Electrospray ionization-mass spectrometry characterization of heterotetrameric sarcosine oxidase

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i. e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 < m/z < 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization.     

Light-mediated inhibition of protein synthesis

The regulation of protein synthesis is vital for a host of cell biological processes, but investigating roles for protein synthesis have been hindered by the inability to selectively interfere with it. To inhibit protein synthesis with spatial and temporal control, we have developed a photo-releasable anisomycin compound, N-([6-bromo-7-hydroxycoumarin-4-yl]methyloxycarbonyl)anisomycin (Bhc-Aniso), that can be removed through exposure to UV light. The area of protein synthesis inhibition can be restricted to a small light-exposed region or, potentially, the volume of two-photon excitation if a pulsed IR laser is the light source. We have tested the compound's effectiveness with an in vitro protein-translation system, CHO cells, HEK293 cells, and neurons. The photo-released anisomycin can inhibit protein synthesis in a spatially restricted manner, which will enable the specific inhibition of protein synthesis in subsets of cells with temporal and spatial precision.     

Tailored thioxanthone‐based photoinitiators for two‐photon‐controllable polymerization and nanolithographic printing

Printing of high‐resolution three‐dimensional nanostructures utilizing two‐photon polymerization has gained significant attention recently. In particular, isopropyl thioxanthone (ITX) has been implemented as a photoinitiator due to its capability of initiating and depleting polymerization on demand, but new photoinitiating materials are still needed in order to reduce the power requirements for the high‐throughput creation of 3D structures. To address this point, a suite of new thioxanthone‐based photoinitiators were synthesized and characterized. Then two‐photon polymerization was performed using the most promising photoinitiating molecule. Importantly, one of the initiators, 2,7‐bis[(4‐(dimethylamino)phenyl ethynyl)‐9H‐thioxanthen‐9‐one] (BDAPT), showed a fivefold improvement in the writing threshold over the commonly used ITX molecule. To elucidate the fundamental mechanism, the excitation and inhibition behavior of the BDAPT molecule were evaluated using density functional theory (DFT) calculations, low‐temperature phosphorescence spectroscopy, ultra‐fast transient absorption spectroscopy, and the two‐photon Z‐scan spectroscopic technique. The improved polymerization threshold of this new photoinitiator presents a clear pathway for the modification of photoinitiators in 3D nanoprinting. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019 , 57, 1462–1475     

Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition

The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na(2)S(2)O(4), 2% HOCH(2)CH(2)SH, 10% HCO(2)H, 95% CF(3)CO(2)H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO(2)H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.     

Increasing the detection speed of an all-electronic real-time biosensor

Biosensor response time, which depends sensitively on the transport of biomolecules to the sensor surface, is a critical concern for future biosensor applications. We have fabricated carbon nanotube field-effect transistor biosensors and quantified protein binding rates onto these nanoelectronic sensors. Using this experimental platform we test the effectiveness of a protein repellent coating designed to enhance protein flux to the all-electronic real-time biosensor. We observe a 2.5-fold increase in the initial protein flux to the sensor when upstream binding sites are blocked. Mass transport modelling is used to calculate the maximal flux enhancement that is possible with this strategy. Our results demonstrate a new methodology for characterizing nanoelectronic biosensor performance, and demonstrate a mass transport optimization strategy that is applicable to a wide range of microfluidic based biosensors.     

Development of vulcanizable elastomers suitable for use in contact with liquid oxygen

For the past few years our research has been concerned with the preparation of polymers which are compatible with and have usable mechanical properties in the presence of liquid oxygen. These are stringent requirements and the need for oxidative stability necessitated investigation of halogen-containing polymers, in particular, polymers containing fluorine. Since only a few fluorocarbon monomers are commercially available, this study required an extensive synthesis program. A comprehensive search of the literature and an attempt to systematically study the effect of polymer structure on low-temperature properties directed our initial work toward the synthesis of some novel, highly fluorinated vinyl monomers and polymers. In this article the effect of structure on polymer glass transition temperatures will be discussed and the synthesis and characterization of some fluorocarbon vinyl ethers and their polymers will be described.     

Human Neuroepithelial Cells Express NMDA Receptors

L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1) cerebral endothelial barrier and 2) cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells) have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR) expression via immunohistochemistry and murine neuroepithelial cell line (V1) were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.     

Design of Photocaged Puromycin for Nascent Polypeptide Release and Spatiotemporal Monitoring of Translation

The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC‐puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome‐bound polypeptide chains was detected in vitro. Upon illumination, cytotoxic activity, in vitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC‐puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1±0.2 % and the NVOC‐puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two‐photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons.     

Synthesis of a novel diarylphosphinic acid: a distorted ground state mimic and transition state analogue for amide hydrolysis

We describe herein the synthesis of a new unsymmetrical diarylphosphinic acid, a hapten aimed to produce catalytic antibodies for the hydrolysis of heterocyclic amides. The phosphinate functionality was selected as a mimic both of the tetrahedral intermediate and the transition state of higher energy along the reaction profile. The phenyl and 2,4,6-(trimethyl)-phenyl groups flanking the phosphinate were chosen in order to impose rotation around the P–C bond, a choice supported by ab initio calculations. This new hapten should elicit catalytic antibodies whose binding site could affect the distortion at nitrogen as well as the twist along the N–C(O) bond for heterocyclic amides. This hapten along with a series of new sterically hindered unsymmetrical phosphinic acid derivatives was prepared by a key palladium-catalysed step.