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- ItemSecondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies(Columbus, Ohio : American Chemical Society, 2016) Davies, Heather S.; Singh, Prabha; Deckert-Gaudig, Tanja; Deckert, Volker; Rousseau, Karine; Ridley, Caroline E.; Dowd, Sarah E.; Doig, Andrew J.; Pudney, Paul D. A.; Thornton, David J.; Blanch, Ewan W.The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
- ItemDetection of Protein Glycosylation Using Tip-Enhanced Raman Scattering(Columbus, Ohio : American Chemical Society, 2016) Cowcher, David P.; Deckert-Gaudig, Tanja; Brewster, Victoria L.; Ashton, Lorna; Deckert, Volker; Goodacre, RoystonThe correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
- ItemHigh-throughput screening Raman microspectroscopy for assessment of drug-induced changes in diatom cells(Cambridge : Royal Society of Chemistry, 2019) Rüger J.; Mondol A.S.; Schie I.W.; Popp J.; Krafft C.High-throughput screening Raman spectroscopy (HTS-RS) with automated localization algorithms offers unsurpassed speed and sensitivity to investigate the effect of dithiothreitol on the diatom Phaedactylum tricornutum. The HTS-RS capability that was demonstrated for this model system can be transferred to unmet analytical applications such as kinetic in vivo studies of microalgal assemblages. © 2019 The Royal Society of Chemistry.
- ItemModified bibenzimidazole ligands as spectator ligands in photoactive molecular functional Ru-polypyridine units? Implications from spectroscopy(Cambridge : RSC, 2014) Meyer-Ilse, J.; Bauroth, S.; Bräutigam, M.; Schmitt, M.; Popp, J.; Beckert, R.; Rockstroh, N.; Pilz, T.D.; Monczak, K.; Heinemann, F.W.; Rau, S.; Dietzek, B.The photophysical properties of Ruthenium-bipyridine complexes bearing a bibenzimidazole ligand were investigated. The nitrogens on the bibenzimidazole-ligand were protected, by adding either a phenylene group or a 1,2-ethandiyl group, to remove the photophysical dependence of the complex on the protonation state of the bibenzimidazole ligand. This protection results in the bibenzimidazole ligand contributing to the MLCT transition, which is experimentally evidenced by (resonance) Raman scattering in concert with DFT calculations for a detailed mode assignment in the (resonance) Raman spectra.
- ItemCharge isomers of myelin basic protein: Structure and interactions with membranes, nucleotide analogues, and calmodulin(San Francisco, CA : Public Library of Science, 2011) Wang, C.; Neugebauer, U.; Bürck, J.; Myllykoski, M.; Baumgärtel, P.; Popp, J.; Kursula, P.As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids - a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.
- ItemLiver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis(San Francisco, CA : Public Library of Science, 2012) Recknagel, P.; Gonnert, F.A.; Westermann, M.; Lambeck, S.; Lupp, A.; Rudiger, A.; Dyson, A.; Carré, J.E.; Kortgen, A.; Krafft, C.; Popp, J.; Sponholz, C.; Fuhrmann, V.; Hilger, I.; Claus, R.A.; Riedemann, N.C.; Wetzker, R.; Singer, M.; Trauner, M.; Bauer, M.Background: Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests. Methods and Findings: In a long-term rat model of faecal peritonitis, biotransformation and hepatobiliary transport were impaired, depending on subsequent disease severity, as early as 6 h after peritoneal contamination. Phosphatidylinositol-3-kinase (PI3K) signalling was simultaneously induced at this time point. At 15 h there was hepatocellular accumulation of bilirubin, bile acids, and xenobiotics, with disturbed bile acid conjugation and drug metabolism. Cholestasis was preceded by disruption of the bile acid and organic anion transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. This was partially confirmed by an increase in plasma bile acids (e.g., chenodeoxycholic acid [CDCA] and taurodeoxycholic acid [TDCA]) observed in 48 patients on the day severe sepsis was diagnosed; unlike bilirubin (area under the receiver-operating curve: 0.59), these bile acids predicted 28-d mortality with high sensitivity and specificity (area under the receiver-operating curve: CDCA: 0.77; TDCA: 0.72; CDCA+TDCA: 0.87). Conclusions: Liver dysfunction is an early and commonplace event in the rat model of sepsis studied here; PI3K signalling seems to play a crucial role. All aspects of hepatic biotransformation are affected, with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reflected by early alterations in plasma bile acids. These observations carry important implications for the diagnosis of liver dysfunction and pharmacotherapy in the critically ill. Further clinical work is necessary to extend these concepts into clinical practice. Please see later in the article for the Editors' Summary.
- ItemOptical photothermal infrared spectroscopy with simultaneously acquired Raman spectroscopy for two-dimensional microplastic identification([London] : Macmillan Publishers Limited, part of Springer Nature, 2022) Böke, Julia Sophie; Popp, Jürgen; Krafft, ChristophIn recent years, vibrational spectroscopic techniques based on Fourier transform infrared (FTIR) or Raman microspectroscopy have been suggested to fulfill the unmet need for microplastic particle detection and identification. Inter-system comparison of spectra from reference polymers enables assessing the reproducibility between instruments and advantages of emerging quantum cascade laser-based optical photothermal infrared (O-PTIR) spectroscopy. In our work, IR and Raman spectra of nine plastics, namely polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate, polycarbonate, polystyrene, silicone, polylactide acid and polymethylmethacrylate were simultaneously acquired using an O-PTIR microscope in non-contact, reflection mode. Comprehensive band assignments were presented. We determined the agreement of O-PTIR with standalone attenuated total reflection FTIR and Raman spectrometers based on the hit quality index (HQI) and introduced a two-dimensional identification (2D-HQI) approach using both Raman- and IR-HQIs. Finally, microplastic particles were prepared as test samples from known materials by wet grinding, O-PTIR data were collected and subjected to the 2D-HQI identification approach. We concluded that this framework offers improved material identification of microplastic particles in environmental, nutritious and biological matrices.
- ItemFiber enhanced Raman spectroscopic analysis as a novel method for diagnosis and monitoring of diseases related to hyperbilirubinemia and hyperbiliverdinemia(Cambridge : Soc., 2016) Yan, Di; Domes, Christian; Domes, Robert; Frosch, Timea; Popp, Jürgen; Pletz, Mathias W.; Frosch, TorstenFiber enhanced resonance Raman spectroscopy (FERS) is introduced for chemically selective and ultrasensitive analysis of the biomolecules hematin, hemoglobin, biliverdin, and bilirubin. The abilities for analyzing whole intact, oxygenated erythrocytes are proven, demonstrating the potential for the diagnosis of red blood cell related diseases, such as different types of anemia and hemolytic disorders. The optical fiber enables an efficient light-guiding within a miniaturized sample volume of only a few micro-liters and provides a tremendously improved analytical sensitivity (LODs of 0.5 μM for bilirubin and 0.13 μM for biliverdin with proposed improvements down to the pico-molar range). FERS is a less invasive method than the standard ones and could be a new analytical method for monitoring neonatal jaundice, allowing a precise control of the unconjugated serum bilirubin levels, and therefore, providing a better prognosis for newborns. The potential for sensing very low concentrations of the bile pigments may also open up new opportunities for cancer research. The abilities of FERS as a diagnostic tool are explored for the elucidation of jaundice with different etiologies including the rare, not yet well understood diseases manifested in green jaundice. This is demonstrated by quantifying clinically relevant concentrations of bilirubin and biliverdin simultaneously in the micro-molar range: for the case of hyperbilirubinemia due to malignancy, infectious hepatitis, cirrhosis or stenosis of the common bile duct (1 μM biliverdin together with 50 μM bilirubin) and for hyperbiliverdinemia (25 μM biliverdin and 75 μM bilirubin). FERS has high potential as an ultrasensitive analytical technique for a wide range of biomolecules and in various life-science applications.
- ItemToward food analytics: fast estimation of lycopene and β-carotene content in tomatoes based on surface enhanced Raman spectroscopy (SERS)(Cambridge : Soc., 2016) Radu, Andreea Ioana; Ryabchykov, Oleg; Bocklitz, Thomas Wilhelm; Huebner, Uwe; Weber, Karina; Cialla-May, Dana; Popp, JürgenCarotenoids are molecules that play important roles in both plant development and in the well-being of mammalian organisms. Therefore, various studies have been performed to characterize carotenoids’ properties, distribution in nature and their health benefits upon ingestion. Nevertheless, there is a gap regarding a fast detection of them at the plant phase. Within this contribution we report the results obtained regarding the application of surface enhanced Raman spectroscopy (SERS) toward the differentiation of two carotenoid molecules (namely, lycopene and β-carotene) in tomato samples. To this end, an e-beam lithography (EBL) SERS-active substrate and a 488 nm excitation source were employed, and a relevant simulated matrix was prepared (by mixing the two carotenoids in defined percentages) and measured. Next, carotenoids were extracted from tomato plants and measured as well. Finally, a combination of principal component analysis and partial least squares regression (PCA-PLSR) was applied to process the data, and the obtained results were compared with HPLC measurements of the same extracts. A good agreement was obtained between the HPLC and the SERS results for most of the tomato samples.
- ItemMicrobial respiration and natural attenuation of benzene contaminated soils investigated by cavity enhanced Raman multi-gas spectroscopy(Cambridge : Soc., 2015) Jochum, Tobias; Michalzik, Beate; Bachmann, Anne; Popp, Jürgen; Frosch, TorstenSoil and groundwater contamination with benzene can cause serious environmental damage. However, many soil microorganisms are capable to adapt and are known to strongly control the fate of organic contamination. Innovative cavity enhanced Raman multi-gas spectroscopy (CERS) was applied to investigate the short-term response of the soil micro-flora to sudden surface contamination with benzene regarding the temporal variations of gas products and their exchange rates with the adjacent atmosphere. 13C-labeled benzene was spiked on a silty-loamy soil column in order to track and separate the changes in heterotrophic soil respiration – involving 12CO2 and O2 – from the natural attenuation process of benzene degradation to ultimately form 13CO2. The respiratory quotient (RQ) decreased from a value 0.98 to 0.46 directly after the spiking and increased again within 33 hours to a value of 0.72. This coincided with the maximum 13CO2 concentration rate (0.63 μmol m−2 s−1), indicating the highest benzene degradation at 33 hours after the spiking event. The diffusion of benzene in the headspace and the biodegradation into 13CO2 were simultaneously monitored and 12 days after the benzene spiking no measurable degradation was detected anymore. The RQ finally returned to a value of 0.96 demonstrating the reestablished aerobic respiration.