N-hydroxysuccinimide Ester Research Articles

Photo-Cross-Linking Mass Spectrometry and Integrative Modeling Enables Rapid Screening of Antigen Interactions Involving Bacterial Transferrin Receptors.

Structure-based approaches to the delineation of immunogens for vaccine development have a throughput requirement that is difficult to meet in practice with conventional methods of structure determination. Here we present a strategy for rapid and accurate structure generation in support of antigen engineering programs. The approach is developed around the modeling of interactions between host transferrin (Tf) and the bacterial vaccine target transferrin binding protein B (TbpB) from Gram-negative pathogens such as Neisseria meningitidis. Using an approach based solely on cross-linking mass spectrometry (XL-MS) data, monomeric structural models, and the Integrative Modeling Platform (IMP), we demonstrate that converged representations of the Tf:TbpB interactions can be returned that accurately reflect the binding interface and the relative orientation of the monomeric units, with the capacity to scale to the analysis of interactions from any number of additional strains. We show that a key element to accurate modeling involves the application of hetero-bifunctional cross-linkers incorporating fast-acting photoactivatable diazirines coupled with conventional amine-targeting N-hydroxysuccinimide esters, and we demonstrate that conventional homo-bifunctional reagents used in cross-linking kinetically trap dynamic states in the ensemble. Therefore, the application of both classes of cross-linker provides an opportunity to empirically detect protein dynamics during integrative structural modeling.

Covalent Attachment of Fibronectin onto Emulsion-Templated Porous Polymer Scaffolds Enhances Human Endometrial Stromal Cell Adhesion, Infiltration, and Function.

A novel strategy for the surface functionalization of emulsion-templated highly porous (polyHIPE) materials as well as its application to in vitro 3D cell culture is presented. A heterobifunctional linker that consists of an amine-reactive N-hydroxysuccinimide ester and a photoactivatable nitrophenyl azide, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), is utilized to functionalize polyHIPE surfaces. The ability to conjugate a range of compounds (6-aminofluorescein, heptafluorobutylamine, poly(ethylene glycol) bis-amine, and fibronectin) to the polyHIPE surface is demonstrated using fluorescence imaging, FTIR spectroscopy, and X-ray photoelectron spectroscopy. Compared to other existing surface functionalization methods for polyHIPE materials, this approach is facile, efficient, versatile, and benign. It can also be used to attach biomolecules to polyHIPE surfaces including cell adhesion-promoting extracellular matrix proteins. Cell culture experiments demonstrated that the fibronectin-conjugated polyHIPE scaffolds improve the adhesion and function of primary human endometrial stromal cells. It is believed that this approach can be employed to produce the next generation of polyHIPE scaffolds with tailored surface functionality, enhancing their application in 3D cell culture and tissue engineering whilst broadening the scope of applications to a wider range of cell types.

Design, Synthesis, and Evaluation of a Diazirine Photoaffinity Probe for Ligand-Based Receptor Capture Targeting G Protein-Coupled Receptors.

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein–coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography–mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P–NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P–binding receptor via liquid chromatography–tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

An accurate TMT-based approach to quantify and model lysine susceptibility to conjugation via N-hydroxysuccinimide esters in a monoclonal antibody

Conjugation of small molecules to proteins through N-hydroxysuccinimide (NHS) esters results in a random distribution of small molecules on lysine residues and the protein N-terminus. While mass spectrometry methods have improved characterization of these protein conjugates, it remains a challenge to quantify the occupancy at individual sites of conjugation. Here, we present a method using Tandem Mass Tags (TMT) that enabled the accurate and sensitive quantification of occupancy at individual conjugation sites in the NIST monoclonal antibody. At conjugation levels relevant to antibody drug conjugates in the clinic, site occupancy data was obtained for 37 individual sites, with average site occupancy data across 2 adjacent lysines obtained for an additional 12 sites. Thus, altogether, a measure of site occupancy was obtained for 98% of the available primary amines. We further showed that removal of the Fc-glycan on the NIST mAb increased conjugation at two specific sites in the heavy chain, demonstrating the utility of this method to identify changes in the susceptibility of individual sites to conjugation. This improved site occupancy data allowed calibration of a bi-parametric linear model for predicting the susceptibility of individual lysines to conjugation from 3D-structure based on their solvent exposures and ionization constants. Trained against the experimental data for lysines from the Fab fragment, the model provided accurate predictions of occupancies at lysine sites from the Fc region and the protein N-terminus (R2 = 0.76). This predictive model will enable improved engineering of antibodies for optimal labeling with fluorophores, toxins, or crosslinkers.

Cy7-Tocilizumab/Fab(Tocilizumab): Near Infrared Fluorescence In Vivo Imaging of Multiple Myeloma

Introduction:Multiple Myeloma (MM) is an incurable bone marrow cancer characterized by the proliferation of malignant plasma cells. Interleukin-6 (IL-6) is a key player molecule related to growth, survival and proliferation of MM cells. Tocilizumab is a humanized monoclonal antibody directed against the soluble and membrane IL-6 receptor. We have previously radiolabeled Tocilizumab and its Fab´s fragments with 99mTechnetium, revealing its potential role as MM radiotracers for targeting IL-6 expression in vivo [1, 2]. Near-infrared (650-900 nm) fluorescence (NIRF) cyanine (Cy) dye has been used for labeling several molecules for optical imaging due to their properties, such as small size, good aqueous solubility, and pH insensitivity between pH 3 and 10.Objectives: Label Tocilizumab and Fab´s (Tocilizumab) with Cy7 and evaluate its potential role as MM imaging agent.Methodology: IL-6R expression in MM cell lines (U266, NCI-H929 and MM1S) was confirmed by laser confocal microscopy. Tocilizumab fragmentation was carried out with papain and, once purified, fragments were identified by MALDI/TOF and SDS-PAGE. Cy7-Tocilizumab / Fab(Tocilizumab) were synthesized through nucleophilic substitution reaction between monofunctional N-hydroxysuccinimide ester (Cy7-NHS) and Tocilizumab / Fab(Tocilizumab) [3]. After purification, the conjugates were characterized by spectrophotometry. For in vivo imaging, Cy7-Tocilizumab/Fab(Tocilizumab) (1 nmol) were injected intravenously in MM1S tumor-bearing Balb/c nude mice and were imaged with near-infrared fluorescence (NIRF) after 0, 1, 2, 6 and 24 h post-injection of Cy7-Tocilizumab and after 0.5, 2, 6, 24, 48 and 72 h post-injection of Cy7-Fab(Tocilizumab).Results: Laser confocal microscopy showed that MM cell lines express high levels of IL-6R. Pure and homogeneous Fab-Tocilizumab fragments were produced. Tocilizumab and Fab (Tocilizumab) were successfully labeled with Cy7 as shown by spectrophotometry. Non-invasive NIRF in vivo imaging of MM tumor-bearing Balb/c nude mice allowed us to distinguish tumors up to 24 h and 72 h post-injection of Cy7-Tocilizumab and Cy7-Fab(Tocilizumab), respectively (Figures A and B).Conclusions: MM cell lines express IL-6R. Cy7 labeled Tocilizumab/Fab(Tocilizumab) has the potential to become optical imaging agents for IL-6R expressing tumors such as MM, being useful to guiding surgical excision of tumors and biopsies, that merits further evaluation.Acknowledgments: Agencia Nacional de Innovación e Investigación - Uruguay (ANII), Roche Laboratories, Pro.In.Bio (Uruguay), PEDECIBA Química (Uruguay)) and Comisión Sectorial de Investigación Científica-Universidad de la República-Uruguay (CSIC, UdelaR) I+D Grupos Oncología Nuclear.Disclosures: No relevant conflicts of interest to declare.

Reactive nanoparticles with activated ester moieties from cellulose acetate phthalate derivatives

Reactive polysaccharide (PS) based nanoparticles (NP) with activated N-hydroxysuccinimide (NHS) ester moieties were prepared by a two-step approach: (i) homogeneous synthesis of activated cellulose ester derivatives followed by (ii) nano-self-assembling of these derivatives into spherical PS-NP. Four different conversions of cellulose acetate phthalate (CAP) into cellulose acetate (N-succinimidyl)phthalate (NHS-CAP) were studied. The carboxyl groups of CAP were converted into NHS-ester in good yield by using N,N′-disuccinimidyl carbonate as reagent. Spherical NP with diameters in the range of 200–400 nm were obtained by self-assembling NHS-CAP derivatives. The NHS moieties were stable in aqueous media and remained reactive. The reactive PS-NP were used for the immobilization of an amine-functionalized fluorescent dye. Under basic conditions, an amide bond was formed, which resulted in high binding efficiencies of up to 90%. On the contrary, only weak non-covalent interaction with the dye was observed for NP derived from CAP and cellulose acetate. The novel NHS-activated PS-NP have great potential for applications in drug delivery and bioimaging.

Detection of Alpha-Fetoprotein in Hepatocellular Carcinoma Patient Plasma with Graphene Field-Effect Transistor.

The detection of alpha-fetoprotein (AFP) in plasma is important in the diagnosis of hepatocellular carcinoma (HCC) in humans. We developed a biosensor to detect AFP in HCC patient plasma and in a phosphate buffer saline (PBS) solution using a graphene field-effect transistor (G-FET). The G-FET was functionalized with 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) for immobilization of an anti-AFP antibody. AFP was detected by assessing the shift in the voltage of the Dirac point (ΔVDirac) after binding of AFP to the anti-AFP-immobilized G-FET channel surface. This anti-AFP-immobilized G-FET biosensor was able to detect AFP at a concentration of 0.1 ng mL−1 in PBS, and the detection sensitivity was 16.91 mV. In HCC patient plasma, the biosensor was able to detect AFP at a concentration of 12.9 ng mL−1, with a detection sensitivity of 5.68 mV. The sensitivity (ΔVDirac) depended on the concentration of AFP in either PBS or HCC patient plasma. These data suggest that G-FET biosensors could have practical applications in diagnostics.

Synthesis and some Pharmacological Properties of an Immunoactive Lysine-Containing Tetrapeptide

The potentially immunoactive lysine-containing tetrapeptide H-Lys-Ala-Val-Gly-OH was synthesized. The value of using a method based on mixed anhydrides and activated esters to obtain tetrapeptide at high yield was demonstrated. The peptide preparation method was improved by replacing p-nitrophenyl esters at the intermediate stage with N-hydroxysuccinimide esters, yielding a crystalline substance at high purity, and replacing the BOC (tert-butyloxycarbonyl) group for protection with a carbobenzoxy group, which provided for a one-step removal of the protection of both amino groups in mild conditions. Comparative assessment of the immunological activity of the tetrapeptide on the background of immunization with sheep erythrocytes in mice with other lysine-containing tetrapeptides showed it to have more marked immunostimulatory activity. Studies of acute and chronic toxicity demonstrated the absence of toxic activity in the lysine-containing tetrapeptide studied here on s.c. administration to experimental animals. The tetrapeptide corresponded to toxicity class 5, i.e., it was essentially nontoxic.

Enhanced Growth Activities of Stem Cell Spheroids Based on a Durable and Chemically Defined Surface Modification Coating

Surface modification layers are needed for the precise definition of surface chemistries and are equally important for durable and stable adhesive properties to ensure long-term stability and effective performance for biotechnological applications. This study demonstrates a robust modification layer that is synthesized based on chemical vapor deposition copolymerization, and the resultant coating layer is composed of the side-by-side presentation of N-hydroxysuccinimide ester and maleimide functionalities with a controlled ratio to define the immobilization accessibility of chitosan and growth factor protein (FGF-2) molecules on the substrate surface for enhancing cellular activities of stem cells. Characterizations of the copolymer modification layer showed excellent durability, including adhesive strength and thermal stability, and the layer is free of concerns for delamination and/or unacceptable deformation/debris formation that can cause potential toxicity to the surrounding biological environment. Modifications using the copolymer layer on the cell culture surface have demonstrated synergistic activity by chitosan to support the formation of spheroids and by FGF-2 to enhance the proliferation of human adipose-derived stem cells (ADSCs) within the spheroids while increasing the spheroid size and cell numbers. Healthy and flourishing growth activities were discovered for ADSCs on the modified culture surfaces, and the results are useful for potential and related stem cell research and the interfaces of biomaterials.

Underestimation of house dust mite–specific IgE with extract-based ImmunoCAPs compared with molecular ImmunoCAPs

Underestimation of house dust mite–specific IgE with extract-based ImmunoCAPs compared with molecular ImmunoCAPs

Measurements of Atmospheric Proteinaceous Aerosol in the Arctic Using a Selective UHPLC/ESI-MS/MS Strategy

In this article, an analytical methodology to investigate the proteinaceous content in atmospheric size-resolved aerosols collected at the Zeppelin observatory (79 °N, 12 °E) at Ny Ålesund, Svalbard, from September to December 2015, is proposed. Quantitative determination was performed after acidic hydrolysis using ultrahigh-performance liquid chromatography in reversed-phase mode coupled to electrospray ionization tandem mass spectrometry. Chromatographic separation, as well as specificity in the identification, was achieved by derivatization of the amino acids with N-butyl nicotinic acid N-hydroxysuccinimide ester prior to the analysis. The chromatographic run was performed within 11 min and instrumental levels of detection (LODs) were between 0.2 and 8.1 pg injected on the column, except for arginine which exhibited an LOD of 37 pg. Corresponding method LODs were between 0.01 and 1.9 fmol/m3, based on the average air sampling volume of 57 m3. The sum of free amino acids and hydrolyzed polyamino acids was shown to vary within 6–2914 and 0.02–1417 pmol/m3 for particles in sizes < 2 and 2–10 μm in equivalent aerodynamic diameter, respectively. Leucine, alanine, and valine were the most abundant among the amino acids in both aerosol size fractions. In an attempt to elucidate source areas of the collected aerosols, 5- to 10-day 3D backward trajectories reaching the sampling station were calculated. Overall, the method described here provides a first time estimate of the proteinaceous content, that is, the sum of free and polyamino acids, in size-resolved aerosols collected in the Arctic.Graphical ᅟ

Dual molecular recognition strategy for highly sensitive electrochemical detection of dopamine based on amplification of DNA–Au bio–bar codes

We had reported an electrochemical assay for dopamine with high sensitivity and selectivity based on dual molecular recognition strategy and amplification of DNA–Au bio–bar codes. In this assay, carbon nanotubes were first immobilized on the glass carbon electrode surface. Subsequently, a boronic acid-functionalized pyrene probe was self-assembled on the sidewalls of carbon nanotubes via π–π stacking interactions as a capture probe. 3,3′-Dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP) and double strands DNA functionalized AuNPs (DSP/AuNPs/dsDNA) were designed as signal probes and characterized by transmission electron microscopy and spectroscopic techniques. Upon the binding of dopamine with boronic acid followed by DSP/AuNPs/dsDNA, the AuNPs were captured on the electrode surface to enhance the signal. RuHex, an electroactive complex, which could bind to the anionic phosphate of DNA strands through electrostatic interaction, serves as signaling transducer. Signal amplification and dual recognition were used to quantify dopamine concentration from 30 pM to 30 nM, with a detection limit of 18 pM. Other small molecules could be measured by this assay after modification with differential recognition molecules.

Carbodiimide-mediated immobilization of acidic biomolecules on reversed-charge zwitterionic sensor chip surfaces.

The carbodiimide-mediated amine coupling of protein ligands to sensor chips coated with anionic polycarboxylate hydrogels, such as carboxymethyl dextran, is the predominant covalent immobilization procedure utilized in optical biosensors, namely surface plasmon resonance (SPR) biosensors. Usually, electrostatic interactions at a slightly acidic pH and low ionic strength are employed to efficiently accumulate neutral and basic ligands on the chip surface, which are then covalently coupled by surface-bound active N-hydroxysuccinimide (NHS) esters. Unfortunately, this approach is not suitable for acidic proteins or other ligands with low isoelectric points (IEPs), such as nucleic acids, because the charge density of the polycarboxylates is greatly reduced at acidic pH or because electrostatic attraction cannot be achieved. To overcome these drawbacks, we have established a charge-reversal approach that allows the preconcentration of acidic proteins above their IEPs. A precisely controlled amount of tertiary amines is applied to reverse the previous anionic surface charge while maintaining carbodiimide compatibility with future protein immobilization. The mechanism of this reversed-charge immobilization approach was demonstrated employing protein A as a model protein and using attenuated total reflectance Fourier transform infrared spectroscopy, dynamic contact angle measurements, colorimetric quantification, and SPR analysis to characterize surface derivatization. Furthermore, even though it had previously proven impossible to preconcentrate DNA electrostatically and to covalently couple it to polyanionic chip surfaces, we demonstrated that our approach allowed DNA to be preconcentrated and immobilized in good yields. Graphical abstract Principle of the covalent immobilization of acidic ligands on reversed-charge zwitterionic sensor chip surfaces.

Investigation on Sugar-Protein Connectivity in Salmonella O-Antigen Glycoconjugate Vaccines.

Invasive nontyphoidal Salmonella disease, for which licensed vaccines are not available, is a leading cause of bloodstream infections in Africa. The O-antigen portion of lipopolysaccharide is a good target for protective immunity. Covalent conjugation of the O-antigen to a carrier protein increases its immunogenicity and O-antigen based glycoconjugate vaccines are currently under investigation at the preclinical stage. We developed a conjugation chemistry for linking O-antigen to CRM197 carrier protein, through sequential insertion of adipic acid dihydrazide (ADH) and adipic acid bis( N-hydroxysuccinimide) ester (SIDEA) as linkers, without impacting O-antigen chain epitopes. Here the resulting sugar-protein connectivity has been investigated in detail. The core portion of the lipopolysaccharide was used as a model molecule to prepare CRM197 conjugates, making structural investigations easier. The first step of reductive amination with ADH involves the terminal 3-deoxy-d- manno-oct-2-ulosonic acid (KDO) residue of the core region. The second reaction step resulted not to be selective, as SIDEA reacted with both ADH and pyrophosphorylethanolamine (PPEtN) of the core region, independently from the pH at which the reaction was performed. Peptide mapping analysis of the deglycosylated core-CRM197 conjugates confirmed that lysine residues of CRM197 were linked to SIDEA not only through KDO-ADH but also through PPEtN. This analysis also confirmed that the conjugation chemistry is random on the protein, involving a large number of lysine residues, particularly the surface exposed ones. The method for core-CRM197 characterization was successfully extended to O-antigen-CRM197 conjugate, confirming the results obtained with the core. This study not only allowed full characterization of OAg-CRM197 conjugates, but can be applied to optimize synthesis and characterization of other OAg-based glycoconjugate vaccines. Analytical methods to investigate saccharide-protein connectivity are also of fundamental importance to study the relationship between glycoconjugate structure and immune response induced.

Multi-Modal Catalytic Cascades on Carbonaceous Scaffolds

Fundamental understanding of the ways organic and/or inorganic catalysts can be incorporated with enzymes to enhance the efficiency of multi-step reactions can lead to knowledge that is crucial for any further improvements in the development of multi-component multi-step catalytic cascades. This knowledge can provide essential clues to the novel design of systems for energy conversion and harvesting as well as for production of advanced materials that require multi-step catalysis. The goal of this project is to develop strategies for spatially organizing three different catalysts (biological, molecular, and inorganic) using 3D-Graphene nanosheets (3D-GNS) as a scaffold and apply it to efficiently catalyze model catalytic cascade: multi-step oxidation of glycerol [1,2]. In this work, we first studied the immobilization of oxalate decarboxylase (OxDC) from Bacillus subtilis, through physical absorption and tethering using 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) on conductive carbonaceous supports such as multi-walled carbon nanotube buckypaper and 3D-GNS. An indirect enzymatic assay using NAD-dependent formate dehydrogenase was performed to determine the kinetic parameters (K M V M) of immobilized OxDC onto these supports. We also employed cyclic voltammetry to study the immobilization of a molecular catalyst, pyrene-tethered TEMPO derivative (TEMPO = 2,2,6,6-tetramethylpiperidine-N-oxyl) on 3D-GNS. Furthermore, we explored optimal strategies to incorporate both OxDC and molecular catalysts in a way that will enable an efficient two-step catalytic cascade - the last two steps in the glycerol oxidation mechanism (Figure). Results of electrochemical studies show that OxDC and pyrene-TEMPO were successfully immobilized on the carbonaceous scaffold. Moreover, it was shown that this hybrid catalyst can catalyze two different reactions sequentially (i.e. conversion of oxalate to formate by OxDC/3D-GNS and oxidation of formate to CO2 by pyrene-TEMPO/3D-GNS). [1] S. Abdellaoui, D. P. Hickey, A. R. Stephens, and S. D. Minteer, Recombinant oxalate decarboxylase: enhancement of a hybrid catalytic cascade for the complete electro-oxidation of glycerol. Chem. Commun., 51, 14330-14333 (2015). [2] S. Abdellaoui, M. S. Chavez, I. Matanovic, A. R. Stephens, P. Atanassov, S. D. Minteer, Hybrid molecular/enzymatic catalytic cascade for complete electro-oxidation of glycerol using a promiscuous NAD-dependent formate dehydrogenase from Candida boidinii. Chem. Commun., 53, 5368-5371 (2016). Figure 1

Quantitative SERS Detection of Dopamine in Cerebrospinal Fluid by Dual-Recognition-Induced Hot Spot Generation

Reliable profiling of the extracellular dopamine (DA) concentration in the central nervous system is essential for a deep understanding of its biological and pathological functions. However, quantitative determination of this neurotransmitter remains a challenge because of the extremely low concentration of DA in the cerebrospinal fluid (CSF) of patients. Herein, on the basis of the specific recognition of boronate toward diol and N-hydroxysuccinimide ester toward the amine group, a simple and highly sensitive strategy was presented for DA detection by using surface-enhanced Raman scattering (SERS) spectroscopy as a signal readout. This was realized by first immobilizing 3,3'-dithiodipropionic acid di( N-hydroxysuccinimide ester) on gold thin film surfaces to capture DA, followed by introducing 3-mercaptophenylboronic acid (3-MPBA)-functionalized silver nanoparticles to generate numerous plasmonic "hot spots" with the nanoparticle-on-mirror geometry. Such a dual-recognition mechanism not only avoids complicated bioelement-based manipulations but also efficiently decreases the background signal. With the direct use of the recognition probe 3-MPBA as a Raman reporter, the "signal-on" SERS method was employed to quantify the concentration of DA from 1 pM to 1 μM with a detection limit of 0.3 pM. Moreover, our dual-recognition-directed SERS assay exhibited a high resistance to cerebral interference and was successfully applied to monitoring of DA in CSF samples of patients.

Mitochondria‐targeting peptide from Hibiscus sabdariffa

Cysteine‐rich peptides (CRPs) are hyperstable, multi‐disulfide‐constrained peptides. Structurally, CRPs contain evolutionary conserved disulfided scaffolds, often with functional intercysteine loops which contribute to their multiple biological activities. Previously, we have identified Hibiscus sabdariffa as a rich source of CRPs. Hibiscus sabdariffa, commonly known as roselle, is a South‐east Asian plant. It is used as a folk medicine for treating age‐related conditions and diseases. The prototypic CRP derived from roselle is roseltide rT1, a 27‐residue cysteine‐rich peptide with six cysteine forming four intercycteine loops. We showed that Loop 1 contributes to the inhibitory activities against human neutrophil elastase. However the functions of other loops, individually or in combination have not been determined. Here we report that roseltide rT1 is a mitochondria‐targeting peptide with bioenergetic properties. We showed that roseltide rT1 interacts with the mitochondrial import receptor TOM20 for its localization to the mitochondria. We prepared the linear precursor of roseltide rT1 using a stepwise solid‐phase method followed by oxidative folding to its native state. The conformation of synthetic rT1 was confirmed using reversed‐phase high‐pressure liquid chromatography and nuclear magnetic resonance spectroscopy. Using N‐hydroxysuccinimide ester, N‐terminal biotin‐ and Cy3‐labeled roseltide rT1 were generated for biological assays. Our results showed that mitochondrial depolarization induced by mitochondria uncoupler carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone does not abolish the mitochondrial localization of Cy3‐rT1. In silico docking suggested that the leucine‐rich loop 2 of roseltide rT1 interacts with TOM20. Using pull‐down assay, biotin‐rT1 was shown to interact with TOM20. Using live‐cell confocal microscopy, Cy3‐rT1 was shown to co‐localize with TOM20‐GFP in stably‐transfected HEK293 cells. Additionally, the localization of Cy3‐rT1 with MitoTracker Green was reduced in TOM20‐knocked‐down HepG2 cells compared to control. Taken together, this study shows that the mitochondrial import receptor TOM20 plays an important role for the localization of roseltide rT1 to the mitochondria.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐ Synzyme and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enantioselective determination of aspartate and glutamate in biological samples by ultrasonic-assisted derivatization coupled with capillary electrophoresis and linked to Alzheimer’s disease progression

A simple cyclodextrin-mediated capillary zone electrophoresis method equipped with a laser-induced fluorescence detector was developed for chiral analysis of the excitatory amino acids aspartate (Asp) and glutamate (Glu). Plasma and cerebrospinal fluid (CSF) samples were pretreated with centrifugal filter devices before analysis to remove high-molecular-weight proteins (molecular weight cut-off: 3000) and then derivatized using 10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester in DMSO under sonication at 25 °C for 2 h. After the derivatization reaction, reacted samples were diluted 100-fold with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (DTAF) solution and then hydrodynamically subjected to capillary electrophoresis (0.5 psi for 5 s, injection volume 8.27 nL). The separation buffer consisted of 50 mM borate buffer (pH 9.0) with 6 mM γ-CD and 0.1% polyvinylpyrrolidone, and the separation voltage was set at 20 kV. In the linearity calculations for the determination of d/l-Asp and d/l-Glu in plasma and CSF, a standard addition method was utilized to spike solutions with 0–20.0 μg mL−1l-Glu and 0–2.0 μg mL−1d-Glu and d/l-Asp to construct calibration curves. Correlation coefficients were above 0.998 for every analyte. The limits of detection (S/N = 3) for d/l-Asp and d/l-Glu standard solutions were 0.85–0.96 μg mL−1. The proposed method was applied successfully to determine d/l-Asp and d/l-Glu concentrations in the plasma and CSF samples of 26 patients with Alzheimer’s disease (AD), and the association between these concentrations and disease severity was investigated. Statistical analysis showed a moderately negative correlation (r = −0.158) between plasma l-Asp concentration and AD severity.

XPlex: An Effective, Multiplex Cross-Linking Chemistry for Acidic Residues.

Cross-linking/Mass spectrometry (XLMS) is a consolidated technique for structural characterization of proteins and protein complexes. Despite its success, the cross-linking chemistry currently used is mostly based on N-hydroxysuccinimide (NHS) esters, which react primarily with lysine residues. One way to expand the current applicability of XLMS into several new areas is to increase the number of cross-links obtainable for a target protein. We introduce a multiplex chemistry (denoted XPlex) that targets Asp, Glu, Lys, and Ser residues. XPlex can generate significantly more cross-links with reactions occurring at lower temperatures and enables targeting proteins that are not possible with NHS ester-based cross-linkers. We demonstrate the effectiveness of our approach in model proteins as well as a target Lys-poor protein, SalBIII. Identification of XPlex spectra requires a search engine capable of simultaneously considering multiple cross-linkers on the same run; to achieve this, we updated the SIM-XL search algorithm with a search mode tailored toward XPlex. In summary, we present a complete chemistry/computational solution for significantly increasing the number of possible distance constraints by mass spectrometry experiments, and thus, we are convinced that XPlex poses as a real complementary approach for structural proteomics studies.

Synthesis of Pentaerythritol-Based Branching Reagents for Modification of Proteins and Nucleic Acids by [2+3] Dipolar Cycloaddition Reaction

Alkylation of pentaerythritol symmetrically substituted with propylene glycol with propargyl bromide afforded compounds containing two or three alkyne moieties. Amidophosphite reagents and solid supports were prepared for the introduction of two and three acetylene fragments into oligonucleotides at the 3'- and 5'-positions and inside the chain under conditions of automated solid-phase oligonucleotide synthesis. Based on the trialkynyl derivative, an N-hydroxysuccinimide ester was obtained which can be used to modify biomolecules attacking the amino group. Conjugates obtained can be used for multiple modifications by [3+2] dipolar cycloaddition reaction.