, however, impractical to provide such in depth coverage of every technique and instrument in the classroom, and even more so in the laboratory. Exploring various instrumental parameters can be time consuming in the lab assuming that hands-on access is even available. One alternative to exploring different instrumental situations in the lab is to use computer simulations to model instrument performance under different conditions. Here, we present a simulation of a time-of-flight (TOF) mass spectrometer using the LabVIEW software platform. LabVIEW is routinely used for instrument interfacing and control and can be used to create simulations of a wide variety of chemical instruments. Because of their relatively high cost, mass spectrome...
- Marty, M. T., Wilcox, K. C., Klein, W. L., & Sligar, S. G. (2013). Nanodisc-solubilized membrane protein library reflects the membrane proteome. Analytical and bioanalytical chemistry, 405(12), 4009-16.More infoThe isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.
- Sloan, C. D., Marty, M. T., Sligar, S. G., & Bailey, R. C. (2013). Interfacing lipid bilayer nanodiscs and silicon photonic sensor arrays for multiplexed protein-lipid and protein-membrane protein interaction screening. Analytical chemistry, 85(5), 2970-6.More infoSoluble proteins are key mediators of many biochemical signaling pathways via direct interaction with the lipid bilayer and via membrane-bound receptors. Components of the cell membrane are involved in many important biological processes, including viral infection, blood clotting, and signal transduction, and as such, they are common targets of therapeutic agents. Therefore, the development of analytical approaches to study interactions at the cell membrane is of critical importance. Herein, we integrate two key technologies, silicon photonic microring resonator arrays and phospholipid bilayer nanodiscs, which together allow multiplexed screening of soluble protein interactions with lipid and membrane-embedded targets. Microring resonator arrays are an intrinsically multiplexable, label-free analysis platform that has previously been applied to studying protein-protein, protein-nucleic acid, and nucleic acid-nucleic acid interactions. Nanodiscs are protein-stabilized lipid assemblies that represent a convenient construct to mimic the native phospholipid bilayer, investigate the effects of membrane composition, and solubilize membrane-embedded targets. Exploiting the natural affinity of nanodisc-supported lipid bilayers for oxide-passivated silicon, we assembled single and multiplex sensor arrays via direct physisorption, characterizing electrostatic effects on the nanodisc attachment. Using model systems, we demonstrate the applicability of this platform for the parallel screening of protein interactions with nanodisc-embedded lipids, glycolipids, and membrane proteins.
- Marty, M. T., Das, A., & Sligar, S. G. (2012). Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs. Analytical and bioanalytical chemistry, 402(2), 721-9.More infoNanodiscs have become a leading technology to solubilize membrane proteins for biophysical, enzymatic, and structural investigations. Nanodiscs are nanoscale, discoidal lipid bilayers surrounded by an amphipathic membrane scaffold protein (MSP) belt. A variety of analytical tools has been applied to membrane proteins in nanodiscs, including several recent mass spectrometry studies. Mass spectrometry of full-length proteins is an important technique for analyzing protein modifications, for structural studies, and for identification of proteins present in binding assays. However, traditional matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry methods for analyzing full-length membrane proteins solubilized in nanodiscs are limited by strong signal from the MSP belt and weak signal from the membrane protein inside the nanodisc. Herein, we show that an optimized ultra-thin layer MALDI sample preparation technique dramatically enhances the membrane protein signal and nearly completely eliminates the MSP signal. First-shot MALDI and MALDI imaging are used to characterize the spots formed by the ultra-thin layer method. Furthermore, the membrane protein enhancement and MSP suppression are shown to be independent of the type of membrane protein and are applicable to mixtures of membrane proteins in nanodiscs.
- Marty, M. T., Sloan, C. D., Bailey, R. C., & Sligar, S. G. (2012). Nonlinear analyte concentration gradients for one-step kinetic analysis employing optical microring resonators. Analytical chemistry, 84(13), 5556-64.More infoConventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes, and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics.
- Marty, M. T., Zhang, H., Cui, W., Blankenship, R. E., Gross, M. L., & Sligar, S. G. (2012). Native mass spectrometry characterization of intact nanodisc lipoprotein complexes. Analytical chemistry, 84(21), 8957-60.More infoWe describe here the analysis of nanodisc complexes by using native mass spectrometry (MS) to characterize their molecular weight (MW) and polydispersity. Nanodiscs are nanoscale lipid bilayers that offer a platform for solubilizing membrane proteins. Unlike detergent micelles, nanodiscs are native-like lipid bilayers that are well-defined and potentially monodisperse. Their mass spectra allow peak assignment based on differences in the mass of a single lipid per complex. Resultant masses agree closely with predicted values and demonstrate conclusively the narrow dispersity of lipid molecules in the nanodisc. Fragmentation with collisionally activated dissociation (CAD) or electron-capture dissociation (ECD) shows loss of a small number of lipids and eventual collapse of the nanodisc with release of the scaffold protein. These results provide a foundation for future studies utilizing nanodiscs as a platform for launching membrane proteins into the gas phase.
Presentations
- Marty, M. T. (2017, April). Combining Nanodiscs with Native Mass Spectrometry to Study the Biophysics of Protein-Lipid Interactions. Biophest.
- Marty, M. T. (2017, February). Nanodiscs and Native Mass Spectrometry: New Tools for Membrane Protein Analysis. Society of Western Analytical Professors.
- Marty, M. T. (2017, July). Dark MS: Studying the Oligomerization, Interactions, and Light Activation of Rhodopsin with Native MS. Advancing Mass Spectrometry for Biophysics and Structural Biology.
- Marty, M. T. (2017, November). Making Membranes Fly:Native Mass Spectrometry of Nanodiscs. Grinnell College Seminar.
Poster Presentations
- Marty, M. T. (2017, June). Engineering Nanodiscs for Membrane Protein Native MS. American Society for Mass Spectrometry Annual Conference.
Reviews
- Townsend, J. A., & Marty, M. T. (2023. What's the defect? Using mass defects to study oligomerization of membrane proteins and peptides in nanodiscs with native mass spectrometry(pp 1-13).More infoMany membrane proteins form functional complexes that are either homo- or hetero-oligomeric. However, it is challenging to characterize membrane protein oligomerization in intact lipid bilayers, especially for polydisperse mixtures. Native mass spectrometry of membrane proteins and peptides inserted in lipid nanodiscs provides a unique method to study the oligomeric state distribution and lipid preferences of oligomeric assemblies. To interpret these complex spectra, we developed novel data analysis methods using macromolecular mass defect analysis. Here, we provide an overview of how mass defect analysis can be used to study oligomerization in nanodiscs, discuss potential limitations in interpretation, and explore strategies to resolve these ambiguities. Finally, we review recent work applying this technique to studying formation of antimicrobial peptide, amyloid protein, and viroporin complexes with lipid membranes.