Ion Mobility Mass Spectrometry-based Disaccharide Analysis of Glycosaminoglycans.

Glycosaminoglycans (GAGs) are linear and acidic polysaccharides. They are ubiquitous molecules, which are involved in a wide range of biological processes. Despite being structurally simple at first glance, with a repeating backbone of alternating hexuronic acid and hexosamine dimers, GAGs display a highly complex structure, which predominantly results from their heterogeneous sulfation patterns. The commonly applied method for compositional analysis of all GAGs is "disaccharide analysis." In this process, GAGs are enzymatically depolymerized into disaccharides, derivatized with a fluorescent label, and then analysed through liquid chromatography. The limiting factor in the high throughput analysis of GAG disaccharides is the time-consuming liquid chromatography. To address this limitation, we here utilized trapped ion mobility-mass spectrometry (TIM-MS) for the separation of isomeric GAG disaccharides, which reduces the measurement time from hours to a few minutes. A full set of disaccharides comprises twelve structures, with eight possessing isomers. Most disaccharides cannot be differentiated by TIM-MS in underivatized form. Therefore, we developed chemical modifications to reduce sample complexity and enhance differentiability. Quantification is performed using stable isotope labelled standards, which are easily available due to the nature of the performed modifications.

Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest.

In recent years, glycosaminoglycans (GAGs) have emerged into the focus of biochemical and biomedical research due to their importance in a variety of physiological processes. These molecules show great diversity, which makes their analysis highly challenging. A promising tool for identifying the structural motifs and conformation of shorter GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this work, the cryogenic gas-phase IR spectra of mass-selected heparan sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to extract vibrational features that are characteristic to structural motifs. The data were augmented with chondroitin sulfate (CS) disaccharide spectra to assemble a training library for random forest (RF) classifiers. These were used to discriminate between GAG classes (CS or HS) and different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With optimized data preprocessing and RF modeling, a prediction accuracy of >97% was achieved for HS tetra- and hexasaccharides based on a training set of only 21 spectra. These results exemplify the importance of combining gas-phase cryogenic IR ion spectroscopy with machine learning to improve the future analytical workflow for GAG sequencing and that of other biomolecules, such as metabolites.

Analytical challenges of glycosaminoglycans at biological interfaces.

The analysis of glycosaminoglycans (GAGs) is a challenging task due to their high structural heterogeneity, which results in diverse GAG chains with similar chemical properties. Simultaneously, it is of high importance to understand their role and behavior in biological systems. It has been known for decades now that GAGs can interact with lipid molecules and thus contribute to the onset of atherosclerosis, but their interactions at and with biological interfaces, such as the cell membrane, are yet to be revealed. Here, analytical approaches that could yield important knowledge on the GAG-cell membrane interactions as well as the synthetic and analytical advances that make their study possible are discussed. Due to recent developments in laser technology, we particularly focus on nonlinear spectroscopic methods, especially vibrational sum-frequency generation spectroscopy, which has the potential to unravel the structural complexity of heterogeneous biological interfaces in contact with GAGs, in situ and in real time.

Protein Secondary Structure Affects Glycan Clustering in Native Mass Spectrometry.

Infection by the humannoroviruses (hNoV), for the vast majority of strains, requires attachment of the viral capsid to histo blood group antigens (HBGAs). The HBGA-binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for the binding of HBGAs to P dimers and, in some cases, disagreed on whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of a reference protein is crucial. We analysed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of ß-sheets has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.

The interaction of chondroitin sulfate with a lipid monolayer observed by using nonlinear vibrational spectroscopy.

The first vibrational sum-frequency generation (VSFG) spectra of chondroitin sulfate (CS) interacting with dipalmitoyl phosphatidylcholine (DPPC) at air-liquid interface are reported here, collected at a laser repetition rate of 100 kHz. By studying the VSFG spectra in the regions of 1050-1450 cm-1, 2750-3180 cm-1, and 3200-3825 cm-1, it was concluded that in the presence of Ca2+ ions, the head groups together with the head-group-bound water molecules in the DPPC monolayer are strongly influenced by the interaction with CS, while the organization of the phospholipid tails remains mostly unchanged. The interactions were observed at a CS concentration below 200 nM, which exemplifies the potential of VSFG in studying biomolecular interactions at low physiological concentrations. The VSFG spectra recorded in the O-H stretching region at chiral polarization combination imply that CS molecules are organized into ordered macromolecular superstructures with a chiral secondary structure.

Probing the Intracellular Bio-Nano Interface in Different Cell Lines with Gold Nanostars.

Gold nanostars are a versatile plasmonic nanomaterial with many applications in bioanalysis. Their interactions with animal cells of three different cell lines are studied here at the molecular and ultrastructural level at an early stage of endolysosomal processing. Using the gold nanostars themselves as substrate for surface-enhanced Raman scattering, their protein corona and the molecules in the endolysosomal environment were characterized. Localization, morphology, and size of the nanostar aggregates in the endolysosomal compartment of the cells were probed by cryo soft-X-ray nanotomography. The processing of the nanostars by macrophages of cell line J774 differed greatly from that in the fibroblast cell line 3T3 and in the epithelial cell line HCT-116, and the structure and composition of the biomolecular corona was found to resemble that of spherical gold nanoparticles in the same cells. Data obtained with gold nanostars of varied morphology indicate that the biomolecular interactions at the surface in vivo are influenced by the spike length, with increased interaction with hydrophobic groups of proteins and lipids for longer spike lengths, and independent of the cell line. The results will support optimized nanostar synthesis and delivery for sensing, imaging, and theranostics.

Polysulfates Block SARS-CoV-2 Uptake through Electrostatic Interactions*.

Here we report that negatively charged polysulfates can bind to the spike protein of SARS-CoV-2 via electrostatic interactions. Using a plaque reduction assay, we compare inhibition of SARS-CoV-2 by heparin, pentosan sulfate, linear polyglycerol sulfate (LPGS) and hyperbranched polyglycerol sulfate (HPGS). Highly sulfated LPGS is the optimal inhibitor, with an IC50 of 67 µg mL-1 (approx. 1.6 µm). This synthetic polysulfate exhibits more than 60-fold higher virus inhibitory activity than heparin (IC50 : 4084 µg mL-1 ), along with much lower anticoagulant activity. Furthermore, in molecular dynamics simulations, we verified that LPGS can bind more strongly to the spike protein than heparin, and that LPGS can interact even more with the spike protein of the new N501Y and E484K variants. Our study demonstrates that the entry of SARS-CoV-2 into host cells can be blocked via electrostatic interactions, therefore LPGS can serve as a blueprint for the design of novel viral inhibitors of SARS-CoV-2.

Intracellular optical probing with gold nanostars.

Gold nanostars are important nanoscopic tools in biophotonics and theranostics. To understand the fate of such nanostructures in the endolysosomal system of living cells as an important processing route in biotechnological approaches, un-labelled, non-targeted gold nanostars synthesized using HEPES buffer were studied in two cell lines. The uptake of the gold nanostructures leads to cell line-dependent intra-endolysosomal agglomeration, which results in a greater enhancement of the local optical fields than those around individual nanostars and near aggregates of spherical gold nanoparticles of the same size. As demonstrated by non-resonant surface-enhanced Raman scattering (SERS) spectra in the presence and absence of aggregation, the spectroscopic signals of molecules are of very similar strength over a wide range of concentrations, which is ideal for label-free vibrational characterization of cells and other complex environments. In 3T3 and HCT-116 cells, SERS data were analyzed together with the properties of the intracellular nanostar agglomerates. Vibrational spectra indicate that the processing of nanostars by cells and their interaction with the surrounding endolysosomal compartment is connected to their morphological properties through differences in the structure and interactions in their intracellular protein corona. Specifically, different intracellular processing was found to result from a different extent of hydrophobic interactions at the pristine gold surface, which varies for nanostars of different spike lengths. The sensitive optical monitoring of surroundings of nanostars and their intracellular processing makes them a very useful tool for optical bionanosensing and therapy.

Fragmentation of Proteins in the Corona of Gold Nanoparticles As Observed in Live Cell Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) can provide information on the structure, composition, and interaction of molecules in the proximity of gold nanoparticles, thereby enabling studies of adsorbed biomolecules in vivo. Here, the processing of the protein corona and the corresponding protein-nanoparticle interactions in live J774 cells incubated with gold nanoparticles was characterized by SERS. Samples of isolated cytoplasm, devoid of active processing, of the same cell line were used as references. The occurrence of the most important SERS signals was compared in both types of samples. The comparison of signal abundances, supported by multivariate assessment, suggests a decreased nanoparticle-peptide backbone interaction and an increased contribution of denatured proteins in endolysosomal compartments, indicating an interaction of protein fragments with the gold nanoparticles in the endolysosome of the living cells. To study the protein fragmentation in a model and to confirm the assignment of specific spectral signatures in the live cell spectra, SERS data were collected from a solution of bovine serum albumin (BSA) digested by trypsin as an enzymatic model and from solutions of intact BSA and trypsin. The spectra from the enzymatic model confirm the strong interaction of protein fragments with the gold nanoparticles in the endolysosomal compartments. By proteomic analysis, using combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry of the extracted hard corona, we directly identified protein fragments, some originating from the culture medium. The results illustrate the use of appropriate models for the validation of SERS spectra and have potential implications for further developments of SERS as an in vivo analytical and biomedical tool.

Mass spectrometric approach for the analysis of the hard protein corona of nanoparticles in living cells.

The diagnostic and therapeutic application of nanoparticles requires comprehensive knowledge of their interaction with the biomolecular surroundings. The formation of the protein corona on nanoparticles that were internalized by living cells is yet to be understood. In this study, we present a robust approach for the electrophoretic and mass spectrometric analysis of the hard protein corona composition formed in living cells on ~30 nm citrate-stabilized gold nanoparticles, i.e., the proteins with the highest affinity towards the gold nanoparticle surface. The gold nanoparticles were internalized by MCF-7 cells for 24 h followed by the extraction of the hard protein corona­gold nanoparticle bioconjugates from living cell cultures. The extracted proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by ESI-Q-TOF-MS, which allowed to identify 108 hard corona proteins. The experiments were repeated with J774 macrophage cells with incubation times of 1.5 h, 3 h, 6 h, and 24 h, and the results showed that the hard protein corona remained unchanged over time. Therefore, the proposed experimental approach proved to be a valuable tool for identifying hard corona proteins of nanoparticles internalized by living cells.

Optical Nanosensing of Lipid Accumulation due to Enzyme Inhibition in Live Cells.

Drugs that influence enzymes of lipid metabolism can cause pathological accumulation of lipids in animal cells. Here, gold nanoparticles, acting as nanosensors that deliver surface-enhanced Raman scattering (SERS) spectra from living cells provide molecular evidence of lipid accumulation in lysosomes after treatment of cultured cells with the three tricyclic antidepressants (TCA) desipramine, amitryptiline, and imipramine. The vibrational spectra elucidate to great detail and with very high sensitivity the composition of the drug-induced lipid accumulations, also observed in fixed samples by electron microscopy and X-ray nanotomography. The nanoprobes show that mostly sphingomyelin is accumulated in the lysosomes but also other lipids, in particular, cholesterol. The observation of sphingomyelin accumulation supports the impairment of the enzyme acid sphingomyelinase. The SERS data were analyzed by random forest based approaches, in particular, by minimal depth variable selection and surrogate minimal depth (SMD), shown here to be particularly useful machine learning tools for the analysis of the lipid signals that contribute only weakly to SERS spectra of cells. SMD is used for the identification of molecular colocalization and interactions of the drug molecules with lipid membranes and for discriminating between the biochemical effects of the three different TCA molecules, in agreement with their different activity. The spectra also indicate that the protein composition is significantly changed in cells treated with the drugs.

SERS Probing of Proteins in Gold Nanoparticle Agglomerates.

The collection of surface-enhanced Raman scattering (SERS) spectra of proteins and other biomolecules in complex biological samples such as animal cells has been achieved with gold nanoparticles that are introduced to the sample. As a model for such a situation, SERS spectra were measured in protein solutions using gold nanoparticles in the absence of aggregating agents, allowing for the free formation of a protein corona. The SERS spectra indicate a varied interaction of the protein molecule with the gold nanoparticles, depending on protein concentration. The concentration-dependent optical properties of the formed agglomerates result in strong variation in SERS enhancement. At protein concentrations that correspond to those inside cells, SERS signals are found to be very low. The results suggest that in living cells the successful collection of SERS spectra must be due to the positioning of the aggregates rather than the crowded biomolecular environment inside the cells. Experiments with DNA suggest the suitability of the applied sample preparation approach for an improved understanding of SERS nanoprobes and nanoparticle-biomolecule interactions in general.

Enhanced virus filtration in hybrid membranes with MWCNT nanocomposite.

Membrane separation is proved to be a powerful tool for several applications such as wastewater treatment or the elimination of various microorganisms from drinking water. In this study, the efficiency of inorganic composite-based multi-walled carbon nanotube (MWCNT) hybrid membranes was investigated in the removal of MS2 bacteriophages from contaminated water. With this object, multi-walled carbon nanotubes were coated with copper(I) oxide, titanium(IV) oxide and iron(III) oxide nanoparticles, respectively, and their virus removal capability was tested in both batch and flow experiments. Considering the possible pH range of drinking water, the filtration tests were carried out at pH 5.0, 7.5 and 9.0 as well. The extent of MS2 removal strongly depended on the pH values for each composite, which can be due to electrostatic interactions between the membrane and the virus. The most efficient removal (greater than or equal to 99.99%) was obtained with the Cu2O-coated MWCNT membrane in the whole pH range. The fabricated nanocomposites were characterized by X-ray diffraction, specific surface area measurement, dynamic light scattering, zeta potential measurement, Raman spectroscopy, transmission electron microscopy and scanning electron microscopy. This study presents a simple route to design novel and effective nanocomposite-based hybrid membranes for virus removal.

Different binding sites of serum albumins in the protein corona of gold nanoparticles.

The interaction of bovine serum albumin (BSA) and human serum albumin (HSA), sharing a sequence similarity of 77.5%, with gold nanoparticles of a size of ∼30 nm was investigated by surface-enhanced Raman scattering (SERS). The spectra provide information on those residues of the proteins in proximity of the nanoparticles. The SERS signals indicate an electrostatic interaction of both proteins with the citrate ligands at the nanoparticle surface via lysine residues. HSA, different from BSA also binds directly to the gold surface by particularly flexible protein segments that were identified by comparison of the vibrational bands with the known amino acid sequence of the molecule. The data suggest that both the direct binding as well as interaction with the citrate ligands determine the interaction, yet to varying extent in the two very similar serum proteins. This has implications for their use in bio-functionalization, and for the application of gold nanostructures in bioanalytics and medicine.