The role of lipid oxidation on electrical properties of planar lipid bilayers and its importance for understanding electroporation.

Electroporation is a useful tool for the manipulation with the cell membrane permeability. Underlying physicochemical processes taking place at the molecular level during electroporation are relatively well studied. However, various processes remain unknown, one of them is lipid oxidation, a chain reaction that causes degradation of lipids, and might explain the long-lasting membrane permeability after the electric field has ceased. The aim of our study was to observe the differences in the electrical properties of planar lipid bilayers, as in vitro cell membrane models, due to lipid oxidation. Phospholipids were chemically oxidized and oxidation products were analysed using mass spectrometry. Electrical properties, resistance R (Ω) and capacitance C (F) were measured using an LCR meter. Using a previously developed measuring device, a linear increasing signal was applied to a stable bilayer in order to measure its breakdown voltage Ubr (V) and lifetime tbr (µs). We observed an increase in conductance and capacitance of the oxidized planar lipid bilayers when compared to their non-oxidized counterparts. With increasing lipid oxidation, the core of the bilayer becomes more polar, and consequently more permeable. Our findings can explain the long-lasting permeability of the cell membrane after electroporation.

Adeno-Associated Virus-like Particles' Response to pH Changes as Revealed by nES-DMA.

Gas-phase electrophoresis on a nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) separates single-charged, native analytes according to the surface-dry particle size. A volatile electrolyte, often ammonium acetate, is a prerequisite for electrospraying. Over the years, nES GEMMA has demonstrated its unique capability to investigate (bio-)nanoparticle containing samples in respect to composition, analyte size, size distribution, and particle numbers. Virus-like particles (VLPs), being non-infectious vectors, are often employed for gene therapy applications. Focusing on adeno-associated virus 8 (AAV8) based VLPs, we investigated the response of these bionanoparticles to pH changes via nES GEMMA as ammonium acetate is known to exhibit these changes upon electrospraying. Indeed, slight yet significant differences in VLP diameters in relation to pH changes are found between empty and DNA-cargo-filled assemblies. Additionally, filled VLPs exhibit aggregation in dependence on the applied electrolyte's pH, as corroborated by atomic force microscopy. In contrast, cryogenic transmission electron microscopy did not relate to changes in the overall particle size but in the substantial particle's shape based on cargo conditions. Overall, we conclude that for VLP characterization, the pH of the applied electrolyte solution has to be closely monitored, as variations in pH might account for drastic changes in particles and VLP behavior. Likewise, extrapolation of VLP behavior from empty to filled particles has to be carried out with caution.

Targeting the Structural Integrity of Extracellular Vesicles via Nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analysis (nES GEMMA).

Extracellular vesicles (EVs) are in the scientific spotlight due to their potential application in the medical field, ranging from medical diagnosis to therapy. These applications rely on EV stability during isolation and purification-ideally, these steps should not impact vesicle integrity. In this context, we investigated EV stability and particle numbers via nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) and nanoparticle tracking analysis (NTA). In nES GEMMA, native, surface-dry analytes are separated in the gas-phase according to the particle size. Besides information on size and particle heterogeneity, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU, 18 October 2011). Likewise, and in contrast to NTA, nES GEMMA enables detection of co-purified proteins. On the other hand, NTA, yielding data on hydrodynamic size distributions, is able to relate particle concentrations, omitting electrolyte exchange (and resulting EV loss), which is prerequisite for nES GEMMA. Focusing on EVs of different origin, we compared vesicles concentrations and stability, especially after electrolyte exchange and size exclusion chromatography (SEC). Co-isolated proteins were detected in most samples, and the vesicle amount varied in dependence on the EV source. We found that depletion of co-purified proteins was achievable via SEC, but was associated with a loss of EVs and-most importantly-with decreased vesicle stability, as detected via a reduced nES GEMMA measurement repeatability. Ultimately, we propose the repeatability of nES GEMMA to yield information on EV stability, and, as a result, we propose that nES GEMMA can yield additional valuable information in EV research.

Quantitative Depth Profiling Using Online-Laser Ablation of Solid Samples in Liquid (LASIL) to Investigate the Oxidation Behavior of Transition Metal Borides.

The increased demand for sustainability requires, among others, the development of new materials with enhanced corrosion resistance. Transition metal diborides are exceptional candidates, as they exhibit fascinating mechanical and thermal properties. However, at elevated temperatures and oxidizing atmospheres, their use is limited due to the fact of their inadequate oxidation resistance. Recently, it was found that chromium diboride doped with silicon can overcome this limitation. Further improvement of this protective coating requires detailed knowledge regarding the composition of the forming oxide layer and the change in the composition of the remaining thin film. In this work, an analytical method for the quantitative measurement of depth profiles without using matrix-matched reference materials was developed. Using this approach, based on the recently introduced online-LASIL technique, it was possible to achieve a depth resolution of 240 nm. A further decrease in the ablation rate is possible but demands a more sensitive detection of silicon. Two chromium diboride samples with different Si contents suffering an oxidation treatment were used to demonstrate the capabilities of this technique. The concentration profiles resembled the pathway of the formed oxidation layers as monitored with transmission electron microscopy. The stoichiometry of the oxidation layers differed strongly between the samples, suggesting different processes were taking place. The validity of the LASIL results was cross-checked with several other analytical techniques.

Fluorescence signal of proteins in birch pollen distorted within its native matrix: Identification of the fluorescence suppressor quercetin-3-O-sophoroside.

The properties of biogenic aerosol strongly depend on the particle's proteinaceous compounds. Proteins from primary biological aerosol particles (PBAPs) can cause allergic reactions in the human respiratory system or act as ice and condensation nuclei in clouds. Consequently, these particles have high impact on human health and climate. The detection of biogenic aerosol is commonly performed with fluorescence-based techniques. However, many PBAPs (i.e., pollen of birch, mugwort, or ragweed) show weak or rather low fluorescence signals in the particular protein region (λex ~ 255-280 nm, λem ~ 280-350 nm). We hypothesize that the fluorescence signal of proteins present in birch pollen is being distorted within its native matrix. In this study, we conducted in vitro quenching experiments and employed UV/Vis spectroscopy, capillary zone electrophoresis (CZE), liquid chromatography (LC), electrospray ionization mass spectrometry (ESI-MS), and multistage MS (MS2 and MS3) to target major components in birch pollen washing water (BPWW) possibly quenching the fluorescence activity of proteins and thus explaining the lack of corresponding protein fluorescent signals. We identified quercetin-3-O-sophoroside (Q3OS, MW 626 g mol-1) to be the main UV/Vis absorbing component in BPWW. Our results point out that Q3OS suppresses the fluorescence of proteins in our samples predominantly due to inner filter effects. In general, when applying fluorescence spectroscopy to analyze and detect PBAPs in the laboratory or the atmosphere, it is important to critically scrutinize the obtained spectra.

Calcium ion effect on phospholipid bilayers as cell membrane analogues.

Ion attachment can modify stability and structure of phospholipid bilayers. Of particular importance is the interaction of phospholipids with divalent cations, such as calcium ions playing an important role in numerous cellular processes. The aim of our study was to determine effects of calcium ions on phospholipid membranes employing two cell membrane analogues, liposomes and planar lipid bilayers, and for the first time the combination of two instrumental setups: gas-phase electrophoresis (nES GEMMA instrumentation) and electrical (capacitance and resistance) measurements. Liposomes and planar lipid bilayers consisted of phosphatidylcholine, cholesterol and phosphatidylethanolamine. Liposomes were prepared from dried lipid films via hydration while planar lipid bilayers were formed using a Mueller-Rudin method. Calcium ions were added to membranes from higher concentrated stock solutions. Changes in phospholipid bilayer properties due to calcium presence were observed for both studied cell membrane analogues. Changes in liposome size were observed, which might either be related to tighter packing of phospholipids in the bilayer or local distortions of the membrane. Likewise, a measurable change in planar lipid bilayer resistance and capacitance was observed in the presence of calcium ions, which can be due to an increased rigidity and tighter packing of the lipid molecules in the bilayer.

A possible role of gas-phase electrophoretic mobility molecular analysis (nES GEMMA) in extracellular vesicle research.

The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Platelet-derived extracellular vesicles (EVs)with/without additional size exclusion chromatographic (SEC) purification were subjected to nanoparticle tracking analysis (NTA) and gas-phase electrophoresis (nES GEMMA). The latter revealed presence of co-purified proteins, targetable via mass spectrometry (MS). MS also revealed that SEC did not influence EV protein content. To conclude, nES GEMMA is a valuable tool for quality control of EV-containing samples under native conditions allowing for detection of co-purified proteins from complex matrices.

Molecular weight determination of adeno-associate virus serotype 8 virus-like particle either carrying or lacking genome via native nES gas-phase electrophoretic molecular mobility analysis and nESI QRTOF mass spectrometry.

Virus-like particles (VLPs) are proteinaceous shells derived from viruses lacking any viral genomic material. Adeno-associated virus (AAV) is a non-enveloped icosahedral virus used as VLP delivery system in gene therapy (GT). Its success as vehicle for GT is due to its selective tropism, high level of transduction, and low immunogenicity. In this study, two preparations of AAV serotype 8 (AAV8) VLPs either carrying or lacking completely genomic cargo (i.e., non-viral ssDNA) have been investigated by means of a native nano-electrospray gas-phase electrophoretic mobility molecular analyzer (GEMMA) (native nES GEMMA) and native nano-electrospray ionization quadrupole reflectron time-of-flight mass spectrometry (MS) (native nESI QRTOF MS). nES GEMMA is based on electrophoretic mobility principles: single-charge nanoparticles (NPs), that is, AAV8 particle, are separated in a laminar sheath flow of dry, particle-free air and a tunable orthogonal electric field. Thus, the electrophoretic mobility diameter (EMD) of a bio-NP (i.e., diameter of globular nano-objects) is obtained at atmospheric pressure, which can be converted into its MW based on a correlation. First is the native nESI QRTOF. MS's goal is to keep the native biological conformation of an analyte during the passage into the vacuum. Subsequently, highly accurate MW values are obtained from multiple-charged species after deconvolution. However, once applied to the analysis of megadalton species, native MS is challenging and requires customized instrumental modifications not readily available on standard devices. Hence, the analysis of AAV8 VLPs via native MS in our hands did not produce a defined charge state assignment, that is, charge deconvolution for exact MW determination was not possible. Nonetheless, the method we present is capable to estimate the MW of VLPs by combining the results from native nES GEMMA and native ESI QRTOF MS. In detail, our findings show a MW of 3.7 and 5.0 MDa for AAV8 VLPs either lacking or carrying an engineered genome, respectively.

nES-DMA with Charge-reduction based on Soft X-ray Radiation: Analysis of a Recombinant Monoclonal Antibody.

Due to the fast growing importance of monoclonal antibodies in biomedical research, bioanalytics and human therapy, sensitive, fast and reliable methods are needed to monitor their production, target their characteristics, and for their final quality control. Application of a nano electrospray (nES) with soft X-ray radiation (SXR) based charge reduction and differential mobility analysis (DMA, aka nano electrospray gas-phase electrophoretic mobility molecular analysis, nES GEMMA) allows the size-separation and detection of macromolecules and (bio-)nanoparticles from a few nm up to several hundreds of nm in diameter in a native-like environment. The current study focuses on the analysis of a 148 kDa recombinant monoclonal antibody (rmAb) with the above mentioned instrumental setup and applying an universal detector, i.e. a water-based condensation particle detector (CPC). Next to the intact rmAb, its aggregates and fragment products after digestion with IdeS protease were analyzed. Additionally, influence of temperature treatment and pH variation on the stability of the rmAb was monitored. In this context, changes in electrophoretic mobility diameter (EMD) values, peak shape, and signal intensity based on particle numbers were of interest. Molecular weights calculated by application of a correlation derived from respective standard protein compounds were compared to mass spectrometric values and were found to be in good accordance. To conclude, we demonstrate that nES-DMA is a valuable tool in the characterization and quality control of rmABs.

Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy.

Adeno-associated virus (AAV)-based virus-like particles (VLPs) are thriving vectors of choice in the biopharmaceutical field of gene therapy. Here, a method to investigate purified AAV serotype 8 (AAV8) batches via a nanoelectrospray gas-phase mobility molecular analyzer (nES GEMMA), also known as an nES differential mobility analyzer, is presented. Indeed, due to AAV's double-digit nanometer scale, nES GEMMA is an excellently suited technique to determine the surface-dry particle size termed electrophoretic mobility diameter of such VLPs in their native state at atmospheric pressure and with particle-number-based detection. Moreover, asymmetric flow field-flow fractionation (AF4, also known as AFFFF) and atomic force microscopy (AFM) techniques were employed as orthogonal techniques for VLP characterization. In addition, AF4 was implemented to size-separate as well as to enrich and collect fractions of AAV8 VLPs after inducing analyte aggregation in the liquid phase. Bionanoparticle aggregation was achieved by a combination of heat and shear stress. These fractions were later analyzed with nES GEMMA (in the gas phase) and AFM (on a solid surface). Both techniques confirm the presence of dimers, trimers, and putative VLP oligomers. Last, AFM reveals even larger AAV8 VLP aggregates, which were not detectable by nES GEMMA because their heterogeneity combined with low abundance was below the limit of detection of the instrument. Hence, the combination of the employed orthogonal sizing methods with the separation technique AF4 allow a comprehensive characterization of AAV8 VLPs applied as vectors.

Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis.

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.

Online hyphenation of size-exclusion chromatography and gas-phase electrophoresis facilitates the characterization of protein aggregates.

Gas-phase electrophoresis yields size distributions of polydisperse, aerosolized analytes based on electrophoretic principles. Nanometer-sized, surface-dry, single-charged particles are separated in a high laminar sheath flow of particle-free air and an orthogonal tunable electric field. Additionally, nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) data are particle-number based. Therefore, small particles can be detected next to larger ones without a bias, for example, native proteins next to their aggregates. Analyte transition from the liquid to the gas phase is a method inherent prerequisite. In this context, nonvolatile sample buffers influence results. In the worst case, the (bio-)nanoparticle signal is lost due to an increased baseline and unspecific clustering of nonvolatile components. We present a novel online hyphenation of liquid chromatography and gas-phase electrophoresis, coupling a size-exclusion chromatography (SEC) column to an advanced nES GEMMA. Via this novel approach, it is possible to (i) separate analyte multimers already present in liquid phase from aggregates formed during the nES process, (ii) differentiate liquid phase and spray-induced multimers, and (iii) to remove nonvolatile buffer components online before SEC-nES GEMMA analysis. Due to these findings, SEC-nES GEMMA has the high potential to help to understand aggregation processes in biological buffers adding the benefit of actual size determination for noncovalent assemblies formed in solution. As detection and characterization of protein aggregation in large-scale pharmaceutical production or sizing of noncovalently bound proteins are findings directly related to technologically and biologically relevant situations, we proposed the presented method to be a valuable addition to LC-MS approaches.

N-terminal VP1 Truncations Favor T = 1 Norovirus-Like Particles.

Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Emerging genotypes, which are divided based on the sequence of the major capsid protein VP1, further enhance this public threat. Self-assembling properties of the human norovirus major capsid protein VP1 are crucial for using virus-like particles (VLPs) for vaccine development. However, there is no vaccine available yet. Here, VLPs from different variants produced in insect cells were characterized in detail using a set of biophysical and structural tools. We used native mass spectrometry, gas-phase electrophoretic mobility molecular analysis, and proteomics to get clear insights into particle size, structure, and composition, as well as stability. Generally, noroviruses have been known to form mainly T = 3 particles. Importantly, we identified a major truncation in the capsid proteins as a likely cause for the formation of T = 1 particles. For vaccine development, particle production needs to be a reproducible, reliable process. Understanding the underlying processes in capsid size variation will help to produce particles of a defined capsid size presenting antigens consistent with intact virions. Next to vaccine production itself, this would be immensely beneficial for bio-/nano-technological approaches using viral particles as carriers or triggers for immunological reactions.

Bipolar Corona Discharge-Based Charge Equilibration for Nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analysis of Bio- and Polymer Nanoparticles.

Separation of polydisperse, single-charged analytes in the nanometer size range in a high laminar sheath flow of particle-free ambient air and a tunable electric field based on the respective particle electrophoretic mobility diameter (EMD) can be achieved via gas-phase electrophoresis. In order to transfer analytes from a volatile electrolyte solution to the gas-phase as a single-charged species, a nano electrospray (nES) process followed by drying of nanodroplets and charge conditioning reaching Boltzmann charge equilibrium is a necessary prerequisite. In the case of a so-called nES gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, also known as nES differential mobility analyzer, nES DMA), charge equilibration is based on bionanoparticle interaction with a bipolar atmosphere induced, e.g., by a radioactive α-particle emitter like 210Po. It was the aim of our investigation to examine whether such a radioactive source can be easily replaced in the same nES housing by a nonradioactive one, i.e., by an AC corona discharge unit. The latter would be significantly easier to handle when compared to radioactive material in laboratory day-to-day business, waste disposal, as well as regulatory confinements. Indeed, we were able to combine a standard nES unit of our nES GEMMA instrument with a commercially available AC corona discharge device in a novel setup via an adapter. Our results show that this replacement yields very good results for a number of chemically different nanoparticles, an exemplary protein, a noncovalent protein complex, a virus-like particle, a polymer, and a liposome sample, when compared to a 210Po based bipolar charge equilibration device.

Nano electrospray differential mobility analysis based size-selection of liposomes and very-low density lipoprotein particles for offline hyphenation to MALDI mass spectrometry.

Gas-phase electrophoresis of single-charged analytes (nanoparticles) enables their separation according to the surface-dry particle size (Electrophoretic Mobility Diameter, EMD), which corresponds to the diameter of spherical shaped particles. Employing a nano Electrospray Differential Mobility Analyzer (nES DMA), also known as nES Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA), allows sizing/size-separation and determination of particle-number concentrations. Separations are based on a constant high laminar sheath flow and a tunable, orthogonal electric field enabling scanning of EMDs in the nanometer size range. Additionally, keeping the voltage constant, only nanoparticles of a given EMD pass the instrument and can be collected on corresponding supporting materials for subsequent nanoparticle analyses applying e.g. microscopic, immunologic or spectroscopic techniques. In our proof-of-concept study we now focus for the first time on mass spectrometric (MS) characterization of DMA size-selected material. We carried out size-selection of liposomes, vesicles consisting of a lipid bilayer and an aqueous lumen employed as carriers in e.g. pharmaceutic, cosmetic or nutritional applications. Particles of 85 nm EMD were collected on gold-coated silicon wafers. Subsequently, matrix was applied and Matrix-Assisted Laser Desorption / Ionization (MALDI) MS carried out. However, we not only focused on plain liposomes but also demonstrated the applicability of our approach for very heterogeneous low density lipoprotein (VLDL) particles, a transporter of lipid metabolism. Our novel offline hyphenation of gas-phase electrophoresis (termed nES DMA or nES GEMMA) and MALDI-MS opens the avenue to the molecular characterization of size-select nanoparticles of complex nature.

Nanoscale Chemical Imaging of Individual, Chemotherapeutic Cytarabine-loaded Liposomal Nanocarriers.

Dosage of chemotherapeutic drugs is a tradeoff between efficacy and side-effects. Liposomes are nanocarriers that increase therapy efficacy and minimize side-effects by delivering otherwise difficult to administer therapeutics with improved efficiency and selectivity. Still, variabilities in liposome preparation require assessing drug encapsulation efficiency at the single liposome level, an information that, for non-fluorescent therapeutic cargos, is inaccessible due to the minute drug load per liposome. Photothermal induced resonance (PTIR) provides nanoscale compositional specificity, up to now, by leveraging an atomic force microscope (AFM) tip contacting the sample to transduce the sample's photothermal expansion. However, on soft samples (e.g. liposomes) PTIR effectiveness is reduced due to the likelihood of tip-induced sample damage and inefficient AFM transduction. Here, individual liposomes loaded with the chemotherapeutic drug cytarabine are deposited intact from suspension via nES-GEMMA (nano-electrospray gas-phase electrophoretic mobility molecular analysis) collection and characterized at the nanoscale with the chemically-sensitive PTIR method. A new tapping-mode PTIR imaging paradigm based on heterodyne detection is shown to be better adapted to measure soft samples, yielding cytarabine distribution in individual liposomes and enabling classification of empty and drug-loaded liposomes. The measurements highlight PTIR capability to detect ≈ 103 cytarabine molecules (≈ 1.7 zmol) label-free and non-destructively.

Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA).

(Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation. Graphical abstract.

Native Nano-electrospray Differential Mobility Analyzer (nES GEMMA) Enables Size Selection of Liposomal Nanocarriers Combined with Subsequent Direct Spectroscopic Analysis.

Gas-phase electrophoresis employing a nano-electrospray differential mobility analyzer (nES DMA), aka gas-phase electrophoretic mobility molecular analyzer (nES GEMMA), enables nanoparticle separation in the gas-phase according to their surface-dry diameter with number-based concentration detection. Moreover, particles in the nanometer size range can be collected after size selection on supporting materials. It has been shown by subsequent analyses employing orthogonal methods, for instance, microscopic or antibody-based techniques, that the surface integrity of collected analytes remains intact. Additionally, native nES GEMMA demonstrated its applicability for liposome characterization. Liposomes are nanometer-sized, biodegradable, and rather labile carriers (nanoobjects) consisting of a lipid bilayer encapsulating an aqueous lumen. In nutritional and pharmaceutical applications, these vesicles allow shielded, targeted transport and sustained release of bioactive cargo material. To date, cargo quantification is based on bulk measurements after bilayer rupture. In this context, we now compare capillary electrophoresis and spectroscopic characterization of vesicles in solution (bulk measurements) to the possibility of spectroscopic investigation of individual, size-separated/collected liposomes after nES GEMMA. Surface-dried, size-selected vesicles were collected intact on calcium fluoride (CaF2) substrates and zinc selenide (ZnSe) prisms, respectively, for subsequent spectroscopic investigation. Our proof-of-principle study demonstrates that the off-line hyphenation of gas-phase electrophoresis and confocal Raman spectroscopy allows detection of isolated, nanometer-sized soft material/objects. Additionally, atomic force microscopy-infrared spectroscopy (AFM-IR) as an advanced spectroscopic system was employed to access molecule-specific information with nanoscale lateral resolution. The off-line hyphenation of nES GEMMA and AFM-IR is introduced to enable chemical imaging of single, i.e., individual, liposome particles.

Size and molecular weight determination of polysaccharides by means of nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA).

Size, size distribution and molecular weight (MW) determination of nanoparticles and that are for example large polymers, are of great interest and pose an analytical challenge. In this context, nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) is a valuable tool with growing impact. Separation of single-charged analytes according to their electrophoretic mobility diameter (EMD) starting from single-digit EMDs up to several hundred nm diameters is possible. In case of spherical analytes, the EMD corresponds to the dry nanoparticle size. Additionally, the instrument is capable of number-based, single-particle detection following the recommendation of the European Commission for nanoparticle characterization (2011/696/EU). In case an EMD/MW correlation for a particular compound class (based on availability of well-defined standards) exists, a nanoparticle's MW can be determined from its EMD. In the present study, we focused on nES GEMMA of linear and branched, water-soluble polysaccharides forming nanoparticles and were able to obtain spectra for both analyte classes regarding single-charged species. Based on EMDs for corresponding analytes, an excellent EMD/MW correlation could be obtained in case of the branched natural polymer (dextran). This enables the determination of dextran MWs from nES GEMMA spectra despite high analyte polydispersity and in a size/MW range, where classical mass spectrometry is limited. EMD/MW correlations based on linear (pullulans, oat-ß-glucans) polymers were significantly different, possibly indicating challenges in the exact MW determination of these compounds by, for example, chromatographic and light scattering means. Despite these observations, nES GEMMA of linear, monosaccharide-based polymers enabled the determination of size and size-distribution of such dry bionanoparticles.

Monolithic anion-exchange chromatography yields rhinovirus of high purity.

For vaccine development, 3D-structure determination, direct fluorescent labelling, and numerous other studies, homogeneous virus preparations of high purity are essential. Working with human rhinoviruses (RVs), members of the picornavirus family and the main cause of generally mild respiratory infections, we noticed that our routine preparations appeared highly pure on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), exclusively showing the four viral capsid proteins (VPs). However, the preparations turned out to contain substantial amounts of contaminating material when analyzed by orthogonal analytical methods including capillary zone electrophoresis, nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA), and negative stain transmission electron microscopy (TEM). Because these latter analyses are not routine to many laboratories, the above contaminations might remain unnoticed and skew experimental results. By using human rhinovirus serotype A2 (RV-A2) as example we report monolithic anion-exchange chromatography (AEX) as a last polishing step in the purification and demonstrate that it yields infective, highly pure, virus (RV-A2 in the respective fractions was confirmed by peptide mass fingerprinting) devoid of foreign material as judged by the above criteria.