Top-down mass spectrometry of native proteoforms and their complexes: a community study.

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.

Structural basis of omega-3 fatty acid transport across the blood-brain barrier.

Docosahexaenoic 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 sources1-6. 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 manner7,8. 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.

Top-Down Characterization of Bacterial Lipopolysaccharides and Lipooligosaccharides Using Activated-Electron Photodetachment Mass Spectrometry.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis. a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis. Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis, revealing a putative hexuronic acid (HexA) conjugated to lipid A.

Dimensional Dependence of Binding Kinetics.

In the context of protein-protein binding, the dissociation constant is used to describe the affinity between two proteins. For protein-protein interactions, most experimentally-measured dissociation constants are measured in solution and reported in units of volume concentration. However, many protein interactions take place on membranes. These interactions have dissociation constants with units of areal concentration, rather than volume concentration. Here, we present a novel, stochastic approach to understanding the dimensional dependence of binding kinetics. Using stochastic exit time calculations, in discrete and continuous space, we derive general reaction rates for protein-protein binding in one, two, and three dimensions and demonstrate that dimensionality greatly affects binding kinetics. Further, we present a formula to transform three-dimensional experimentally-measured dissociation constants to two-dimensional dissociation constants. This conversion can be used to mathematically model binding events that occur on membranes.

Investigating the Lipid Selectivity of Membrane Proteins in Heterogeneous Nanodiscs.

The 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 Escherichia coli 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.

Initiation and entrainment of multicellular automaticity via diffusion limited extracellular domains.

Electrically excitable cells often spontaneously and synchronously depolarize in vitro and in vivo preparations. It remains unclear how cells entrain and autorhythmically activate above the intrinsic mean activation frequency of isolated cells with or without pacemaking mechanisms. Recent studies suggest that cyclic ion accumulation and depletion in diffusion-limited extracellular volumes modulate electrophysiology by ephaptic mechanisms (nongap junction or synaptic coupling). This report explores how potassium accumulation and depletion in a restricted extracellular domain induces spontaneous action potentials in two different computational models of excitable cells without gap junctional coupling: Hodgkin-Huxley and Luo-Rudy. Importantly, neither model will spontaneously activate on its own without external stimuli. Simulations demonstrate that cells sharing a diffusion-limited extracellular compartment can become autorhythmic and entrained despite intercellular electrical heterogeneity. Autorhythmic frequency is modulated by the cleft volume and potassium fluxes through the cleft. Additionally, inexcitable cells can suppress or induce autorhythmic activity in an excitable cell via a shared cleft. Diffusion-limited shared clefts can also entrain repolarization. Critically, this model predicts a mechanism by which diffusion-limited shared clefts can initiate, entrain, and modulate multicellular automaticity in the absence of gap junctions.

The PafBC-mediated response sensitizes a bistable DNA damage response in Mycobacteria.

Due to the genotoxically challenging environments in which they live in, Mycobacteria have a complex DNA damage repair system that is governed by two major DNA damage responses, namely, the LexA/RecA-dependent response and the newly characterized PafBC-mediated response (Müller et al., 2018). The LexA/RecA-dependent response is a well-known bistable response found in different types of bacteria, and the Mycobacteria-specific PafBC-mediated response interacts with and modifies the LexA/RecA-dependent response (Müller et al., 2018). The interaction between the LexA/RecA-dependent response and the PafBC-mediated response has not been characterized mathematically. Our analysis shows that the addition of the PafBC-mediated response sensitizes the overall DNA damage response, effectively lowering the DNA damage rate threshold for activation.

A mathematical model of flagellar gene regulation and construction in Salmonella enterica.

Millions of people worldwide develop foodborne illnesses caused by Salmonella enterica (S. enterica) every year. The pathogenesis of S. enterica depends on flagella, which are appendages that the bacteria use to move through the environment. Interestingly, populations of genetically identical bacteria exhibit heterogeneity in the number of flagella. To understand this heterogeneity and the regulation of flagella quantity, we propose a mathematical model that connects the flagellar gene regulatory network to flagellar construction. A regulatory network involving more than 60 genes controls flagellar assembly. The most important member of the network is the master operon, flhDC, which encodes the FlhD4C2 protein. FlhD4C2 controls the construction of flagella by initiating the production of hook basal bodies (HBBs), protein structures that anchor the flagella to the bacterium. By connecting a model of FlhD4C2 regulation to a model of HBB construction, we investigate the roles of various feedback mechanisms. Analysis of our model suggests that a combination of regulatory mechanisms at the protein and transcriptional levels induce bistable FlhD4C2 levels and heterogeneous numbers of flagella. Also, the balance of regulatory mechanisms that become active following HBB construction is sufficient to provide a counting mechanism for controlling the total number of flagella produced.

Red Light and the Dormancy-Germination Decision in Arabidopsis Seeds.

The Arabidopsis dormancy-germination transition is known to be environmentally cued and controlled by the competing hormones abscisic acid (ABA) and gibberellin (GA) produced by the seed. Recently, new molecular details have emerged concerning the propagation of red light through a complex gene regulatory network involving PhyB, PIF1, and RVE1. This network influences the formation of the PIF1-RVE1 complex [1,2]. The PIF1-RVE1 complex is a transcription factor that regulates the production of ABA and GA and helps shift the balance to high concentration of ABA and low concentration of GA, which corresponds to a dormant seed state. This newly discovered gene regulatory network has not been analyzed mathematically. Our analysis shows that this gene regulatory network exhibits switch-like bistability as a function of the red light input and makes a suite of biologically testable predictions concerning seed dormancy and germination in response to the amplitude and periodicity of an oscillatory red light input.

Bifurcation Analysis of a Heat-Sensitive Epigenetic Regulatory Network.

An epigenetic regulatory network that influences transgenerational inheritance of a heat-altered phenotype was recently discovered in Arabidopsis. Our analysis shows that transgenerational inheritance of the heat-altered phenotype operates in a switch-like manner and can be turned on or off as a function of heat. We also show that trans-acting small interfering RNAs act as an "inverse amplifier" of HTT5, the protein that controls the heat-altered phenotype by a currently unknown mechanism. Our analysis uses the resultant to find an analytic expression for a cusp curve in parameter space and to find a parameter bound on switch-like behavior.

Native Mass Spectrometry Reveals the Simultaneous Binding of Lipids and Zinc to Rhodopsin.

Rhodopsin, 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.

A systematic search for switch-like behavior in type II toxin-antitoxin systems.

Bistable switch-like behavior is a ubiquitous feature of gene regulatory networks with decision-making capabilities. Type II toxin-antitoxin (TA) systems are hypothesized to facilitate a bistable switch in toxin concentration that influences the dormancy transition in persister cells. However, a series of recent retractions has raised fundamental questions concerning the exact mechanism of toxin propagation in persister cells and the relationship between type II TA systems and cellular dormancy. Through a careful modeling search, we identify how sp: bistablilty can emerge in type II TA systems by systematically modifying a basic model for the RelBE system with other common biological mechanisms. Our systematic search uncovers a new combination of mechanisms influencing bistability in type II TA systems and explores how toxin bistability emerges through synergistic interactions between paired type II TA systems. Our analysis also illustrates how Descartes' rule of signs and the resultant can be used as a powerful delineator of bistability in mathematical systems regardless of application.

Measuring Remodeling of the Lipid Environment Surrounding Membrane Proteins with Lipid Exchange and Native Mass Spectrometry.

Due 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 E. coli ammonium transporter AmtB prefers phosphatidylglycerol lipids overall but has a minor affinity for phosphatidylcholine lipids.

A Mathematical Model for Inheritance of DNA Methylation Patterns in Somatic Cells.

DNA methylation is an essential epigenetic mechanism used by cells to regulate gene expression. Interestingly, DNA replication, a function necessary for cell division, disrupts the methylation pattern. Since perturbed methylation patterns are associated with aberrant gene expression and many diseases, including cancer, restoration of the correct pattern following DNA replication is crucial. However, the exact mechanisms of this restoration remain under investigation. DNA methyltransferases (Dnmts) perform methylation by adding a methyl group to cytosines at CpG sites in the DNA. These CpG sites are found in regions of high density, termed CpG islands (CGIs), and regions of low density in the genome. Nearly, every CpG site in a CGI has the same state, either methylated or unmethylated, and almost all CpG sites in regions of low CpG density are methylated. We propose a stochastic model for the dynamics of the post-replicative restoration of methylation patterns. The model considers the recruitment of Dnmts and demethylating enzymes to regions of hyper- and hypomethylation, respectively. The model also includes the interaction between Dnmt1 and PCNA, an enzyme that localizes Dnmt1 to the replication complex. Using our model, we predict that the methylation of regions of DNA can be bistable. Further, we predict that recruitment mechanisms maintain methylation in CGIs, whereas the Dnmt1-PCNA interaction maintains methylation in low-density regions.

Chemical Additives Enable Native Mass Spectrometry Measurement of Membrane Protein Oligomeric State within Intact Nanodiscs.

Membrane 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 previous 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 the 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.

Imidazole Derivatives Improve Charge Reduction and Stabilization for Native Mass Spectrometry.

Noncovalent 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.

Enhanced Nucleocytoplasmic Transport due to Competition for Elastic Binding Sites.

Nuclear pore complexes (NPCs) control all traffic into and out of the cell nucleus. NPCs are molecular machines that simultaneously achieve high selectivity and high transport rates. The biophysical details of how cargoes rapidly traverse the pore remain unclear but are known to be mediated by interactions between cargo-binding chaperone proteins and natively unstructured nucleoporin proteins containing many phenylalanine-glycine repeats (FG nups) that line the pore's central channel. Here, we propose a specific and detailed physical mechanism for the high speed of nuclear import based on the elasticity of FG nups and on competition between individual chaperone proteins for FG nup binding. We develop a mathematical model to support our proposed mechanism. We suggest that the recycling of nuclear import factors back to the cytoplasm is important for driving high-speed import and predict the existence of an optimal cytoplasmic concentration of cargo for enhancing the rate of import over a purely diffusive rate.

Rapid LC-MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins.

Therapeutic 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.

The computation of biomarkers in pharmacokinetics with the aid of singular perturbation methods.

In pharmacokinetics, exact solutions to one-compartment models with nonlinear elimination kinetics cannot be found analytically, if dosages are assumed to be administered repetitively through extravascular routes (Tang and Xiao in J Pharmacokinet Pharmacodyn 34(6):807-827, 2007). Hence, for the corresponding impulsed dynamical system, alternative methods need to be developed to find approximate solutions. The primary purpose of this paper is to use the method of matched asymptotic expansions (Holmes Introduction to Perturbation Methods, vol 20. Springer Science & Business Media, Berlin, 2012), a singular perturbation method (Holmes, Introduction to Perturbation Methods, vol 20. Springer Science & Business Media, Berlin, 2012; Keener Principles of Applied Mathematics, Addison-Wesley, Boston, 1988), to obtain approximate solutions. With this method, we are able to rigorously determine conditions under which there is a stable periodic solution of the model equations. Furthermore, typical important biomarkers that enable the design of practical, efficient and safe drug delivery protocols, such as the time the drug concentration reaches the peak and the peak concentrations, are theoretically estimated by the perturbation method we employ.

A physics-based model for maintenance of the pH gradient in the gastric mucus layer.

It is generally accepted that the gastric mucus layer provides a protective barrier between the lumen and the mucosa, shielding the mucosa from acid and digestive enzymes and preventing autodigestion of the stomach epithelium. However, the precise mechanisms that contribute to this protective function are still up for debate. In particular, it is not clear what physical processes are responsible for transporting hydrogen protons, secreted within the gastric pits, across the mucus layer to the lumen without acidifying the environment adjacent to the epithelium. One hypothesis is that hydrogen may be bound to the mucin polymers themselves as they are convected away from the mucosal surface and eventually degraded in the stomach lumen. It is also not clear what mechanisms prevent hydrogen from diffusing back toward the mucosal surface, thereby lowering the local pH. In this work we investigate a physics-based model of ion transport within the mucosal layer based on a Nernst-Planck-like equation. Analysis of this model shows that the mechanism of transporting protons bound to the mucus gel is capable of reproducing the trans-mucus pH gradients reported in the literature. Furthermore, when coupled with ion exchange at the epithelial surface, our analysis shows that bicarbonate secretion alone is capable of neutralizing the epithelial pH, even in the face of enormous diffusive gradients of hydrogen. Maintenance of the pH gradient is found to be robust to a wide array of perturbations in both physiological and phenomenological model parameters, suggesting a robust physiological control mechanism.NEW & NOTEWORTHY This work combines modeling techniques based on physical principles, as well as novel numerical simulations to test the plausibility of one hypothesized mechanism for proton transport across the gastric mucus layer. Results show that this mechanism is able to maintain the extreme pH gradient seen in in vivo experiments and suggests a highly robust regulation mechanism to maintain this gradient in the face of dynamic lumen composition.