Subunits of highly fluorescent protein R-phycoerythrin as probes for cell imaging and single-molecule detection

R-phycoerythrin (R-PE) subunits and enzymatic digests were characterized by high-performance liquid chromatography (HPLC), capillary and gel electrophoresis, and HPLC-electrospray ionization mass spectrometry. Subunits were isolated from R-PE by HPLC and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). Favorable spectroscopic characteristics of R-PE subunits and digest peptides in the visible region of spectrum originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores. High absorption coefficients and fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra, and low molecular weights make these molecules suitable for fluorescence labeling of biomolecules and cells.;Fluorescent proteins were further formed both in vitro and in vivo by attachment of PEB to recombinant apo-subunits of R-PE and their genetic fusions to maltose binding protein (MBP). Apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii into bacterium Escherichia coli (E. coli). Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were formed after incubation of E. coli cells with PEB. Holo-subunits contained both PEB and urobilin (UB) chromophores. Fluorescence and differential interference contrast (DIC) microscopy localized holo-subunits at poles of E. coli cells. Proteins formed by attachment of PEB to MBP-subunit fusions were soluble, displayed high fluorescence, contained only PEB, and were located either throughout cells or at cell poles. As measured by flow cytometry, cells containing fluorescent holo-subunits or MBP-subunit fusions were up to ten times brighter than control cells. Proteins formed by attachment of PEB to R-PE apo-subunits can be used as florescence reporters of gene expression and protein localization in cells, and in flow cytometry.;Finally, a high-throughput method was demonstrated which recorded emission fluorescence spectra of individual E. coli cells containing fluorescent proteins. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera recorded simultaneously the zero order and the first order spectrum for each cell. Demonstrated method could have a higher throughput, better sensitivity, and better spectral resolution compared to spectral flow cytometry

Neuroprotective peptide ADNF-9 in fetal brain of C57BL/6 mice exposed prenatally to alcohol

<p>Abstract</p> <p>Background</p> <p>A derived peptide from activity-dependent neurotrophic factor (ADNF-9) has been shown to be neuroprotective in the fetal alcohol exposure model. We investigated the neuroprotective effects of ADNF-9 against alcohol-induced apoptosis using TUNEL staining. We further characterize in this study the proteomic architecture underlying the role of ADNF-9 against ethanol teratogenesis during early fetal brain development using liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS).</p> <p>Methods</p> <p>Pregnant C57BL/6 mice were exposed from embryonic days 7-13 (E7-E13) to a 25% ethanol-derived calorie [25% EDC, Alcohol (ALC)] diet, a 25% EDC diet simultaneously administered i.p. ADNF-9 (ALC/ADNF-9), or a pair-fed (PF) liquid diet. At E13, fetal brains were collected from 5 dams from each group, weighed, and frozen for LC-MS/MS procedure. Other fetal brains were fixed for TUNEL staining.</p> <p>Results</p> <p>Administration of ADNF-9 prevented alcohol-induced reduction in fetal brain weight and alcohol-induced increases in cell death. Moreover, individual fetal brains were analyzed by LC-MS/MS. Statistical differences in the amounts of proteins between the ALC and ALC/ADNF-9 groups resulted in a distinct data-clustering. Significant upregulation of several important proteins involved in brain development were found in the ALC/ADNF-9 group as compared to the ALC group.</p> <p>Conclusion</p> <p>These findings provide information on potential mechanisms underlying the neuroprotective effects of ADNF-9 in the fetal alcohol exposure model.</p

Energy Transfer From Fluorescent Proteins To Metal Nanoparticles

Energy transfer plays a significant role in numerous chemical, physical, and biological processes. While the use of fluorescent proteins in Forster resonance energy transfer (FRET) studies of biomolecules is common, energy transfer between fluorescent proteins and inorganic nanoparticles has not been explored in detail. In this study, energy transfer from fluorescent phycobiliproteins to noble metal nanoparticles was analyzed. Solutions of B-phycoerythrin (B-PE) were mixed with colloidal Au and Ag nanoparticles and were characterized by steady-state and time-resolved fluorescence spectroscopy to determine the magnitude and mechanism of the energy transfer. It was found that the protein fluorescence was quenched after the addition of metal nanoparticles. Electron microscopy and absorption spectroscopy confirmed that B-PE was adsorbed onto the nanoparticles, creating a favorable geometry for quenching. Time-resolved fluorescence spectroscopy showed that B-PE fluorescence lifetimes decreased from 2.2 ns to 0.5 and 0.6 ns upon adsorption onto Au and Ag nanoparticles, respectively, corresponding to energy transfer efficiencies of \u3e70%. Our results, which include lifetimes, efficiencies, and energy transfer distances, show that energy was transferred via the surface energy transfer (SET) mechanism, rather than FRET

N-linked glycan profiling of GGTA1/CMAH knockout pigs identifies new potential carbohydrate xenoantigens

BACKGROUND: The temporary or long-term xenotransplantation of pig organs into people would save thousands of lives each year if not for the robust human antibody response to pig carbohydrates. Genetically engineered pigs deficient in galactose α1,3 galactose (gene modified: GGTA1) and N-glycolylneuraminic acid (gene modified: CMAH) have significantly improved cell survival when challenged by human antibody and complement in vitro. There remains, however, a significant portion of human antibody binding. METHODS: To uncover additional xenoantigens, we compared the asparagine-linked (N-linked) glycome from serum proteins of humans, domestic pigs, GGTA1 knockout pigs, and GGTA1/CMAH knockout pigs using mass spectrometry. Carbohydrate structures were determined with assistance from GlycoWorkbench, Cartoonist, and SimGlycan software by comparison to existing database entries and collision-induced dissociation fragmentation data. RESULTS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of reduced and solid-phase permethylated glycans resulted in the detection of high-mannose, hybrid, and complex type N-linked glycans in the 1000-4500 m/z ion range. GGTA1/CMAH knockout pig samples had increased relative amounts of high-mannose, incomplete, and xylosylated N-linked glycans. All pig samples had significantly higher amounts of core and possibly antennae fucosylation. CONCLUSIONS: We provide for the first time a comparison of the serum protein glycomes of the human, domestic pig, and genetically modified pigs important to xenotransplantation

Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were formed after incubation of E. coli cells with PEB. Spectroscopic characterization of holo-subunits confirmed that the attachment of PEB chromophore to apo-subunits yielded holo-subunits containing both PEB and urobilin (UB). Fluorescence and differential interference contrast (DIC) microscopy showed polar location of holo-subunit inclusion bodies in E. coli cells. In another example, R-PE apo-subunits were genetically fused to cytoplasmic and periplasmic versions of E. coli maltose binding protein (MBP). Fluorescent proteins formed after attachment of PEB to MBP-subunit fusions in vitro and in vivo contained PEB as the sole chromophore, were soluble, and displayed high orange fluorescence. Fluorescence microscopy showed that fusions are located either throughout cells or at cell poles. In addition, cells containing fluorescent holo-subunits or MBP-subunit fusions were up to ten times brighter than control cells as measured by flow cytometry. Results show that the fluorescent proteins formed after non-enzymatic attachment of PEB to R-PE subunit fusions could be used as reporters of gene expression and protein localization in cells as well as fluorescence labels in flow cytometry. Finally, we demonstrated a high-throughput method able to record emission fluorescence spectra of individual cells containing fluorescent proteins. Upon excitation with a 488 mn argon-ion laser many bacterial cells were imaged by a 20X microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera simultaneously recorded the zero and the first orders of the fluorescence from each cell. Single-cell fluorescence spectra were reconstructed from the distance between zero-order and first-order maxima as well as the length and the pixel intensity distribution of the first-order images. By using this approach, the emission spectrum of E. coli cells expressing green fluorescent protein (GFP) was reconstructed. Also, fluorescence spectra of E. coli cells expressing apo-subunits of R-PE were recorded after incubation of the cells with PEB. The fluorescence spectra are in good agreement with results obtained on the same cells using a fluorescence spectrometer and a fluorescence microscope. When spectra are to be acquired, this approach could have a higher throughput, better sensitivity, and better spectral resolution compared to spectral flow cytometry

Subunits of highly fluorescent protein R-phycoerythrin as probes for cell imaging and single-molecule detection

R-phycoerythrin (R-PE) subunits and enzymatic digests were characterized by high-performance liquid chromatography (HPLC), capillary and gel electrophoresis, and HPLC-electrospray ionization mass spectrometry. Subunits were isolated from R-PE by HPLC and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). Favorable spectroscopic characteristics of R-PE subunits and digest peptides in the visible region of spectrum originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores. High absorption coefficients and fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra, and low molecular weights make these molecules suitable for fluorescence labeling of biomolecules and cells.;Fluorescent proteins were further formed both in vitro and in vivo by attachment of PEB to recombinant apo-subunits of R-PE and their genetic fusions to maltose binding protein (MBP). Apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii into bacterium Escherichia coli (E. coli). Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were formed after incubation of E. coli cells with PEB. Holo-subunits contained both PEB and urobilin (UB) chromophores. Fluorescence and differential interference contrast (DIC) microscopy localized holo-subunits at poles of E. coli cells. Proteins formed by attachment of PEB to MBP-subunit fusions were soluble, displayed high fluorescence, contained only PEB, and were located either throughout cells or at cell poles. As measured by flow cytometry, cells containing fluorescent holo-subunits or MBP-subunit fusions were up to ten times brighter than control cells. Proteins formed by attachment of PEB to R-PE apo-subunits can be used as florescence reporters of gene expression and protein localization in cells, and in flow cytometry.;Finally, a high-throughput method was demonstrated which recorded emission fluorescence spectra of individual E. coli cells containing fluorescent proteins. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera recorded simultaneously the zero order and the first order spectrum for each cell. Demonstrated method could have a higher throughput, better sensitivity, and better spectral resolution compared to spectral flow cytometry.</p

Removal of Microcystins and Nodularin-R from Water by Corncobs Studied Using LC-MS

Microcystins (MCs) and nodularins are released during harmful algal blooms (HABs) in fresh and brackish water. Consumption of water contaminated with MCs or nodularins can promote health risks for humans and animals. In this study, The Andersons corncobs were acid refluxed and heat treated to remove MCs and nodularin-R from water. Simultaneous sorption of six common MC congeners (MC-RR, MC-YR, MC-LR, MC-LA, MC-LW, and MC-LF) and nodularin-R onto treated corncobs was determined in suspension and filter mode. An LC-Orbitrap-MS system was used to measure the percent removal of MCs and NOD-R in water. Results from preliminary sorption experiments showed that a sorbent (S-5), which was acid refluxed and heat treated, has the highest removal efficiency. When a mixture containing six MCs and nodularin-R (10 µg/L each) was incubated with 50 mg of sorbent, all analytes were completely removed. Even after increasing the concentration of analytes ten times, removal for all analytes by sorbent was ≥ 98.1%. When S-5 was incubated with Lake Erie water collected during the 2020 HAB, it was able to remove most of the MC variants completely. LC-MS was used to show that treated corncobs are promising sorbent materials to remove MCs and nodularin-R from drinking water

Multimodal Spectral Imaging of Cells using a Transmission Diffraction Grating on a Light Microscope

A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield or DIC spectral microscopy, respectively. The presented spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens