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.

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.

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.

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.

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.

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.

Enhanced and Selective MALDI-MS Detection of Peptides via the Nanomaterial-Dependent Coffee Ring Effect.

Nanomaterials have been explored as alternative matrices in MALDI-MS to overcome some of the limitations of conventional matrices. Recently, we demonstrated a new means by which nanomaterials can improve peptide ionization and detection in MALDI-MS analyses by exploiting the tendency of nanomaterials to form "coffee rings" upon drying from liquids. In the current work, we investigate how nanomaterial size and composition affect the signal enhancement of peptides through the coffee-ring effect. From studies of eight different types of nanomaterials ranging in size and composition, we find that most nanomaterials can provide signal enhancement ranging from 2- to 10-fold for individual peptides, as long as a coffee ring is formed. However, when a mixture of peptides is present in a sample, the signal enhancement is the greatest for peptides whose net charge is complementary to the nanomaterial's surface charge. These results suggest that careful design of NM surface properties could allow for selective, enhanced MALDI-MS detection of specific peptides in complex mixtures.

The Cleavage Profile of Protein Substrates by ClpXP Reveals Deliberate Starts and Pauses.

Cells rely on protein degradation by AAA+ proteases. A well-known example is the hexameric ClpX unfoldase, which captures ATP hydrolysis to feed substrates into the oligomeric ClpP peptidase. Recent studies show that an asymmetric ClpX spiral cycles protein translocation upon ATP hydrolysis. However, how this cycle affects peptide products is less explored in part because ClpP cleavage is thought to be solely defined by sequence constraints. Here, we comprehensively characterize peptides from Caulobacter crescentus ClpXP degradation of three different substrates using high-resolution mass spectrometry and find that cleavage of translocated substrates is driven by factors other than sequence. We report that defined locations in a translocated protein are especially sensitive to cleavage spaced on average every 10-13 residues. These sites are not exclusively controlled by sequence and are independent of bulk changes in catalytic peptidase sites, ATP hydrolysis, or the efficiency of initial recognition. These results fit a model in which processive translocation through ClpX starts at a specific location in a polypeptide and pauses during reset of the ClpX hexamer after a cycle of translocation. Our work suggests that defined peptides, which could be used as signaling molecules, can be generated from a given substrate by a nonspecific peptidase.

Epigallocatechin-3-gallate Inhibits Cu(II)-Induced ß-2-Microglobulin Amyloid Formation by Binding to the Edge of Its ß-Sheets.

Epigallocatechin-3-gallate (EGCG) is a catechin found in green tea that can inhibit the amyloid formation of a wide variety of proteins. EGCG's ability to prevent or redirect the amyloid formation of so many proteins may reflect a common mechanism of action, and thus, greater molecular-level insight into how it exerts its effect could have broad implications. Here, we investigate the molecular details of EGCG's inhibition of the protein ß-2-microglobulin (ß2m), which forms amyloids in patients undergoing long-term dialysis treatment. Using size-exclusion chromatography and a collection of mass spectrometry-based techniques, we find that EGCG prevents Cu(II)-induced ß2m amyloid formation by diverting the normal progression of preamyloid oligomers toward the formation of spherical, redissolvable aggregates. EGCG exerts its effect by binding with a micromolar affinity (Kd ≈ 5 µM) to the ß2m monomer on the edge of two ß-sheets near the N-terminus. This interaction destabilizes the preamyloid dimer and prevents the formation of a tetramer species previously shown to be essential for Cu(II)-induced ß2m amyloid formation. EGCG's binding at the edge of the ß-sheets in ß2m is consistent with a previous hypothesis that EGCG generally prevents amyloid formation by binding cross-ß-sheet aggregation intermediates.

Polymeric nanoassemblies for enrichment and detection of peptides and proteins in human breast milk.

Human breast milk is an understudied biological fluid that may be useful for early detection of breast cancer. Methods for enriching and detecting biomarkers in human breast milk, however, are not as well-developed as compared with other biological fluids. In this work, we demonstrate a new enrichment method based on polymeric nanoassemblies that is capable of enhancing the mass spectrometry-based detection of peptides and proteins in human breast milk. In this method, positively charged nanoassemblies are used to selectively deplete abundant proteins in milk based on electrostatic interactions, which simplifies the mixture and enhances detection of positively charged peptides and proteins. Negatively charged nanoassemblies are used in a subsequent enrichment step to further enhance the detection and quantification of trace-level peptides and proteins. Together the depletion and enrichment steps allow model biomarkers to be detected at low nM levels, which are close to instrumental limits of detection. This new method not only demonstrates the ability to detect proteins in human breast milk but also provides an alternative approach for targeted protein detection in complex biological matrices. Graphical abstract.

Rod-shape theranostic nanoparticles facilitate antiretroviral drug biodistribution and activity in human immunodeficiency virus susceptible cells and tissues.

Human immunodeficiency virus theranostics facilitates the development of long acting (LA) antiretroviral drugs (ARVs) by defining drug-particle cell depots. Optimal drug formulations are made possible based on precise particle composition, structure, shape and size. Through the creation of rod-shaped particles of defined sizes reflective of native LA drugs, theranostic probes can be deployed to measure particle-cell and tissue biodistribution, antiretroviral activities and drug retention. Methods: Herein, we created multimodal rilpivirine (RPV) 177lutetium labeled bismuth sulfide nanorods (177LuBSNRs) then evaluated their structure, morphology, configuration, chemical composition, biological responses and adverse reactions. Particle biodistribution was analyzed by single photon emission computed tomography (SPECT/CT) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging. Results: Nanoformulated RPV and BSNRs-RPV particles showed comparable physicochemical and cell biological properties. Drug-particle pharmacokinetics (PK) and biodistribution in lymphoid tissue macrophages proved equivalent, one with the other. Rapid particle uptake and tissue distribution were observed, without adverse reactions, in primary blood-derived and tissue macrophages. The latter was seen within the marginal zones of spleen. Conclusions: These data, taken together, support the use of 177LuBSNRs as theranostic probes as a rapid assessment tool for PK LA ARV measurements.

Structural Heterogeneity in the Preamyloid Oligomers of ß-2-Microglobulin.

In dialysis patients, the protein ß2-microglobulin (ß2m) forms amyloid fibrils in a condition known as dialysis-related amyloidosis. To understand the early stages of the amyloid assembly process, we have used native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble preamyloid oligomers. ESI-IM-MS reveals the presence of multiple conformers for the dimer, tetramer, and hexamer that precede the Cu(II)-induced amyloid assembly process, results which are distinct from ß2m oligomers formed at low pH. Experimental and computational results indicate that the predominant dimer is a Cu(II)-bound structure with an antiparallel side-by-side configuration. In contrast, tetramers exist in solution in both Cu(II)-bound and Cu(II)-free forms. Selective depletion of Cu(II)-bound species results in two primary conformers-one that is compact and another that is more expanded. Molecular modeling and molecular dynamics simulations identify models for these two tetrameric conformers with unique interactions and interfaces that enthalpically compensate for the loss of Cu(II). Unlike with other amyloid systems in which conformational heterogeneity is often associated with different amyloid morphologies or off-pathway events, conformational heterogeneity in the tetramer seems to be a necessary aspect of Cu(II)-induced amyloid formation by ß2m. Moreover, the Cu(II)-free models represent a new advance in our understanding of Cu(II) release in Cu(II)-induced amyloid formation, laying a foundation for further mechanistic studies as well as development of new inhibition strategies.

A programmable chemical switch based on triggerable Michael acceptors.

Developing an engineerable chemical reaction that is triggerable for simultaneous chemical bond formation and cleavage by external cues offers tunability and orthogonality which is highly desired in many biological and materials applications. Here, we present a chemical switch that concurrently captures these features in response to chemically and biologically abundant and important cues, viz., thiols and amines. This thiol/amine-triggerable chemical switch is based on a Triggerable Michael Acceptor (TMAc) which bears good leaving groups at its ß-position. The acceptor undergoes a "trigger-to-release" process where thiol/amine addition triggers cascaded release of leaving groups and generates a less activated acceptor. The newly generated TMAc can be further reversed to liberate the original thiol/amine by a second nucleophile trigger through a "trigger-to-reverse" process. Within the small molecular volume of the switch, we have shown five locations that can be engineered to achieve tunable "trigger-to-release" kinetics and tailored reversibility. The potential of the engineerable bonding/debonding capability of the chemical switch is demonstrated by applications in cysteine-selective and reversible protein modification, universal self-immolative linkers, and orthogonally addressable hydrogels.

Supramolecular Polymeric Assemblies for the Selective Depletion of Abundant Acidic Proteins in Serum.

The analysis of low-level protein biomarkers in serum is precluded by the presence of other highly abundant serum proteins. Hence, the preliminary removal of serum albumin along with other abundant proteins in serum (i.e., immunoglobulins, transferrin, haptoglobin, α-2-macroglobulin, and apolipoproteins) is often a requirement prior to any biomarker analysis. In this work, we take advantage of the low isoelectric points (pI's) of these highly abundant proteins to selectively deplete them from serum by extraction using functionalized amphiphilic polymeric nanoassemblies. The selectivity of extraction is dependent on the pI of the protein and the extraction pH, which holds true even for extremely complex protein mixtures like serum. High extraction capacity is achieved by optimizing the extraction conditions and is found to be comparable to currently available methods for depletion. Depletion of these abundant acidic proteins allows for the enhanced detection of higher pI proteins and enables a 3 orders of magnitude increase in detection sensitivity for a putative cancer biomarker, demonstrating the utility of these polymeric assemblies for enhancing the analysis of the serum proteome.

Molecular Features Influencing the Release of Peptides from Amphiphilic Polymeric Reverse Micelles.

Efficient and controlled release of peptides bound to polymeric reverse micelle assemblies can be achieved through the cooperative effects of disassembly and disruption of charge-charge interactions. Through the examination of various peptides and polymer architectures, we have identified the factors that affect the release efficiency of the electrostatically bound peptides. Peptide guests and polymers with a greater number of complementary charges result in less efficient release than peptides and polymers with lower numbers of charges. Interestingly, we find that the presence of adjacent charged groups on the monomeric unit of the polymer exhibits exceptionally low release efficiency, perhaps because of a chelate-like effect, even when the total polymer charge is lower. Overall, our findings inform the design principles for catch-and-release systems based on polymeric reverse micelles, which offer great versatility and tunability.

Matrix Metalloproteinase-9-Responsive Nanogels for Proximal Surface Conversion and Activated Cellular Uptake.

Here, we have exploited the heightened extracellular concentration of matrix metalloproteinase-9 (MMP-9) to induce surface-conversional properties of nanogels with the aim of tumor-specific enhanced cellular uptake. A modular polymeric nanogel platform was designed and synthesized for facile formulation and validation of MMP-9-mediated dePEGylation and generation of polyamine-type surface characteristics through peptide N-termini. Nanogels containing MMP-9-cleavable motifs and different poly(ethylene glycol) corona lengths (350 and 750 g/mol) were prepared, and enzymatic surface conversional properties were validated by MALDI characterization of cleaved byproducts, fluorescamine assay amine quantification, and zeta potential. The nanogel with a shorter PEG length, mPEG350-NG, exhibited superior surface conversion in response to extracellular concentrations of MMP-9 compared to that of the longer PEG length, mPEG750-NG. Confocal microscopy images of HeLa cells incubated with both fluorescein-labeled nanogels and DiI-encapsulated nanogels demonstrated greater uptake following MMP-9 "activation" for mPEG350-NG compared to its nontreated "passive" mPEG350-NG parent, demonstrating the versatility of such systems to achieve stimuli-responsive uptake in response to cancer-relevant proteases.

Gas-Phase Protein Salt Bridge Stabilities from Collisional Activation and Electron Transfer Dissociation.

The gas phase structures of several proteins have been studied by electron transfer dissociation (ETD) with and without prior collisional heating after electrospraying these proteins from native-like solutions into a quadrupole ion trap mass spectrometer. Without prior collisional heating, we find that ETD fragmentation is mostly limited to regions of the protein that are not spanned by the salt bridges known to form in solution. When protein ions are collisionally heated before ETD, new product ions are observed, and in almost all cases, these new ions arise from protein regions that are spanned by the salt bridges. Together these results confirm the existence of salt bridges in protein ions and demonstrate that a sufficient amount energy is required to disrupt these salt bridges in the gas phase. More interestingly, we also show that different salt bridges require different collisional activation voltages to be disrupted, suggesting that they have variable stabilities in the gas phase. These stabilities appear to be influenced by the gas-phase basicities of the involved residues and the presence of nearby charged residues. We also find that higher collisional activation voltages are needed to enable the formation of new product from sites spanned by multiple salt bridges.

Altering the Peptide Binding Selectivity of Polymeric Reverse Micelle Assemblies via Metal Ion Loading.

Supramolecular reverse micelle assemblies, formed by amphiphilic copolymers, can selectively encapsulate molecules in their interiors depending on the functional groups present in the polymers. Altering the binding selectivity of these materials typically requires the synthesis of alternate functional groups. Here, we demonstrate that the addition of Zr(IV) ions to the interiors of reverse micelles having phosphonate functional groups transforms the supramolecular materials from ones that selectively bind positively charged peptides into materials that selectively bind phosphorylated peptides. We also show that the binding selectivity of these reverse micelle assemblies can be further tuned by varying the fractions of phosphonate groups in the copolymer structure. The optimized reverse micelle materials can selectively transfer and bind phosphorylated peptides from aqueous solutions over a wide range of pH conditions and can selectively enrich phosphorylated peptides even in complicated mixtures.