In vitro and in vivo applications of a universal and synthetic thermo-responsive drug delivery hydrogel platform.

A synthetic and thermo-responsive polymer, poly(N-isopropylacrylamide)-co-(polylactide/2-hydroxy methacrylate)-co-(oligo (ethylene glycol)), is used to formulate a universal carrier platform for sustained drug release. The enabling carrier, denoted as TP, is prepared by dissolving the polymer in an aqueous solution at a relatively neutral pH. A wide range of therapeutic moieties can be incorporated without the need for the addition of surfactants, organic solvents, and other reagents to the carrier system. The resulting solution is flowable through fine gauge needle, allowing accurate administration of TP to the target site. After injection, TP carrier undergoes a coil to globe phase transition to form a hydrogel matrix at the site. The benign nature of the polymer carrier and its physical gelation process are essential to preserve the biological activity of the encapsulated compounds while the adhesive hydrogel nature of the matrix allows sustained elusion and controlled delivery of the incorporated therapeutics. The TP carrier system has been shown to be non-toxic and elicits a minimal inflammatory response in multiple in vitro studies. These findings suggest the suitability of TP as an enabling carrier of therapeutics for localized and sustained drug delivery. To confirm this hypothesis, the capabilities of TP to encapsulate and effectively deliver multiple therapeutics of different physicochemical characteristics was evaluated. Specifically, a broad range of compounds were tested, including ciprofloxacin HCl, tumor necrosis factor-alpha (TNF-α), transforming growth factor beta 1 (TGF-ß1), and recombinant human bone morphogenetic protein 2 (BMP2). In vitro studies confirmed that TP carrier is able to control the release of the encapsulated drugs over an extended period of time and mitigate their burst release regardless of the compounds' physiochemical properties for the majority of the loaded therapeutics. Importantly, in vitro and in vivo animal studies showed that the released drugs from the TP hydrogel matrix remained potent and bioactive, confirming the high potential of the TP polymer system as an enabling carrier.

Immunoaffinity extraction followed by enzymatic digestion for the isolation and identification of proteins employing automated µSPE reactors and mass spectrometry.

This work describes a novel automated and rapid method for bottom-up proteomics combining protein isolation with a micro-immobilised enzyme reactor (IMER). Crosslinking chemistry based on 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling was exploited to immobilise trypsin and antibodies onto customisable silica particles coated with carboxymethylated dextran (CMD). This novel silica-CMD solid-phase extraction material was characterised using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), conductometric titrations and enzymatic colorimetric assays. Micro-solid-phase extraction (µSPE) cartridges equipped with the modified CMD material were employed and integrated into an automated and repeatable workflow using a sample preparation workstation to achieve rapid and repeatable protein isolation and pre-concentration, followed by tryptic digestion producing peptide fragments that were identified by liquid chromatography mass spectrometry (LC-MS).

Thermoresponsive and Injectable Hydrogel for Tissue Agnostic Regeneration.

Injectable hydrogels can support the body's innate healing capability by providing a temporary matrix for host cell ingrowth and neovascularization. The clinical adoption of current injectable systems remains low due to their cumbersome preparation requirements, device malfunction, product dislodgment during administration, and uncontrolled biological responses at the treatment site. To address these challenges, a fully synthetic and ready-to-use injectable biomaterial is engineered that forms an adhesive hydrogel that remains at the administration site regardless of defect anatomy. The product elicits a negligible local inflammatory response and fully resorbs into nontoxic components with minimal impact on internal organs. Preclinical animal studies confirm that the engineered hydrogel upregulates the regeneration of both soft and hard tissues by providing a temporary matrix to support host cell ingrowth and neovascularization. In a pilot clinical trial, the engineered hydrogel is successfully administered to a socket site post tooth extraction and forms adhesive hydrogel that stabilizes blood clot and supports soft and hard tissue regeneration. Accordingly, this injectable hydrogel exhibits high therapeutic potential and can be adopted to address multiple unmet needs in different clinical settings.

Coated glass capillaries as SPME devices for DART mass spectrometry.

RATIONALE: Solid-phase microextraction (SPME) provides high-throughput sample cleanup and pre-concentration. Here we demonstrate coated glass capillaries (CGCs) as SPME devices for specific applications in direct analysis in real time (DART) mass spectrometry, referred to as "CGC-DART", for rapid screening of environmental contaminants at low parts-per-trillion detection limits and with accurate identification of analytes. METHODS: The extraction is performed in a one-step process in minutes by dipping the CGC in solutions containing the analytes, and then placing the CGC in a DART source for analysis. CGCs are disposable and relatively inexpensive in comparison with SPME devices, and can be prepared with hydrophobic, hydrophilic or mixed-mode materials similar to SPME. CGCs were prepared by adsorption coating with incubation of capillaries in saturated solutions of octadecylamine or covalent activation of silanes. RESULTS: Quantitation is shown with perfluorooctanoic acid (PFOA) at 1 ppt to 100 ppb, with the lowest detection at 500 parts-per quadrillion (ppq) in tap water. One-step extraction of contaminated groundwater from Northern Queensland, Australia, revealed perfluorooctane sulfonate (PFOS) and perfluorohexanesulfonamide as well as C4-C8 perfluoroalkyl carboxylic acids. A soil sample taken near a former military air base (New Hampshire, USA) revealed the presence of perfluorononanoic acid (PFNA) at 1 ppb and traces of perfluoroheptanoic acid. CONCLUSIONS: CGC-DART enabled one-step extraction of PFASs in minutes with mL sample volumes at low concentrations as shown for the standards and contaminated soil and water samples. DART-MS combined with Kendrick mass defect analysis enabled accurate identification of PFASs without chromatography steps, as fluorinated compounds are mass deficient and easily distinguished over background signal.

Protein Footprinting with Radical Probe Mass Spectrometry- Two Decades of Achievement.

BACKGROUND: Radical Probe Mass Spectrometry (RP-MS) describes a pioneering methodology in structural biology that enables the study of protein structures, their interactions, and dynamics on fast timescales (down to sub-milliseconds). Hydroxyl radicals (•OH) generated directly from water within aqueous solutions induce the oxidation of reactive, solvent accessible amino acid side chains that are then analyzed by mass spectrometry. Introduced in 1998 at the American Society for Mass Spectrometry annual conference, RP-MS was first published on in 1999. OBJECTIVE: This review article describes developments and applications of the RP-MS methodology over the past two decades. METHODS: The RP-MS method has been variously referred to as synchrotron X-ray radiolysis footprinting, Hydroxyl Radical Protein Footprinting (HRPF), X-ray Footprinting with Mass Spectrometry (XF-MS), Fast Photochemical Oxidation of Proteins (FPOP), oxidative labelling, covalent oxidative labelling, and even the Stability of Proteins from Rates of Oxidation (SPROX). RESULTS: The article describes the utility of hydroxyl radicals as a protein structural probe, the advantages of RP-MS in comparison to other MS-based approaches, its proof of concept using ion mobility mass spectrometry, its application to protein structure, folding, complex and aggregation studies, its extension to study the onset of protein damage, its implementation using a high throughput sample loading approach, and the development of protein docking algorithms to aid with data analysis and visualization. CONCLUSION: RP-MS represents a powerful new structural approach that can aid in our understanding of the structure and functions of proteins, and the impact of sustained oxidation on proteins in disease pathogenesis.

A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures.

A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.

Stability of the ßB2B3 crystallin heterodimer to increased oxidation by radical probe and ion mobility mass spectrometry.

Ion mobility mass spectrometry was employed to study the structure of the ßB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of ßB3 crystallin across residues 115-126 and 152-166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.

Advances in radical probe mass spectrometry for protein footprinting in chemical biology applications.

Radical Probe Mass Spectrometry (RP-MS), first introduced in 1999, utilizes hydroxyl radicals generated directly within aqueous solutions using synchrotron radiolysis, electrical discharge, and photochemical laser sources to probe protein structures and their interactions. It achieves this on millisecond and submillisecond timescales that can be used to capture protein dynamics and folding events. Hydroxyl radicals are ideal probes of solvent accessibility as their size approximates a water molecule. Their high reactivity results in oxidation at a multitude of amino acid side chains providing greater structural information than a chemical cross-linker that reacts with only one or few residues. The oxidation of amino acid side chains occurs at rates in accord with the solvent accessibility of the residue so that the extent of oxidation can be quantified to reveal a three-dimensional map or footprint of the protein's surface. Mass spectrometry is central to this analysis of chemical oxidative labelling. This tutorial review, some 15 years on from the first reports, highlights the development and significant growth of the application of RP-MS including its validation and utility with ion-mobility mass spectrometry (IM-MS), the use of RP-MS data to help model protein complexes, studies of the onset of oxidative damage, and more recent advances that enable high throughput applications through simultaneous protein oxidation and on-plate deposition. The accessibility of the RP-MS technology, by means of a modified electrospray ionization source, enables the approach to be implemented in many laboratories to address a wide range of applications in chemical biology.

On-plate deposition of oxidized proteins to facilitate protein footprinting studies by radical probe mass spectrometry.

The on-plate deposition of oxidized proteins is described to advance footprinting applications by radical probe mass spectrometry (RP-MS). An electrospray ionization (ESI) needle assembly mounted vertically over a 384-target matrix-assisted laser desorption/ionization (MALDI) plate enabled the limited oxidation of proteins as they were released in the charged droplets ahead of their deposition on the plate. This method combined with on-plate proteolytic digestion protocols expedites the analysis of proteins oxidized by RP-MS, and avoids the need to collect and reconstitute samples prior to analysis by MALDI mass spectrometry. Oxidation of peptides from solutions in water as well as an ammonium bicarbonate solution was investigated to test the optimal conditions required for on-plate oxidation of proteins. These comprised of peptides with a wide range of reactive amino acids including Phe, Tyr, Pro, His, Leu, Met and Lys that were previously shown to oxidize in both electrospray discharge and synchrotron radiolysis based footprinting experiments. The on-plate deposition of lysozyme oxidized at electrospray needle voltages of 6 and 9 kV were carried out to demonstrate conditions suitable for footprinting experiments as well as those that induce the onset of protein damage.

Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry.

The effect of hydroxyl radical induced oxidation on the collision cross-sections of hen egg lysozyme and bovine ubiquitin was investigated by travelling wave ion mobility mass spectrometry for the first time. The oxidized ions of lysozyme and ubiquitin share common collision cross-sections with their unoxidized counterparts suggesting that they share common structures that were unaffected by limited oxidation. In the case of bovine ubiquitin, two distinct conformers were detected for the protein in its unoxidized and oxidized states though no change in the levels of each was observed upon oxidation. This supports the validity of Radical Probe Mass Spectrometry (RP-MS) using an electrical discharge source for protein footprinting experiments. Travelling wave ion mobility mass spectrometry has been used for the first time to confirm that limited oxidation does not have an impact on the global structure of proteins.

Temperature-dependent release of volatile organic compounds of eucalypts by direct analysis in real time (DART) mass spectrometry.

A method is described for the rapid identification of biogenic, volatile organic compounds (VOCs) emitted by plants, including the analysis of the temperature dependence of those emissions. Direct analysis in real time (DART) enabled ionization of VOCs from stem and leaf of several eucalyptus species including E. cinerea, E. citriodora, E. nicholii and E. sideroxylon. Plant tissues were placed directly in the gap between the DART ionization source skimmer and the capillary inlet of the time-of-flight (TOF) mass spectrometer. Temperature-dependent emission of VOCs was achieved by adjusting the temperature of the helium gas into the DART ionization source at 50, 100, 200 and 300 degrees C, which enabled direct evaporation of compounds, up to the onset of pyrolysis of plant fibres (i.e. cellulose and lignin). Accurate mass measurements facilitated by TOF mass spectrometry provided elemental compositions for the VOCs. A wide range of compounds was detected from simple organic compounds (i.e. methanol and acetone) to a series of monoterpenes (i.e. pinene, camphene, cymene, eucalyptol) common to many plant species, as well as several less abundant sesquiterpenes and flavonoids (i.e. naringenin, spathulenol, eucalyptin) with antioxidant and antimicrobial properties. The leaf and stem tissues for all four eucalypt species showed similar compounds. The relative abundances of methanol and ethanol were greater in stem wood than in leaf tissue suggesting that DART could be used to investigate the tissue-specific transport and emissions of VOCs.

Stylopeptide 2, a proline-rich cyclodecapeptide from the sponge Stylotella sp.

A new proline-rich cyclodecapeptide, designated stylopeptide 2, has been isolated from a cytotoxic extract of the Papua New Guinea marine sponge Stylotella sp. and found to correspond to structure 1. The structural assignment was based on HRMS collision-induced dissociation tandem mass spectrometry (CID MS/MS), NMR spectroscopic data, and amino acid analysis, which led to assignment of the absolute configuration.

Oxidation inhibits amyloid fibril formation of transthyretin.

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.

Onset of oxidative damage in alpha-crystallin by radical probe mass spectrometry.

The early onset oxidative damage within segments of the protein alpha-crystallin is examined by radical probe mass spectrometry by its treatment with reactive oxygen species under low-, moderate-, and high-oxidizing conditions. Five regions comprising the first 11 residues of the N-termini of the A and B subunits (A/B:1-11), central domains from each subunit B:57-69 and A:104-112, and a C-terminal segment of the A subunit A:120-145 were found to be the initial sites of oxidation. The susceptibility of each segment to oxidation and oxidative damage is investigated by subjecting the intact protein to different oxidation conditions within the ion source of an electrospray ionization mass spectrometer. LC-MS of the oxidized protein digests enables the sites and levels of oxidation to be monitored. The onset of oxidative damage and the levels of oxidation observed before damage occurs differ across the protein surface. The regions comprising residues A/B:1-11 and A:104-112 are shown to be more susceptible to oxidative damage than those comprising residues B:57-69 and A:120-145. The results are discussed in the context of available experimental and homology-modeled theoretical structures for the subunits of alpha-crystallin.

Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry.

The calcium-dependent interaction of calmodulin and melittin is studied through the application of a radical probe approach in which solutions of the protein and peptide and protein alone are subjected to high fluxes of hydroxyl and other oxygen radicals on millisecond timescales. These radicals are generated by an electrical discharge within an electrospray ion source of a mass spectrometer. Condensation of the electrosprayed droplets followed by proteolytic digestion of both calmodulin and melittin has identified residues in both which participate in the interaction and/or are shielded from solvent within the protein complex. Consistent with other theoretical models and available experimental data, the tryptophan residue of melittin at position 19 is shown to be critical to the formation of the complex with the C-terminal domain of peptide enveloped by and protected from oxidation upon binding to the protein. Furthermore, the N-terminal domain (to residue 36) and tyrosine at position 99 in calmodulin are significantly protected from limited oxidation upon the binding of melittin while exposing the phenylalanine residue at position 92 of the flexible loop domain. The N-terminus (through residue 36) of calmodulin is shown to lie in closer proximity to the melittin helix than its C-terminal counterpart (residues 127-148) based upon the protection levels measured at reactive residues within these segments of the protein.

Charge ratio analysis method: approach for the deconvolution of electrospray mass spectra.

A new method to interpret electrospray mass spectral data based on calculating the ratio of mass-to-charge (m/z) values of multiply charged ions is described. The mass-to-charge ratios of any two multiply charged ions corresponding to a single compound are unique numbers that enable the charge states for each ion to be unequivocally identified. The multiply charged ions in electrospray mass spectra originate from the addition or abstraction of protons, cations, or anions to and from a compound under analysis. In contrast to existing deconvolution processes, the charge ratio analysis method (CRAM), identifies the charge states of multiply charged ions without any prior knowledge of the nature of the charge-carrying species. In the case of high-resolution electrospray mass spectral data, in which multiply charged ions are resolved to their isotopic components, the CRAM is capable of correlating the isotope peaks of different multiply charged ions that share the same isotopic composition. This relative ratio method is illustrated here for electrospray mass spectral data of lysozyme and oxidized ubiquitin recorded at low- to high-mass resolution on quadrupole ion trap and Fourier transform ion cyclotron mass spectrometers, and theoretical data for the protein calmodulin based upon a reported spectrum recorded on the latter.

Photochemical and electrophysical production of radicals on millisecond timescales to probe the structure, dynamics and interactions of proteins.

The reaction of hydroxyl and other oxygen-based radicals with the side chains of proteins on millisecond timescales has been used to probe the structure of proteins, their dynamics in solution and interactions with other macromolecules. Radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources and react with proteins on timescales that are less than those often attributed to structural reorganisation and folding. The oxygen-based radicals generated in aqueous solution react with proteins to effect limited oxidation at specific amino acids throughout the sequence of the protein. The extent of oxidation at these residue markers is highly influenced by the accessibility of the reaction site to the bulk solvent. The extent of oxidation allows protection levels to be measured based on the degree to which a reaction occurs. A map of a protein's three-dimensional structure is subsequently assembled as in a footprinting experiment. Protein solutions that contain various concentrations of substrates that either promote or disrupt structural transitions can be investigated to facilitate site-specific equilibrium and time-resolved studies of protein folding. The radical-based strategies can also be employed in the study of protein-protein interactions to provide a new avenue for investigating protein complexes and assemblies with high structural resolution. The urea-induced unfolding of apomyoglobin, and the binding domains within the ribonuclease S and calmodulin-melittin protein-peptide complexes are presented to illustrate the approach.

Study of the ribonuclease-S-protein-peptide complex using a radical probe and electrospray ionization mass spectrometry.

The interaction between ribonuclease (RNase) S-protein and S-peptide is examined by studying their limited oxidation within the RNase-S complex and free forms using radicals. The limited oxidation of the RNase-S complex and each component is effected through their reaction with a high flux of oxygen-based radicals generated by an electrical discharge within an electrospray ion source. Their exposure to radicals occurs on short millisecond time scales and has been consistently found not to cause any measurable structural damage or conformational change to proteins in a number of published reports. Consistent with these studies, S-peptide is preferentially protected from reactions with radicals under conditions in which it is bound to S-protein. Conversely, a region of S-protein comprising residues 96-100 constitutes the S-peptide binding domain based on its diminished reactivity with radicals within the RNase-S complex over the free S-protein. The results, for the first time, demonstrate the use of radicals generated by an electrical discharge to study protein complexes.

Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry.

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the surface of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions with synchrotron light for several milliseconds result in the reaction of amino acid side chains. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side chains to the bulk solvent. The aromatic and sulfur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for each of the proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates have been found to correlate closely with a theoretical measure of the accessibility of residue side chains to the bulk solvent in the native protein structure. The synchrotron-based approach is able to distinguish the relative accessibility of the tryptophan residue side chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues based on their rates of oxidation.