Reactive Amino Research Articles

Active Targeting of Cancer Cells by Nanobody Decorated Polypeptide Micelle with Bio-orthogonally Conjugated Drug.

Polypeptides are promising carriers for chemotherapeutics: they have minimal toxicity, can be recombinantly synthesized with precise control over molecular weight, and enhance drug pharmacokinetics as self-assembled nanoparticles. Polypeptide-based systems also provide the ability to achieve active targeting with genetically encoded targeting ligands. While passive targeting promotes accumulation of nanocarriers in solid tumors, active targeting provides an additional layer of tunable control and widens the therapeutic window. However, fusion of most targeting proteins to polypeptide carriers exposes the limitations of this approach: the residues that are used for drug attachment are also promiscuously distributed on protein surfaces. We present here a universal methodology to solve this problem by the site-specific attachment of extrinsic moieties to polypeptide drug delivery systems without cross-reactivity to fused targeting domains. We incorporate an unnatural amino acid, p-acetylphenylalanine, to provide a biorthogonal ketone for attachment of doxorubicin in the presence of reactive amino acids in a nanobody-targeted, elastin-like polypeptide nanoparticle. These nanoparticles exhibit significantly greater cytotoxicity than nontargeted controls in multiple cancer cell lines.

Enantiospecific cyclization of methyl N-(tert-butoxycarbonyl)-N-(3-chloropropyl)-D-alaninate to 2-methylproline derivative via ‘memory of chirality’ in flow

We report for the very first time a continuous-flow route to perform the intramolecular cyclization of haloalkyl-substituted α-amino esters via memory of chirality (MoC), using lithium bis(trimethylsilyl)amine as a base and methyl N-(tert-butoxycarbonyl)-N-(3-chloropropyl)-D-alaninate as a model reactant. The various reaction parameters, such as temperature, residence time, reactant stoichiometry, or type and concentration of the base were optimized to maximize the yield of the cyclized product and its enantiomeric excess. At the conditions identified, the reaction was eventually scaled up, reaching a productivity of 11 g h−1. Compared to the standard batch protocols available in the literature, the use of a microreactor enables a better control of the exothermicity associated with the addition of the organolithium reagent to the reaction mixture, resulting in operations at more practical temperatures, with high enantiospecificity and full conversion of the reactive amino ester within a few seconds of residence time.

Edge enriched self-assembly of Au nanoparticles: Coffee-ring effect during microcontact printing via agarose stamps

Amino functionalized surfaces were selectively modified via the combination of a wet agarose stamping technique and microcontact printing technique. Because of the specific reaction environments and diffusion of HNO2 confined in agarose stamp, the reaction of amino groups in the edge of the strip pattern was much more intense than other areas. The modified amino groups in the edge areas show higher affinity to Au-NPs than other areas, consequently, edge enriched Au-NPs patterns were observed after the self-assembly of Au-NPs. A “cylindrical droplet” model, in a manner analogous to “coffee-ring” effect, was proposed to describe the diffusion of HNO2 from the bulk to the edge in agarose stamp. By using such density varied Au-NPs patterns as templates for the growth of ZnO nanorods, we observed high density of Au-NPs resulting in high density and highly (0 0 1) oriented ZnO nanorods. In contrast, sparse and non-oriented ZnO nanorods were grew on low density of Au-NPs areas. Our findings might open new routes for the fabrication of gradient patterns and extend applications of Au-NPs patterns in surface enhanced Raman scattering and catalysis.

Methionine oxidation by hydrogen peroxide in peptides and proteins: A theoretical and Raman spectroscopy study

The oxidation of proteins results in their deterioration via the oxidation of reactive amino acids. Oxidation of the amino acid, methionine plays an important role during biological conditions of oxidative stress, and equally a role in protein stability. In this study the oxidation of the methionine residue using the tripeptide GlyMetGly with respect to hydrogen peroxide has been studied using both Raman spectroscopy and DFT calculations. Spectral modifications following the formation of methionine sulfoxide are shown with the appearance of the SO vibration whilst there is also the modification of the CS vibrations at approximately 700 cm−1. The changes in the intensity of the CS stretching band were used to calculate the kinetic rate constant as 7.9 ± 0.6 × 10−3 dm3 mol−1 s−1. The energy barrier for the reaction. is determined both experimentally and using DFT calculations. The reaction of the dairy protein beta-lactoglobulin with hydrogen peroxide is equally studied using the same technique. The solvent accessible surface area of the methionine residues within the protein were also determined and a comparison of the reaction rate constant and the energy barriers of reaction for the oxidation of the tripeptide and for the protein respectively thus, provides information about the role of the protein environment in the oxidation process.

Elucidation of chemical modifier reactivity towards peptides and proteins and the analysis of specific fragmentation by matrix-assisted laser desorption/ionization collision-induced dissociation tandem mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of covalent 5-lipoxygenase inhibitors is challenging due to unknown amino acid specificity and low posttranslational modification (PTM)-identification rates. The analysis of the amino-acid specificity and of the characteristic fragmentation of chemically modified peptides is considered to improve knowledge for the analysis of chemically modified peptides and proteins by MALDI-MS. Various compounds were used to investigate the modification of synthetic peptides carrying reactive amino acid residues. Mass spectra were recorded using a MALDI-LTQ Orbitrap XL for high-resolution mass spectrometry and ion trap MALDI-MS2 . UV-Vis-based reduction and radical scavenging analysis was conducted. The on-plate digestion method described by Rühl et al was utilized for modification-site analysis at 5-lipoxygenase. The analysis of amino-acid-specific reactivity revealed the reactivity of quinones towards cysteine residues and the potential occurrence of a subsequent oxidative process was observed by an UV-Vis-based reduction assay. MALDI collision-induced dissociation tandem mass spectrometry (CID-MS2 ) indicated a prominent fragmentation mechanism of modified cysteine and histidine residues. Fragmentation included highly abundant neutral-loss signals which could be used to identify new modifications induced by chemical modifiers at the cysteine-159 residue of 5-lipoxygenase. Specificity and fragmentation analysis provides crucial information for the analysis of chemically modified cysteines and histidines by MALDI-MS. Elucidation of binding sites by MALDI-MS has been significantly improved using an easy-to-run peptide assay and gives background information for the analysis in the case of chemically modified 5-lipoxygenase.

Immobilization and characterization of PLGA nanoparticles on polyethylene terephthalate cardiovascular grafts for local drug therapy of associated graft complications

The cardiovascular graft associated complications limit the long-term patency of the graft and restrict the patient's life quality. In the present study, PLGA nanoparticles were covalently immobilized onto the woven form of crimped polyethylene terephthalate (PET, Dacron®) cardiovascular graft to treat the early thrombosis, inflammation, or bacterial infection via local delivery of therapeutic agents. PET surface was firstly functionalized to produce reactive amino groups used as anchor sites for covalent immobilization of PLGA nanoparticles by end-point technique. The functionalized surface characterized by electro kinetic analyzer showed marked negative surface potential values up to −41 mV at high pH value indicating the presence of amino groups on PET surface. The scanning electron microscopy observations of nano-coated PET showed topographic architecture of homogenously distributed monolayer of PLGA nanoparticles on the PET surfaces. Profoundly, the immobilized nanoparticles manifested stability under blood flow-mimetic conditions for 24 h. The cytotoxicity and biocompatibility of the nano-coated PET in mouse L929 fibroblasts revealed adequate biocompatibility in terms of the cellular adhesion and growth pattern without remarkable cytotoxicity.

Investigation of modified water glass as adhesive for wood and particleboard: mechanical, thermal and flame retardant properties

Water glass (WG) is an inorganic binder with excellent fire resistance in porous materials but has limited application to wood bonding. In this work, modified WG adhesive was developed and the effect of modification on the properties of adhesive and particleboard bonded with it was investigated. The bonding strength of WG adhesives increased by the modification with boric acid and aminofunctional silane. Stable siloxane linkage can lead to enhanced bonding. Particleboards with modified WG were manufactured with a target density of 750 kg m−3. The silane was selected as the best modifier; it was the only one to meet the requirements of EN 312 (Type 2). The reaction of amino groups onto the surface of wood was confirmed by Fourier-transform infrared spectrum. All particleboards produced with WG adhesives showed resistance to fire comparable to those with urea formaldehyde in the combustion test for 30 min. As a result of this investigation, WG modified with silane could be a desirable alternative to current synthetic adhesives for wood composites and fire resistance application.

Engineering Heterodimeric Kinesins through Genetic Incorporation of Noncanonical Amino Acids.

Kinesins are commonly homodimers with two identical heavy chains (protomers) and play indispensable roles in many intracellular processes. Engineered heterodimeric kinesins with two distinct protomers are important tools for dissecting coordination and regulation of naturally occurring kinesin homodimers. Here, we report a chemical-biology-based approach that generates kinesin heterodimers by combining genetic incorporation of reactive noncanonical amino acids and small-molecule-based cross-linking. We verified using yeast kinesin-8/Kip3 as a model system that our method yields kinesin heterodimers of desired properties without introducing unintended motility disruption. To demonstrate the utility of our method, we engineered a crippled Kip3 heterodimer that contains both a wild-type-like protomer and a catalytically inactive one, and our results revealed that the resulting heterodimer moves on the microtubule with a significant reduction in velocity but not processivity. Due to its versatility, we expect that our method can be broadly adopted to create novel heterodimers for other kinesins and will thus greatly expand the studies on kinesin mechanisms.

Chitosan Nanocapsules for pH‐Triggered Dual Release Based on Corrosion Inhibitors as Model Study

AbstractChitosan (CS) is known for its uniqueness in terms of containing reactive functional groups, i.e., amino and hydroxyl groups, along the chain which offer the noncovalent bonds and chemical modifications. At pH 3 in emulsion system, the CS chains are under charge‐charge repulsive force, leading the polysaccharide chains to align as the hollow nanospheres and at that time, the crosslink leads to the nanocapsules. The CS nanocapsules allow the model cargos, i.e., corrosion inhibitors, entrapment not only via the noncovalent bond based on the weak interaction in the core for 2‐mercaptobenzothiazole (MBT) cargo but also via the chemical bonds based on the amino group remaining on CS structure to result in either amide or Schiff base linkage in case of 3‐nitrosalicylic acid (3NiSA) cargo. The release studies indicate that CS nanocapsules can release two cargoes upon pH change due to the acidic/basic‐triggered cleavage of bonds between cargoes and nanocapsules. Once corrosion occurs, resulting in variation of the local pH value, the CS nanocapsules release MBT in fast and 3NiSA in a slow, sustained manner. The efficiency of the approach is demonstrated using the nanocapsules for hindering the corrosion of copper as measured by electrochemical quartz crystal microbalance.

Development of a Novel Sulfoxide-Containing MS-Cleavable Homobifunctional Cysteine-Reactive Cross-Linker for Studying Protein-Protein Interactions.

Cross-linking mass spectrometry (XL-MS) has become an emerging technology for defining protein-protein interactions (PPIs) and elucidating architectures of large protein complexes. Up to now, the most widely used cross-linking reagents target lysines. Although such reagents have been successfully applied to map PPIs at the proteome-wide scale, comprehensive PPI profiling would require additional cross-linking chemistries. Cysteine is one of the most reactive amino acids and an attractive target for cross-linking owing to its unique role in protein structures. Although sulfhydryl-reactive cross-linkers are commercially available, their applications in XL-MS studies remain sparse, likely due to the difficulty in identifying cysteine cross-linked peptides. Previously, we developed a new class of sulfoxide-containing MS-cleavable cross-linkers to enable fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MS n). Here, we present the development of a new sulfoxide-containing MS-cleavable homobifunctional cysteine-reactive cross-linker, bismaleimide sulfoxide (BMSO). We demonstrate that BMSO-cross-linked peptides display the same characteristic fragmentation pattern during collision-induced dissociation (CID) as other sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MS n. Additionally, we show that BMSO can complement amine- and acidic-residue-reactive reagents for mapping protein-interaction regions. Collectively, this work not only enlarges the toolbox of MS-cleavable cross-linkers with diverse chemistries, but more importantly expands our capacity and capability of studying PPIs in general.

Site-Specific Conjugation of Polymers to Proteins.

Conjugation of polymer to protein has been widely employed in therapeutics, medicine, biotechnology, and enzymatic catalysis. The synergistic effect benefits both counterparts and potentially overcomes their inherent limitations. This article reviews the strategies for the site-specific synthesis of well-defined protein-polymer conjugates, aiming to provide a toolbox for the community. First, it is essential to set a definite reactive site on the protein because the position of the reaction site can directly influence the reaction activity and the bioactivity of the protein after modification. The origins of the specific functional groups on protein include the utilization of the unique natural amino acid, mutagenesis to introduce a sole reactive amino acid, chemical modification, noncanonical amino acid incorporation, and enzyme-mediated introduction of functional groups. Second, the main conjugation methods, i.e., "grafting to" and "grafting from" methods, are summarized and compared with each other. In the "grafting to" method, a comprehensive investigation on the reactions used to attach an end functional polymer chain to a protein is conducted according to the position of the target site and its nature. In the "grafting from" method, a comparison between the commonly used controlled polymerization, i.e., atom transfer radical polymerization (ATRP) and reversible addition-fragmentation transfer (RAFT), is surveyed. Further, a special case where a noncovalent bond is adopted to link the protein and polymer together is investigated due to its high specificity and reversibility, typically biotin-(strept)avidin-based interactions and metal-mediated conjugation. Finally, applications of protein-polymer conjugates in drug delivery, biomedicine, biosensor, and the disease-related protein self-assembly are illustrated. This precise review on the conjugation of polymer chain to protein to form well-defined protein-polymer conjugates summarizes the representative strategies and may provide useful cues in the areas of biotechnology, therapeutic drugs, and biomedicine.

PLA and single component silicone rubber blends for sub-zero temperature blown film packaging applications

Abstract The poly(lactic acid) (PLA) blend with single component silicone rubber in the presence of reactive amino silane coupling agent and polyester polyols plasticizer were studied. The manufacturing of film packaging for sub-zero temperature applications from the PLA blend was the main objective. The mechanical properties, especially the impact strengths, of PLA/silicone blends were significantly depended on the silicone loading. The outstanding impact strengths, tested at sub-zero temperature, of the blend having silicone content of 8.0 phr was achieved. It was chosen as the best candidate for the processability improvement. Adding the talc filler into the PLA/silicone blend to enhance the rheological properties was investigated. The ductility of the talc filled blends were decreased with increasing the filler contents. However, the shear viscosity of the blend was raised with talc loading. The blend loaded with 40 phr of talc filler was justified as the optimal formula for the blown film process testing and it was successfully performed with a few difficulties. The obtained blown film showed relative good flexibility in comparison with LDPE but it has low transparency.

A Novel Colorimetric Fluorescent Probe for SO₂ and Its Application in Living Cells Imaging.

A novel chromenylium-based fluorescent probe was exploited for sulphur dioxide (SO2) detecting. The probe displayed a remarkable fluorescence turn-on response towards SO2 based on the nucleophilic addition reaction to the carbon-carbon double bond with 105 nm Stock shift. The probe was successfully applied for the quantification of SO2.The linear detection range was from 0–160 μM with the detection limit as low as 99.27 nM. It also exhibited high selectivity for SO2 than other reactive species and amino acids. Furthermore, cell staining experiments indicated that the probe was cell membrane permeable and could be used for high-performance imaging of SO2 in living cells. The superior properties of the probe made it highly promising for use in chemical and biological applications.

Vapor-Phase Nanopatterning of Aminosilanes with Electron Beam Lithography: Understanding and Minimizing Background Functionalization.

Electron beam lithography (EBL) is a highly precise, serial method for patterning surfaces. Positive tone EBL resists enable patterned exposure of the underlying surface, which can be subsequently functionalized for the application of interest. In the case of widely used native oxide-capped silicon surfaces, coupling an activated silane with electron beam lithography would enable nanoscale chemical patterning of the exposed regions. Aminoalkoxysilanes are extremely useful due to their reactive amino functionality but have seen little attention for nanopatterning silicon surfaces with an EBL resist due to background contamination. In this work, we investigated three commercial positive tone EBL resists, PMMA (950k and 495k) and ZEP520A (57k), as templates for vapor-phase patterning of two commonly used aminoalkoxysilanes, 3-aminopropyltrimethoxysilane (APTMS) and 3-aminopropyldiisopropylethoxysilane (APDIPES). The PMMA resists were susceptible to significant background reaction within unpatterned areas, a problem that was particularly acute with APTMS. On the other hand, with both APTMS and APDIPES exposure, unpatterned regions of silicon covered by the ZEP520A resist emerged pristine, as shown both with SEM images of the surfaces of the underlying silicon and through the lack of electrostatically driven binding of negatively charged gold nanoparticles. The ZEP520A resist allowed for the highly selective deposition of these alkoxyaminosilanes in the exposed areas, leaving the unpatterned areas clean, a claim also supported by contact angle measurements with four probe liquids and X-ray photoelectron spectroscopy (XPS). We investigated the mechanistic reasons for the stark contrast between the PMMA resists and ZEP520A, and it was found that the efficacy of resist removal appeared to be the critical factor in reducing the background functionalization. Differences in the molecular weight of the PMMA resists and the resulting influence on APTMS diffusion through the resist films are unlikely to have a significant impact. Area-selective nanopatterning of 15 nm gold nanoparticles using the ZEP520A resist was demonstrated, with no observable background conjugation noted in the unexposed areas on the silicon surface by SEM.

Macrophage polarization, inflammatory signaling, and NF-κB activation in response to chemically modified titanium surfaces.

Functionalization of titanium devices with various bioactive molecules enhances many of their properties as implants, including biocompatibility, which is typically assessed by macrophage activation and inflammation. However, functionalization requires prior introduction of reactive groups, to which bioactive agents can then be grafted. Thus, we investigated the inflammatory properties of titanium pretreated with NaOH, titanium pretreated with NaOH and then with 3-aminopropyl triethoxysilane, and titanium pretreated with dopamine. Inflammation, macrophage polarization, and activation of NF-κB signaling were assessed by real-time PCR and western blotting. The data demonstrate that silanized titanium is the least inflammatory, and promotes macrophage M2 polarization with modest engagement of the NF-κB signaling pathway. Importantly, silanization introduces a reactive amino group, providing more opportunities for further functionalization.

Reagent enables new protein modification

Chemists modify protein sidechains to modulate the biomolecules’ functions. Researchers are usually stuck modifying cysteines because the amino acid’s sulfide side chain makes it the most reactive amino acid. But cysteines are relatively rare, representing ~1.9% of the amino acids in human proteins. Cysteines can be installed using genetic engineering, but researchers would like to modify fully functional native proteins. An international team of researchers led by Goncalo J. L. Bernardes of the University of Cambridge and the Institute of Molecular Medicine Lisbon and Gonzalo Jimenez-Oses of the University of Rioja has now discovered a sulfonyl acrylate reagent that should give researchers more options for modifying proteins. The reagent selectively targets only the most reactive lysine in a protein—not other lysines or buried cysteines (J. Am. Chem. Soc. 2018, DOI: 10.1021/jacs.7b12874). Lysines are nucleophilic and much more abundant than cysteines, representing ~5.9% of the amino acids in human protei...

Designing Recognition Molecules and Tailoring Functional Surfaces for In Vivo Monitoring of Small Molecules in the Brain

The in vivo analysis of chemical signals in brain extracellular fluid (ECF) using implanted electrochemical biosensors is a vital way to study brain functions and brain activity mapping. This approach offers excellent spatial (10-200 μm) and temporal (approximately second) resolution and the major advantage of long-term stability. By implantation of a microelectrode in a specific brain region, changes in the concentration of a variety of ECF chemical species can be monitored through applying a suitable electrical signal and, typically, recording the resulting Faradaic current. However, the high performance requirements for in vivo biosensors greatly limit our understanding of the roles that biomolecules play in the brain. Since a large number of biological species, including reactive oxygen species (ROS), metal ions, amino acids, and proteins, coexist in the brain and interact with each other, developing in vivo biosensors with high selectivity is a great challenge. Meanwhile, it is difficult to quantitatively determine target molecules in the brain because of the variation in the distinct environments for monitoring biomolecules in vitro and in vivo. Thus, there are large errors in the quantification of concentrations in the brain using calibration curves obtained in artificial cerebrospinal fluid (aCSF). More importantly, to gain a full understanding of the physiological and pathological processes in the brain, the development of novel approaches for the simultaneous determination of multiple species in vivo is urgently needed. This Account provides insight into the basic design principles and criteria required to convert chemical/electrochemical reactions into electric signals, while satisfying the increasing requirements, including high selectivity, sensitivity, and accuracy, for the in vivo analysis of biomolecules in the brain. Recent developments in designing various functional surfaces, such as self-assembled monolayers, gold nanostructures, and nanostructured semiconductors for facilitating electron transfer from specific enzymes, including superoxide dismutase (SOD), and further application to an O2•- biosensor are summarized. This Account also aims to highlight the design principles for the selective biosensing of Cu2+ and pH in the brain through the rational design and synthesis of specific recognition molecules. Additionally, electrochemical ratiometric biosensors with current signal output have been constructed to correct the effect of distinct environments in a timely manner, thus greatly improving the accuracy of the determination of Cu2+ in the live brain. This method of using a built-in element has been extended to biosensors with the potential signal output for in vivo pH analysis. More importantly, the new concept of both current and potential signal outputs provides an avenue to simultaneously determine dual species in the brain. The extension of the design principles and developed strategy demonstrated in this Account to other biomolecules, which may be closely correlated to the biological processes of brain events, is promising. The final section of this Account outlines potential future directions in tailoring functional surfaces and designing recognition molecules based on recent advances in molecular science, nanoscience and nanotechnology, and biological chemistry for the design of advanced devices with multiple target species to map the molecular imaging of the brain. There are still opportunities to engineer surfaces that improve on this approach by constructing implantable, multifunctional nanodevices that promise to combine the benefits of multiple sensing and therapeutic modules.

Alpha-Synuclein Glycation and the Action of Anti-Diabetic Agents in Parkinson's Disease.

Parkinson’s disease (PD) is a neurodegenerative disorder with complex etiology and variable pathology. While a subset of cases is associated with single-gene mutations, the majority originates from a combination of factors we do not fully understand. Thus, understanding the underlying causes of PD is indispensable for the development of novel therapeutics. Glycation, the non-enzymatic reaction between reactive dicarbonyls and amino groups, gives rise to a variety of different reaction products known as advanced glycation end products (AGEs). AGEs accumulate over a proteins life-time, and increased levels of glycation reaction products play a role in diabetic complications. It is now also becoming evident that PD patients also display perturbed sugar metabolism and protein glycation, including that of alpha-synuclein, a key player in PD. Here, we hypothesize that anti-diabetic drugs targeting the levels of glycation precursors, or promoting the clearance of glycated proteins may also prove beneficial for PD patients.

Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization.

Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools, including X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM, and traditional low-resolution methods, such as circular dichroism, UV-vis, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure. The methodology relies on "painting" the reactive and solvent-exposed amino acid residues with chemical tags and using the pattern of modifications as footprints from analysis by bottom-up MS-based proteomics to deduce protein higher order structures. The outcome can refer to proteins in solution or even in cells and is complementary to those of X-ray crystallography and NMR. It is particularly useful in mapping protein-ligand interfaces and conformational changes resulting from ligand binding, mutation, and aggregation. Fast photochemical oxidation of proteins (FPOP), in its original conception, is a type of hydroxyl-radical-based protein footprinting that utilizes a pulsed KrF laser (248 nm) to trigger hydrolysis of hydrogen peroxide to produce solution hydroxyl radicals, which subsequently modify the protein in situ. The platform is expanding to adopt other reactive species including carbenes. The reactivity of the probe depends on the intrinsic reactivity of the radical with the residue side chain and the solvent accessibility of the residue as a function of the tertiary/quaternary structures. By introducing an appropriate scavenger to compete with hydroxyl radical self-quenching, the lifetime of the primary radicals is remarkably shortened to approximately microsecond. Thus, the sampling time scale of FPOP is much faster than hydrogen-deuterium exchange and other covalent labeling methods relying on nonradical reactions. The short footprinting time scale of FPOP offers two major advantages for protein structure elucidation: (1) it allows the protein to be interrogated in its native or near-native state with minimum structural perturbation; (2) it exhibits high sensitivity toward alterations in protein higher order structures because its sampling time is short with respect to protein conformational changes and dynamic motion. In addition, the covalent and irreversible oxidation by the hydroxyl radical provides more flexibility in the downstream proteomics workflow and MS analysis, permitting high spatial resolution with residue-specific information. Since its invention in 2005 by Hambly and Gross, FPOP has developed from proof-of-concept to a valuable biophysical tool for interrogating protein structure. In this Account, we summarize the principles and experimental design of FPOP that enable its fast labeling and describe the current and unique capabilities of the technique in protein higher order structure elucidation. Application examples include characterization of amyloid β self-assembly, protein-ligand interactions with a special emphasis on epitope mapping for protein therapeutics (e.g., antibody, Fab, and adnectin), protein folding detailed to residue-specific folding kinetics, and protein flexibility/dynamics. Additionally, the utility of FPOP-based oxidative footprinting should grow with our continuing developments of novel reagents (e.g., sulfate radical anion, carbene diradical, and trifluoromethyl radical). These reactive reagents are compatible with the current FPOP platform and offer different reactivity and selectivity toward various types of amino acid residues, providing complementary insights into protein higher order structures for soluble proteins and ultimately for membrane-bound proteins.

The Fate of a Hapten - From the Skin to Modification of Macrophage Migration Inhibitory Factor (MIF) in Lymph Nodes

Skin (contact) allergy, the most prevalent form of immunotoxicity in humans, is caused by low molecular weight chemicals (haptens) that penetrate stratum corneum and modify endogenous proteins. The fate of haptens after cutaneous absorption, especially what protein(s) they react with, is largely unknown. In this study the fluorescent hapten tetramethylrhodamine isothiocyanate (TRITC) was used to identify hapten-protein conjugates in the local lymph nodes after topical application, as they play a key role in activation of the adaptive immune system. TRITC interacted with dendritic cells but also with T and B cells in the lymph nodes as shown by flow cytometry. Identification of the most abundant TRITC-modified protein in lymph nodes by tandem mass spectrometry revealed TRITC-modification of the N-terminal proline of macrophage migration inhibitory factor (MIF) – an evolutionary well-conserved protein involved in cell-mediated immunity and inflammation. This is the first time a hapten-modified protein has been identified in lymph nodes after topical administration of the hapten. Most haptens are electrophiles and can therefore modify the N-terminal proline of MIF, which has an unusually reactive amino group under physiological conditions; thus, modification of MIF by haptens may have an immunomodulating role in contact allergy as well as in other immunotoxicity reactions.