Suborgan Level Quantitation of Proteins in Tissues Delivered by Polymeric Nanocarriers.

Amidst the rapid growth of protein therapeutics as a drug class, there is an increased focus on designing systems to effectively deliver proteins to target organs. Quantitative monitoring of protein distributions in tissues is essential for optimal development of delivery systems; however, existing strategies can have limited accuracy, making it difficult to assess suborgan dosing. Here, we describe a quantitative imaging approach that utilizes metal-coded mass tags and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to quantify the suborgan distributions of proteins in tissues that have been delivered by polymeric nanocarriers. Using this approach, we measure nanomole per gram levels of proteins as delivered by guanidinium-functionalized poly(oxanorborneneimide) (PONI) polymers to various tissues, including the alveolar region of the lung. Due to the multiplexing capability of the LA-ICP-MS imaging, we are also able to simultaneously quantify protein and polymer distributions, obtaining valuable information about the relative excretion pathways of the protein cargo and carrier. This imaging approach will facilitate quantitative correlations between nanocarrier properties and protein cargo biodistributions.

Proteins Can Withstand More Extensive Labeling while Providing Accurate Structural Information in Covalent Labeling-Mass Spectrometry.

Diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry (CL-MS) has been extensively utilized to study protein structure and interactions owing to its ease of use, commercial availability, and broad labeling of nucleophilic residues. During typical CL-MS experiments with DEPC, the extent of labeling is kept low to avoid any structural perturbations resulting from covalent modification of the protein. In this study, we demonstrate that proteins can be labeled more extensively via DEPC and still provide accurate structural information. To show this, we modeled labeling kinetics over a range of DEPC concentrations and used molecular dynamics simulations to investigate the molecular-level effects of extensive labeling on the protein structure. Our results indicate that higher extents of DEPC labeling do not significantly perturb the protein structure and can lead to improved precision, detectability of labeled peptides, and protein structural resolution. Furthermore, higher extents of labeling enable better identification of protein-ligand binding sites where lower extents of modification provide ambiguous results.

Quantitative imaging of the sub-organ distributions of nanomaterials in biological tissues via laser ablation inductively coupled plasma mass spectrometry.

Nanomaterials have been employed in many biomedical applications, and their distributions in biological systems can provide an understanding of their behavior in vivo. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) can be used to determine the distributions of metal-based NMs in biological systems. However, LA-ICP-MS has not commonly been used to quantitatively measure the cell-specific or sub-organ distributions of nanomaterials in tissues. Here, we describe a new platform that uses spiked gelatin standards with control tissues on top to obtain an almost perfect tissue mimic for quantitative imaging purposes. In our approach, gelatin is spiked with both nanomaterial standards and an internal standard to improve quantitation and image quality. The value of the developed approach is illustrated by determining the sub-organ distributions of different metal-based and metal-tagged polymeric nanomaterials in mice organs. The LA-ICP-MS images reveal that the chemical and physical properties of the nanomaterials cause them to distribute in quantitatively different extents in spleens, kidneys, and tumors, providing new insight into the fate of nanomaterials in vivo. Furthermore, we demonstrate that this approach enables quantitative co-localization of nanomaterials and their cargo. We envision this method being a valuable tool in the development of nanomaterial drug delivery systems.

Synergistic Treatment of Multidrug-Resistant Bacterial Biofilms Using Silver Nanoclusters Incorporated into Biodegradable Nanoemulsions.

Multidrug resistance (MDR) in bacteria is a critical global health challenge that is exacerbated by the ability of bacteria to form biofilms. We report a combination therapy for biofilm infections that integrates silver nanoclusters (AgNCs) into polymeric biodegradable nanoemulsions (BNEs) incorporating eugenol. These Ag-BNEs demonstrated synergistic antimicrobial activity between the AgNCs and the BNEs. Microscopy studies demonstrated that Ag-BNEs penetrated the dense biofilm matrix and effectively disrupted the bacterial membrane. The Ag-BNE vehicle also resulted in more effective silver delivery into the biofilm than AgNCs alone. This combinacional system featured disruptionof biofilms by BNEs and enhanced delivery of AgNCs for synergy to provide highly efficient killing of MDR biofilms.

Multisite Labeling of Proteins Using the Ligand-Directed Reactivity of Triggerable Michael Acceptors.

Targeted modification of endogenous proteins without genetic manipulation of protein expression machinery has a range of applications from chemical biology to drug discovery. Despite being demonstrated to be effective in various applications, target-specific protein labeling using ligand-directed strategies is limited by stringent amino acid selectivity. Here, we present highly reactive ligand-directed triggerable Michael acceptors (LD-TMAcs) that feature rapid protein labeling. Unlike previous approaches, the unique reactivity of LD-TMAcs enables multiple modifications on a single target protein, effectively mapping the ligand binding site. This capability is attributed to the tunable reactivity of TMAcs that enable the labeling of several amino acid functionalities via a binding-induced increase in local concentration while remaining fully dormant in the absence of protein binding. We demonstrate the target selectivity of these molecules in cell lysates using carbonic anhydrase as the model protein. Furthermore, we demonstrate the utility of this method by selectively labeling membrane-bound carbonic anhydrase XII in live cells. We envision that the unique features of LD-TMAcs will find use in target identification, investigation of binding/allosteric sites, and studying membrane proteins.

Membrane Protein Binding Interactions Studied in Live Cells via Diethylpyrocarbonate Covalent Labeling Mass Spectrometry.

Membrane proteins are vital in the human proteome for their cellular functions and make up a majority of drug targets in the U.S. However, characterizing their higher-order structures and interactions remains challenging. Most often membrane proteins are studied in artificial membranes, but such artificial systems do not fully account for the diversity of components present in cell membranes. In this study, we demonstrate that diethylpyrocarbonate (DEPC) covalent labeling mass spectrometry can provide binding site information for membrane proteins in living cells using membrane-bound tumor necrosis factor α (mTNFα) as a model system. Using three therapeutic monoclonal antibodies that bind TNFα, our results show that residues that are buried in the epitope upon antibody binding generally decrease in DEPC labeling extent. Additionally, serine, threonine, and tyrosine residues on the periphery of the epitope increase in labeling upon antibody binding because of a more hydrophobic microenvironment that is created. We also observe changes in labeling away from the epitope, indicating changes to the packing of the mTNFα homotrimer, compaction of the mTNFα trimer against the cell membrane, and/or previously uncharacterized allosteric changes upon antibody binding. Overall, DEPC-based covalent labeling mass spectrometry offers an effective means of characterizing structure and interactions of membrane proteins in living cells.

Bioorthogonal nanozymes for breast cancer imaging and therapy.

Bioorthogonal catalysis via transition metal catalysts (TMCs) enables the generation of therapeutics locally through chemical reactions not accessible by biological systems. This localization can enhance the efficacy of anticancer treatment while minimizing off-target effects. The encapsulation of TMCs into nanomaterials generates "nanozymes" to activate imaging and therapeutic agents. Here, we report the use of cationic bioorthogonal nanozymes to create localized "drug factories" for cancer therapy in vivo. These nanozymes remained present at the tumor site at least seven days after a single injection due to the interactions between cationic surface ligands and negatively charged cell membranes and tissue components. The prodrug was then administered systemically, and the nanozymes continuously converted the non-toxic molecules into active drugs locally. This strategy substantially reduced the tumor growth in an aggressive breast cancer model, with significantly reduced liver damage compared to traditional chemotherapy.

Diethylpyrocarbonate-Based Covalent Labeling Mass Spectrometry of Protein Interactions in a Membrane Complex System.

Membrane-associated proteins are important because they mediate interactions between a cell's external and internal environment and they are often targets of therapeutics. Characterizing their structures and binding interactions, however, is challenging because they typically must be solubilized using artificial membrane systems that can make measurements difficult. Mass spectrometry (MS) is emerging as a valuable tool for studying membrane-associated proteins, and covalent labeling MS has unique potential to provide higher order structure and binding information for these proteins in complicated membrane systems. Here, we demonstrate that diethylpyrocarbonate (DEPC) can be effectively used as a labeling reagent to characterize the binding interactions between a membrane-associated protein and its binding partners in an artificial membrane system. Using chemotaxis histidine kinase (CheA) as a model system, we demonstrate that DEPC-based covalent labeling MS can provide structural and binding information about the ternary complex of CheA with two other proteins that is consistent with structural models of this membrane-associated chemoreceptor system. Despite the moderate hydrophobicity of DEPC, we find that its reactivity with proteins is not substantially influenced by the presence of the artificial membranes. However, correct structural information for this multiprotein chemoreceptor system requires measurements of DEPC labeling at multiple reagent concentrations to enable an accurate comparison between CheA and its ternary complex in the chemoreceptor system. In addition to providing structural information that is consistent with the model of this complex system, the labeling data supplements structural information that is not sufficiently refined in the chemoreceptor model.

Gas-Phase Unfolding of Protein Complexes Distinguishes Conformational Isomers.

Proteins can adopt different conformational states that are important for their biological function and, in some cases, can be responsible for their dysfunction. The essential roles that proteins play in biological systems make distinguishing the structural differences between these conformational states both fundamentally and practically important. Here, we demonstrate that collision-induced unfolding (CIU), in combination with ion mobility-mass spectrometry (IM-MS) measurements, distinguish subtly different conformational states for protein complexes. Using the open and closed states of the ß-lactoglobulin (ßLG) dimer as a model, we show that these two conformational isomers unfold during collisional activation to generate distinct states that are readily separated by IM-MS. Extensive molecular modeling of the CIU process reproduces the distinct unfolding intermediates and identifies the molecular details that explain why the two conformational states unfold in distinct ways. Strikingly, the open conformational state forms new electrostatic interactions upon collisional heating, while the closed state does not. These newly formed electrostatic interactions involve residues on the loop differentially positioned in the two ßLG conformational isomers, highlighting that gas-phase unfolding pathways reflect aspects of solution structure. This combination of experiment and theory provides a path forward for distinguishing subtly different conformational isomers for protein complexes via gas-phase unfolding experiments. Our results also have implications for understanding how protein complexes dissociate in the gas phase, indicating that current models need to be refined to explain protein complex dissociation.

Complementary Structural Information for Antibody-Antigen Complexes from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry.

Characterizing antibody-antigen interactions is necessary for properly developing therapeutic antibodies, understanding their mechanisms of action, and patenting new drug molecules. Here, we demonstrate that hydrogen-deuterium exchange (HDX) mass spectrometry (MS) measurements together with diethylpyrocarbonate (DEPC) covalent labeling (CL) MS measurements provide higher order structural information about antibody-antigen interactions that is not available from either technique alone. Using the well-characterized model system of tumor necrosis factor α (TNFα) in complex with three different monoclonal antibodies (mAbs), we show that two techniques offer a more complete overall picture of TNFα's structural changes upon binding different mAbs, sometimes providing synergistic information about binding sites and changes in protein dynamics upon binding. Labeling decreases in CL generally occur near the TNFα epitope, whereas decreases in HDX can span the entire protein due to substantial stabilization that occurs when mAbs bind TNFα. Considering both data sets together clarifies the TNFα regions that undergo a decrease in solvent exposure due to mAb binding and that undergo a change in dynamics due to mAb binding. Moreover, the single-residue level resolution of DEPC-CL/MS can clarify HDX/MS data for long peptides. We feel that the two techniques should be used together when studying the mAb-antigen interactions because of the complementary information they provide.

Multiplexed Analysis of the Cellular Uptake of Polymeric Nanocarriers.

Polymeric nanocarriers (PNCs) are versatile drug delivery vehicles capable of delivering a variety of therapeutics. Quantitatively monitoring their uptake in biological systems is essential for realizing their potential as next-generation delivery systems; however, existing quantification strategies are limited due to the challenges of detecting polymeric materials in complex biological samples. Here, we describe a metal-coded mass tagging approach that enables the multiplexed quantification of the PNC uptake in cells using mass spectrometry (MS). In this approach, PNCs are conjugated with ligands that bind strongly to lanthanide ions, allowing the PNCs to be sensitively quantitated by inductively coupled plasma-MS. The metal-coded tags have little effect on the properties or toxicity of the PNCs, making them biocompatible. We demonstrate that the conjugation of different metals to the PNCs enables the multiplexed analysis of cellular uptake of multiple distinct PNCs at the same time. This multiplexing capability should improve the design and optimization of PNCs by minimizing biological variability and reducing analysis time, effort, and cost.

Covalent Labeling-Mass Spectrometry Provides a Molecular Understanding of Noncovalent Polymer-Protein Complexation.

The delivery of functional proteins to the intracellular space offers tremendous advantages for the development of new therapeutics but is limited by the passage of these large polar biomacromolecules through the cell membrane. Noncovalent polymer-protein binding that is driven by strong carrier-cargo interactions, including electrostatics and hydrophobicity, has previously been explored in the context of delivery of functional proteins. Appropriately designed polymer-based carriers can take advantage of the heterogeneous surface of protein cargoes, where multiple types of physical binding interactions with polymers can occur. Traditional methods of assessing polymer-protein binding, including dynamic light scattering, circular dichroism spectroscopy, and fluorescence-based assays, are useful in the study of new polymer-based carriers but face a number of limitations. We implement for the first time the method of covalent labeling-mass spectrometry (CL-MS) to probe intermolecular surface interactions within noncovalent polymer-protein complexes. We demonstrate the utility of CL-MS for establishing binding of an amphiphilic block copolymer to negatively charged and hydrophobic surface patches of a model protein, superfolder green fluorescent protein (sfGFP), using diethylpyrocarbonate as a pseudo-specific labeling reagent. In addition, we utilize this method to explore differences at the intermolecular surface as the ratio of polymer to protein increases, particularly in the context of defining effective protein delivery regimes. By promoting an understanding of the intermolecular interactions in polymer-protein binding and identifying sites where polymers bind to protein surfaces, noncovalent polymer carriers can be more effectively designed for protein delivery applications.

Multiplatform Metabolomics Studies of Human Cancers With NMR and Mass Spectrometry Imaging.

The status of metabolomics as a scientific branch has evolved from proof-of-concept to applications in science, particularly in medical research. To comprehensively evaluate disease metabolomics, multiplatform approaches of NMR combining with mass spectrometry (MS) have been investigated and reported. This mixed-methods approach allows for the exploitation of each individual technique's unique advantages to maximize results. In this article, we present our findings from combined NMR and MS imaging (MSI) analysis of human lung and prostate cancers. We further provide critical discussions of the current status of NMR and MS combined human prostate and lung cancer metabolomics studies to emphasize the enhanced metabolomics ability of the multiplatform approach.

Accounting for Neighboring Residue Hydrophobicity in Diethylpyrocarbonate Labeling Mass Spectrometry Improves Rosetta Protein Structure Prediction.

Covalent labeling mass spectrometry allows for protein structure elucidation via covalent modification and identification of exposed residues. Diethylpyrocarbonate (DEPC) is a commonly used covalent labeling reagent that provides insight into structure through the labeling of lysine, histidine, serine, threonine, and tyrosine residues. We recently implemented a Rosetta algorithm that used binary DEPC labeling data to improve protein structure prediction efforts. In this work, we improved on our modeling efforts by accounting for the level of hydrophobicity of neighboring residues in the microenvironment of serine, threonine, and tyrosine residues to obtain a more accurate estimate of the hydrophobic neighbor count. This was incorporated into Rosetta functionality, along with considerations for solvent-exposed histidine and lysine residues. Overall, our new Rosetta score term successfully identified best scoring models with less than 2 Å root-mean-squared deviations (RMSDs) for five of the seven benchmark proteins tested. We additionally developed a confidence metric to measure prediction success for situations in which a native structure is unavailable.

Distinguishing Histidine Tautomers in Proteins Using Covalent Labeling-Mass Spectrometry.

In this work, we use diethylpyrocarbonate (DEPC)-based covalent labeling together with LC-MS/MS analysis to distinguish the two sidechain tautomers of histidine residues in peptides and proteins. From labeling experiments on model peptides, we demonstrate that DEPC reacts equally with both tautomeric forms to produce chemically different products with distinct dissociation patterns and LC retention times, allowing the ratios of the two tautomers to be determined in peptides and proteins. Upon measuring the tautomer ratios of several histidine residues in myoglobin, we find good agreement with previous 2D NMR data on this protein. Because our DEPC labeling/MS approach is simpler, faster, and more precise than 2D NMR, our method will be a valuable way to determine how protein structure enforces histidine sidechain tautomerization. Because the tautomeric state of histidine residues is often important for protein structure and function, the ability of DEPC labeling/MS to distinguish histidine tautomers should equip researchers with a tool to understand the histidine residue structure and function more deeply in proteins.

MEMBRANE PROTEIN STRUCTURES AND INTERACTIONS FROM COVALENT LABELING COUPLED WITH MASS SPECTROMETRY.

Membrane proteins are incredibly important biomolecules because they mediate interactions between a cell's external and internal environment. Obtaining information about membrane protein structure and interactions is thus important for understanding these essential biomolecules. Compared with the analyses of water-soluble proteins, the structural analysis of membrane proteins is more challenging owing to their unique chemical properties and the presence of lipid components that are necessary to solubilize them. The combination of covalent labeling (CL) and mass spectrometry (MS) has recently been applied with great success to study membrane protein structure and interactions. These studies have demonstrated the many advantages that CL-MS methods have over other traditional biophysical techniques. In this review, we discuss both amino acid-specific and non-specific labeling approaches and the special considerations needed to address the unique challenges associated with interrogating membrane proteins. This review highlights the aspects of this approach that require special care to be applied correctly and provides a comprehensive review of the membrane protein systems that have been studied by CL-MS. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Epitope Mapping with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

Antigen-antibody epitope mapping is essential for understanding binding mechanisms and developing new protein therapeutics. In this study, we investigate diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry as a means of analyzing antigen-antibody interactions using the well-characterized model system of TNFα in complex with three different antibodies. Results show that residues buried in the epitope undergo substantial decreases in labeling, as expected. Interestingly, serine, threonine, and tyrosine residues at the edges of the epitope undergo unexpected increases in labeling. The increased labeling of these weakly nucleophilic residues is caused by the formation of hydrophobic pockets upon antibody binding that presumably increase local DEPC concentrations. Residues that are distant from the epitope generally do not undergo changes in labeling extent; however, some that do change experience variations in their local microenvironment due to side-chain reorganization or stabilization of the TNFα trimer that occurs upon binding. Overall, DEPC labeling of antigen-antibody complexes is found to depend on both changes in solvent exposure and changes to the residue microenvironment.

LA-ICP-MS and MALDI-MS image registration for correlating nanomaterial biodistributions and their biochemical effects.

Laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) imaging and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) are complementary methods that measure distributions of elements and biomolecules in tissue sections. Quantitative correlations of the information provided by these two imaging modalities requires that the datasets be registered in the same coordinate system, allowing for pixel-by-pixel comparisons. We describe here a computational workflow written in Python that accomplishes this registration, even for adjacent tissue sections, with accuracies within ±50 µm. The value of this registration process is demonstrated by correlating images of tissue sections from mice injected with gold nanomaterial drug delivery systems. Quantitative correlations of the nanomaterial delivery vehicle, as detected by LA-ICP-MS imaging, with biochemical changes, as detected by MALDI-MSI, provide deeper insight into how nanomaterial delivery systems influence lipid biochemistry in tissues. Moreover, the registration process allows the more precise images associated with LA-ICP-MS imaging to be leveraged to achieve improved segmentation in MALDI-MS images, resulting in the identification of lipids that are most associated with different sub-organ regions in tissues.

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry.

Characterizing a protein's higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein's structure by MS, specific chemical reactions are performed in solution that encode a protein's structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as ß2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

Nanodelivery vehicles induce remote biochemical changes in vivo.

Nanomaterial-based platforms are promising vehicles for the controlled delivery of therapeutics. For these systems to be both efficacious and safe, it is essential to understand where the carriers accumulate and to reveal the site-specific biochemical effects they produce in vivo. Here, a dual-mode mass spectrometry imaging (MSI) method is used to evaluate the distributions and biochemical effects of anti-TNF-α nanoparticle stabilized capsules (NPSCs) in mice. It is found that most of the anticipated biochemical changes occur in sub-organ regions that are separate from where the nanomaterials accumulate. In particular, TNF-α-specific lipid biomarker levels change in immune cell-rich regions of organs, while the NPSCs accumulate in spatially isolated filtration regions. Biochemical changes that are associated with the nanomaterials themselves are also observed, demonstrating the power of matrix-assisted laser desorption/ionization (MALDI) MSI to reveal markers indicating possible off-target effects of the delivery agent. This comprehensive assessment using MSI provides spatial context of nanomaterial distributions and efficacy that cannot be easily achieved with other imaging methods, demonstrating the power of MSI to evaluate both expected and unexpected outcomes associated with complex therapeutic delivery systems.