Defining proteoform-specific interactions for drug targeting in a native cell signalling environment.

Understanding the dynamics of membrane protein-ligand interactions within a native lipid bilayer is a major goal for drug discovery. Typically, cell-based assays are used, however, they are often blind to the effects of protein modifications. In this study, using the archetypal G protein-coupled receptor rhodopsin, we found that the receptor and its effectors can be released directly from retina rod disc membranes using infrared irradiation in a mass spectrometer.

Structural insights into the high basal activity and inverse agonism of the orphan receptor GPR6 implicated in Parkinson's disease.

GPR6 is an orphan G protein-coupled receptor with high constitutive activity found in D2-type dopamine receptor-expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson's disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson's disease in clinical trials. In addition, we obtained a cryo-electron microscopy structure of the signaling complex between GPR6 and its cognate G heterotrimer.

New Processes for Ionizing Nonvolatile Compounds in Mass Spectrometry: The Road of Discovery to Current State-of-the-Art.

This covers discovery and mechanistic aspects as well as initial applications of novel ionization processes for use in mass spectrometry that guided us in a series of subsequent discoveries, instrument developments, and commercialization. matrix-assisted ionization on an intermediate pressure matrix-assisted laser desorption/ionization source the use of a laser, high voltages, or any other added energy was simply unbelievable, at first. Individually and as a whole, the various discoveries and inventions started to paint, , an exciting new picture and outlook in mass spectrometry from which key developments grew that were at the time unimaginable, and continue to surprise us in its simplistic preeminence.

Thermal Remodeling of Human HDL Particles Reveals Diverse Subspecies.

High-density lipoproteins (HDL) are micelle-like particles consisting of a core of triglycerides and cholesteryl esters surrounded by a shell of phospholipid, cholesterol, and apolipoproteins. HDL is considered "good" cholesterol, and its concentration in plasma is used clinically in assessing cardiovascular health. However, these particles vary in structure, composition, and therefore function, and thus can be resolved into subpopulations, some of which have specific cardioprotective properties.

ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT). Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS).

Phospholipids Differentially Regulate Ca Binding to Synaptotagmin-1.

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap.

ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data.

Native mass spectrometry has recently moved alongside traditional structural biology techniques in its ability to provide clear insights into the composition of protein complexes. However, to date, limited software tools are available for the comprehensive analysis of native mass spectrometry data on protein complexes, particularly for experiments aimed at elucidating the composition of an intact protein complex. Here, we introduce ProSight Native as a start-to-finish informatics platform for analyzing native protein and protein complex data.

Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein -acyltransferase.

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein -acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap.

Uncovering the Role of -Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry.

Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of -glycan occupancy on the assembly of multiple Spike-ACE2 complexes.

The next decade of protein structure.

With recent dramatic advances in various techniques used for protein structure research, we asked researchers to comment on the next exciting questions for the field and about how these techniques will advance our knowledge not only about proteins but also about human health and diseases.

Calcium Contributes to Polarized Targeting of HIV Assembly Machinery by Regulating Complex Stability.

Polarized or precision targeting of protein complexes to their destinations is fundamental to cellular homeostasis, but the mechanism underpinning directional protein delivery is poorly understood. Here, we use the uropod targeting HIV synapse as a model system to show that the viral assembly machinery Gag is copolarized with the intracellular calcium (Ca) gradient and binds specifically with Ca. Conserved glutamic/aspartic acids flanking endosomal sorting complexes required for transport binding motifs are major Ca binding sites.

Multiple Roles of SARS-CoV-2 N Protein Facilitated by Proteoform-Specific Interactions with RNA, Host Proteins, and Convalescent Antibodies.

The SARS-CoV-2 nucleocapsid (N) protein is a highly immunogenic viral protein that plays essential roles in replication and virion assembly. Here, using native mass spectrometry, we show that dimers are the functional unit of ribonucleoprotein assembly and that N protein binds RNA with a preference for GGG motifs, a common motif in coronavirus packaging signals. Unexpectedly, proteolytic processing of N protein resulted in the formation of additional proteoforms.

Thermal Analysis of a Mixture of Ribosomal Proteins by vT-ESI-MS: Toward a Parallel Approach for Characterizing the .

The thermal stabilities of endogenous, intact proteins and protein assemblies in complex mixtures were characterized in parallel by means of variable-temperature electrospray ionization coupled to mass spectrometry (vT-ESI-MS). The method is demonstrated by directly measuring the melting transitions of seven proteins from a mixture of proteins derived from ribosomes. A proof-of-concept measurement of a fraction of an lysate is provided to extend this approach to characterize the thermal stability of a proteome.

Heterogeneity of Glycan Processing on Trimeric SARS-CoV-2 Spike Protein Revealed by Charge Detection Mass Spectrometry.

The heterogeneity associated with glycosylation of the 66 N-glycan sites on the protein trimer making up the spike (S) region of the SARS-CoV-2 virus has been assessed by charge detection mass spectrometry (CDMS). CDMS allows simultaneous measurement of the mass-to-charge ratio and charge of individual ions, so that mass distributions can be determined for highly heterogeneous proteins such as the heavily glycosylated S protein trimer. The CDMS results are compared to recent glycoproteomics studies of the structure and abundance of glycans at specific sites.

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.

The SARS-CoV-2 main protease (M ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. M is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of M .

New mass spectrometry concepts for characterization of synthetic polymers and additives.

Rationale: New ionization processes have been developed for biological mass spectrometry (MS) in which the matrix lifts the nonvolatile analyte into the gas phase as ions without any additional energy input. We rationalized that additional fundamental knowledge is needed to assess analytical utility for the field of synthetic polymers and additives.

Methods: Different mass spectrometers (Thermo Orbitrap (Q-)Exactive (Focus); Waters SYNAPT G2(S)) were employed.

Lot-to-Lot Variation in Adeno-Associated Virus Serotype 9 (AAV9) Preparations.

Viral vectors are complex drugs that pose a particular challenge for manufacturing. Previous studies have shown that, unlike small-molecule drugs, vector preparations do not yield a collection of identical particles. Instead, a mixture of particles that vary in capsid stoichiometry and impurities is created, which may differ from lot to lot.

Resolution of Lipoprotein Subclasses by Charge Detection Mass Spectrometry.

Lipoproteins are micelle-like assemblies that are key players in the pathogenesis of atherosclerosis. High-density lipoprotein (HDL), low-density lipoproteins (LDL), and very low density lipoprotein (VLDL) are the three major classes present in fasting plasma. Within each class, there is a broad size distribution with wide variations in protein and lipid content.

Multiple Pathways in Capsid Assembly.

For a three-dimensional structure to spontaneously self-assemble from many identical components, the steps on the pathway must be kinetically accessible. Many virus capsids are icosahedral and assembled from hundreds of identical proteins, but how they navigate the assembly process is poorly understood. Capsid assembly is thought to involve stepwise addition of subunits to a growing capsid fragment.

Unprecedented Ionization Processes in Mass Spectrometry Provide Missing Link between ESI and MALDI.

In the field of mass spectrometry, producing intact, highly-charged protein ions from surfaces is a conundrum with significant potential payoff in application areas ranging from biomedical to clinical research. Here, we report on the ability to form intact, highly-charged protein ions on high vacuum time-of-flight mass spectrometers in the linear and reflectron modes achievable using experimental conditions that allow effective matrix removal from both the sample surfaces and from the charged clusters formed by the laser ablation event. The charge states are the highest reported on high vacuum mass spectrometers, yet they remain at only around a third of the highest charge obtained using laser ablation with a suitable matrix at atmospheric pressure.

Hepatitis B Virus Capsid Completion Occurs through Error Correction.

Understanding capsid assembly is important because of its role in virus lifecycles and in applications to drug discovery and nanomaterial development. Many virus capsids are icosahedral, and assembly is thought to occur by the sequential addition of capsid protein subunits to a nucleus, with the final step completing the icosahedron. Almost nothing is known about the final (completion) step because the techniques usually used to study capsid assembly lack the resolution.

Matrix-Assisted Ionization-Ion Mobility Spectrometry-Mass Spectrometry: Selective Analysis of a Europium-PEG Complex in a Crude Mixture.

The analytical utility of a new and simple to use ionization method, matrix-assisted ionization (MAI), coupled with ion mobility spectrometry (IMS) and mass spectrometry (MS) is used to characterize a 2-armed europium(III)-containing poly(ethylene glycol) (Eu-PEG) complex directly from a crude sample. MAI was used with the matrix 1,2-dicyanobenzene, which affords low chemical background relative to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MAI provides high ion abundance of desired products in comparison to ESI and MALDI.