Native Mass Spectrometry Studies Research Articles

BRD4354 Is a Potent Covalent Inhibitor against the SARS-CoV-2 Main Protease.

Numerous organic molecules are known to inhibit the main protease (MPro) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of MPro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of MPro. The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against MPro with an IC50 (concentration that inhibits activity by 50%) of 0.72 ± 0.04 μM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a KI of 1.9 ± 0.5 μM followed by a second slow inactivation step, kinact,max of 0.040 ± 0.002 min-1. Native mass spectrometry studies indicate that a covalent intermediate is formed where the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of MPro. Based on these data, a Michael-addition reaction mechanism between MPro C145 and BRD4354 was proposed. These results suggest that both preclinical testing of BRD4354 and structure-activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.

Polyamine detergents tailored for native mass spectrometry studies of membrane proteins

Native mass spectrometry (MS) is a powerful technique for interrogating membrane protein complexes and their interactions with other molecules. A key aspect of the technique is the ability to preserve native-like structures and noncovalent interactions, which can be challenging depending on the choice of detergent. Different strategies have been employed to reduce charge on protein complexes to minimize activation and preserve non-covalent interactions. Here, we report the synthesis of a class of polyamine detergents tailored for native MS studies of membrane proteins. These detergents, a series of spermine covalently attached to various alkyl tails, are exceptional charge-reducing molecules, exhibiting a ten-fold enhanced potency over spermine. Addition of polyamine detergents to proteins solubilized in maltoside detergents results in improved, charge-reduced native mass spectra and reduced dissociation of subunits. Polyamine detergents open new opportunities to investigate membrane proteins in different detergent environments that have thwarted previous native MS studies.

Microflow size exclusion chromatography to preserve micromolar affinity complexes and achieve subunit separations for native state mass spectrometry

For high throughput native mass spectrometry (MS) protein characterization, it is advantageous to desalt and separate proteins by size exclusion chromatography (SEC). Sensitivity, resolution, and speed in these methods remain limited by standard SEC columns. Moreover, the efficient packing of small bore columns is notoriously difficult. SEC sensitivity is inherently limited because solutes are not focused into concentrated bands and low affinity native complexes may dissociate on column. Recent work evaluated the suitability of crosslinked gel media in small bore formats for online desalting. Here, small bore format online SEC for native MS studies is again investigated but with alternative materials. We systematically studied the utility of diol and hydroxy terminated polyethylene oxide (PEO) bonded 1.7 µm organosilica particles as packed into 1 mm ID stainless steel (SS) hardware and hardware treated with hydrophilic hybrid surface technology (h-HST). For the equivalent diol-bonded particle and hardware, UV limits of detection (LODs) were reduced 32 to 89% with a microflow separation (15 µL/min) on a 1 × 50 mm column as compared to a 4.6 × 150 mm high-flow separation (300 µL/min) at the same linear velocity. Run times were also shortened by 45%. A switch from SS to h-HST hardware led to a significant reduction in secondary interactions and a corresponding improvement in detection limits for trastuzumab, myoglobin, IgG and albumin for both UV and MS. Coupling of the small bore columns to multichannel microflow emitters resulted in 10 to 100-fold gains in MS sensitivity, depending on the analyte. MS LOD values were significantly reduced into the low attomole ranges. Columns were then evaluated for their effects on the preservation of complexes, including concanavalin A, in its apo and ligand-bound states, and three therapeutically relevant noncovalent systems previously undetected on large column formats. The results suggest that the detection of large complexes by SEC is not just a function of sensitivity but is directly affected by chemical secondary interactions. The ability to detect 0.1 to 1 MDa complexes, with between 1 and 40 micromolar dissociation constants, represents a critical advancement for high-throughput native MS workflows as applied to the analysis of therapeutics.

Probing the structure of nanodiscs using surface-induced dissociation mass spectrometry.

In 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.

Expanding the Types of Lipids Amenable to Native Mass Spectrometry of Lipoprotein Complexes.

Native 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.

Generation of Charge-Reduced Ions of Membrane Protein Complexes for Native Ion Mobility Mass Spectrometry Studies.

Recent advances in native mass spectrometry (MS) have enabled the elucidation of how small molecule binding to membrane proteins modulates their structure and function. The protein-stabilizing osmolyte, trimethylamine oxide (TMAO), exhibits attractive properties for native MS studies. Here, we report significant charge reduction, nearly threefold, for three membrane protein complexes in the presence of this osmolyte without compromising mass spectral resolution. TMAO improves the ability to resolve individual lipid-binding events to the ammonia channel (AmtB) by over 200% compared to typical native conditions. The generation of ions with compact structure and access to a larger number of lipid-binding events through the incorporation of TMAO increases the utility of IM-MS for structural biology studies. Graphical Abstract.

Native Mass Spectrometry Characterizes the Photosynthetic Reaction Center Complex from the Purple Bacterium Rhodobacter sphaeroides.

Native mass spectrometry (MS) is an emerging approach to study protein complexes in their near-native states and to elucidate their stoichiometry and topology. Here, we report a native MS study of the membrane-embedded reaction center (RC) protein complex from the purple photosynthetic bacterium Rhodobacter sphaeroides. The membrane-embedded RC protein complex is stabilized by detergent micelles in aqueous solution, directly introduced into a mass spectrometer by nano-electrospray (nESI), and freed of detergents and dissociated in the gas phase by collisional activation. As the collision energy is increased, the chlorophyll pigments are gradually released from the RC complex, suggesting that native MS introduces a near-native structure that continues to bind pigments. Two bacteriochlorophyll a pigments remain tightly bound to the RC protein at the highest collision energy. The order of pigment release and their resistance to release by gas-phase activation indicates the strength of pigment interaction in the RC complex. This investigation sets the stage for future native MS studies of membrane-embedded photosynthetic pigment-protein and related complexes.Graphical Abstract.