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Here we used native mass spectrometry (native MS) to probe a SARS-CoV protease, PLpro, which plays critical roles in coronavirus disease by affecting viral protein production and antagonizing host antiviral responses. Ultraviolet photodissociation (UVPD) and variable temperature electrospray ionization (vT ESI) were used to localize binding sites of PLpro inhibitors and revealed the stabilizing effects of inhibitors on protein tertiary structure. We compared PLpro from SARS-CoV-1 and SARS-CoV-2 in terms of inhibitor and ISG15 interactions to discern possible differences in protease function. A PLpro mutant lacking a single cysteine was used to localize inhibitor binding, and thermodynamic measurements revealed that inhibitor PR-619 stabilized the folded PLpro structure. These results will inform further development of PLpro as a therapeutic target against SARS-CoV-2 and other emerging coronaviruses.

The main protease (M pro ) of SARS-CoV-2 is an essential enzyme for coronaviral maturation and is the target of Paxlovid, which is currently the standard-of-care treatment for COVID-19. There remains a need to identify new inhibitors of M pro as viral resistance to Paxlovid emerges. Here, we report the use of native mass spectrometry coupled with 193 nm ultraviolet photodissociation (UVPD) and integrated with other biophysical tools to structurally characterize M pro and its interactions with potential covalent inhibitors. The overall energy landscape was obtained using variable temperature nanoelectrospray ionization (vT-nESI), thus providing quantitative evaluation of inhibitor binding on the stability of M pro . Thermodynamic parameters extracted from van't Hoff plots revealed that the dimeric complexes containing each inhibitor showed enhanced stability through increased melting temperatures as well as overall lower average charge states, giving insight into the basis for inhibition mechanisms.

Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans, including microheterogeneity of some of the glycosites (amino acid residues). Various mass spectrometry (MS) strategies have proven tremendously successful for localizing and identifying glycans, typically utilizing a bottom-up workflow in which glycoproteins are digested to create glycopeptides to facilitate analysis. An emerging alternative is top-down MS that aims to characterize intact glycoproteins to allow precise identification and localization of glycans. The most comprehensive characterization of intact glycoproteins requires integration of a suitable separation method and high performance tandem mass spectrometry to provide both protein sequence information and glycosite localization. Here, we couple ultraviolet photodissociation and hydrophilic interaction chromatography with high resolution mass spectrometry to advance the characterization of intact glycoproteins ranging from 15 to 34 kDa, offering site localization of glycans, providing sequence coverages up to 93%, and affording relative quantitation of individual glycoforms.

Gram-negative bacteria develop and exhibit resistance to antibiotics, owing to their highly asymmetric outer membrane maintained by a group of six proteins comprising the Mla (maintenance of lipid asymmetry) pathway. Here, we investigate the lipid binding preferences of one Mla protein, MlaC, which transports lipids through the periplasm. We used ultraviolet photodissociation (UVPD) to identify and characterize modifications of lipids endogenously bound to MlaC expressed in three different bacteria strains. UVPD was also used to localize lipid binding to MlaC residues 130-140, consistent with the crystal structure reported for lipid-bound MlaC. The impact of removing the bound lipid from MlaC on its structure was monitored based on collision cross section measurements, revealing that the protein unfolded prior to release of the lipid. The lipid selectivity of MlaC was evaluated based on titrimetric experiments, indicating that MlaC-bound lipids in various classes (sphingolipids, glycerophospholipids, and fatty acids) as long as they possessed no more than two acyl chains.

GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal β-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual β-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack β-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3's role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3's differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients., Competing Interests: Conflict of interest The author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The measurement of collision cross sections (CCS, σ) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the modified hard collision model, thus deviating from the prior FT-MS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision induced unfolding and tandem mass spectrometry experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.

Measurement of collision cross section (CCS), a parameter reflecting an ion's size and shape, alongside high-resolution mass analysis extends the depth of molecular analysis by providing structural information beyond molecular mass alone. Although these measurements are most commonly undertaken using a dedicated ion mobility cell coupled to a mass spectrometer, alternative methods have emerged to extract CCSs directly by analysis of the decay rates of either time-domain transient signals or the FWHM of frequency domain peaks in FT mass analyzers. This information is also accessible from FTMS mass spectra obtained in commonly used workflows directly without the explicit access to transient or complex Fourier spectra. Previously, these experiments required isolation of individual charge states of ions prior to CCS analysis, limiting throughput. Here we advance Orbitrap CCS measurements to more users and applications by determining CCSs from commonly available mass spectra files as well as estimating CCS for multiple charge states simultaneously and showcase these methods by the measurement of CCSs of fragment ions produced from collisional activation of proteins.

The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.

The structural diversity of phospholipids plays a critical role in cellular membrane dynamics, energy storage, and cellular signaling. Despite its importance, the extent of this diversity has only recently come into focus, largely owing to advances in separation science and mass spectrometry methodology and instrumentation. Characterization of glycerophospholipid (GP) isomers differing only in their acyl chain configurations and locations of carbon-carbon double bonds (C═C) remains challenging due to the need for both effective separation of isomers and advanced tandem mass spectrometry (MS/MS) technologies capable of double-bond localization. Drift tube ion mobility spectrometry (DTIMS) coupled with MS can provide both fast separation and accurate determination of collision cross section (CCS) of molecules but typically lacks the resolving power needed to separate phospholipid isomers. Ultraviolet photodissociation (UVPD) can provide unambiguous double-bond localization but is challenging to implement on the timescales of modern commercial drift tube time-of-flight mass spectrometers. Here, we present a novel method for coupling DTIMS with a UVPD-enabled Orbitrap mass spectrometer using absorption mode Fourier transform multiplexing that affords simultaneous localization of double bonds and accurate CCS measurements even when isomers cannot be fully resolved in the mobility dimension. This method is demonstrated on two- and three-component mixtures and shown to provide CCS measurements that differ from those obtained by individual analysis of each component by less than 1%.

Glycerophospholipids (GPLs), one of the main components of bacterial cell membranes, exhibit high levels of structural complexity that are directly correlated with biophysical membrane properties such as permeability and fluidity. This structural complexity arises from the substantial variability in the individual GPL structural components such as the acyl chain length and headgroup type and is further amplified by the presence of modifications such as double bonds and cyclopropane rings. Here we use liquid chromatography coupled to high-resolution and high-mass-accuracy ultraviolet photodissociation mass spectrometry for the most in-depth study of bacterial GPL modifications to date. In doing so, we unravel a diverse array of unexplored GPL modifications, ranging from acyl chain hydroxyl groups to novel headgroup structures. Along with characterizing these modifications, we elucidate general trends in bacterial GPL unsaturation elements and thus aim to decipher some of the biochemical pathways of unsaturation incorporation in bacterial GPLs. Finally, we discover aminoacyl-PGs not only in Gram-positive bacteria but also in Gram-negative C. jejuni , advancing our knowledge of the methods of surface charge modulation that Gram-negative organisms may adopt for antibiotic resistance.