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.

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.

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.

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.

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.

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.

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.

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.

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.

Microchip capillary gel electrophoresis combined with lectin affinity enrichment employing magnetic beads for glycoprotein analysis.

Due to the constant search for reliable methods to investigate glycoproteins in complex biological samples, an alternative approach combining affinity enrichment with rapid and sensitive analysis on-a-chip is presented. Glycoproteins were specifically captured by lectin-coated magnetic beads, eluted by competitive sugars, and investigated with microchip capillary gel electrophoresis (MCGE), i.e., CGE-on-a-chip. We compared our results to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data, which turned out to be in very good agreement. While SDS-PAGE offers the possibility of subsequent mass spectrometric analysis of captured and separated analytes, MCGE scores with time savings, higher throughput, and lower sample consumption as well as quality control (QC) and process analytical technology (PAT) applicability. Due to these advantages, a lectin-based glycoprotein capture protocol can easily be optimized. In our case, two different types of magnetic beads were tested and compared regarding lectin binding. The selectivity of our strategy was demonstrated with a set of model glycoproteins, as well as with human serum and serum depleted from high-abundance proteins. The specificity of the capturing method was investigated revealing to a certain degree an unspecific binding between each sample and the beads themselves, which has to be considered for any specific enrichment and data interpretation. In addition, two glycoproteins from Trichoderma atroviride, a fungus with mycoparasitic activity and only barely studied glycoproteome, were enriched by means of a lectin and so identified for the first time. Graphical abstract Glycoproteins from biological samples were detected by microchip capillary gel electrophoresis after lectin affinity enrichment using magnetic beads and elution with respective competitive monosaccharides.

Nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) of liposomes: applicability of the technique for nano vesicle batch control.

Liposomes are biodegradable nanoparticle vesicles consisting of a lipid bilayer encapsulating an aqueous core. Entrapped cargo material is shielded from the extra-vesicular medium and sustained release of encapsulated material can be achieved. However, application of liposomes as nano-carriers demands their characterization concerning size and size distribution, particle-number concentration, occurrence of vesicle building blocks in solution and determination of the resulting vesicle encapsulation capacity. These questions can be targeted via gas-phase electrophoretic mobility molecular analysis (GEMMA) based on a nano electrospray (nES) charge-reduction source. This instrument separates single-charged nanoparticles in the gas-phase according to size in a high-laminar sheath-flow by means of an orthogonal, tunable electric field. nES GEMMA analysis enables to confirm liposome integrity after passage through the instrument (in combination with atomic force microscopy) as well as to exclude vesicle aggregation. Additionally, nanoparticle diameters at peak apexes and size distribution data are obtained. Differences of hydrodynamic and dry particle diameter values, as well as the effect of number- and mass-based concentration data analysis on obtained liposome diameters are shown. Furthermore, the repeatability of liposome preparation is studied, especially upon incorporation of PEGylated lipids in the bilayer. Finally, the instruments applicability to monitor mechanical stress applied to vesicles is demonstrated.

In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis.

Liquid-phase electrophoresis either in the classical capillary format or miniaturized (chip CE) is a valuable tool for quality control of virus preparations and for targeting questions related to conformational changes of viruses during infection. We present an in vitro assay to follow the release of the RNA genome from a human rhinovirus (common cold virus) by using a molecular beacon (MB) and chip CE. The MB, a probe that becomes fluorescent upon hybridization to a complementary sequence, was designed to bind close to the 3' end of the viral genome. Addition of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known additive for reduction of bleaching and blinking of fluorophores in fluorescence microscopy, to the background electrolyte increased the sensitivity of our chip CE set-up. Hence, a fast, sensitive and straightforward method for the detection of viral RNA is introduced. Additionally, challenges of our assay will be discussed. In particular, we found that (i) desalting of virus preparations prior to analysis increased the recorded signal and (ii) the MB-RNA complex signal decreased with the time of virus storage at -70 °C. This suggests that 3'-proximal sequences of the viral RNA, if not the whole genome, underwent degradation during storage and/or freezing and thawing. In summary, we demonstrate, for two independent virus batches, that chip electrophoresis can be used to monitor MB hybridization to RNA released upon incubation of the native virus at 56 °C. Graphical Abstract Schematic of the study strategy: RNA released from HRV-A2 is detected by chip electrophoresis through the increase in fluorescence after genom complexation to a cognate molecular beacon.

Analysis of a common cold virus and its subviral particles by gas-phase electrophoretic mobility molecular analysis and native mass spectrometry.

Gas-phase electrophoretic mobility molecular analysis (GEMMA) separates nanometer-sized, single-charged particles according to their electrophoretic mobility (EM) diameter after transition to the gas-phase via a nano electrospray process. Electrospraying as a soft desorption/ionization technique preserves noncovalent biospecific interactions. GEMMA is therefore well suited for the analysis of intact viruses and subviral particles targeting questions related to particle size, bioaffinity, and purity of preparations. By correlating the EM diameter to the molecular mass (Mr) of standards, the Mr of analytes can be determined. Here, we demonstrate (i) the use of GEMMA in purity assessment of a preparation of a common cold virus (human rhinovirus serotype 2, HRV-A2) and (ii) the analysis of subviral HRV-A2 particles derived from such a preparation. (iii) Likewise, native mass spectrometry was employed to obtain spectra of intact HRV-A2 virions and empty viral capsids (B-particles). Charge state resolution for the latter allowed its Mr determination. (iv) Cumulatively, the data measured and published earlier were used to establish a correlation between the Mr and EM diameter for a range of globular proteins and the intact virions. Although a good correlation resulted from this analysis, we noticed a discrepancy especially for the empty and subviral particles. This demonstrates the influence of genome encapsulation (preventing analytes from shrinking upon transition into the gas-phase) on the measured analyte EM diameter. To conclude, GEMMA is useful for the determination of the Mr of intact viruses but needs to be employed with caution when subviral particles or even empty viral capsids are targeted. The latter could be analyzed by native MS.

Challenges of glycoprotein analysis by microchip capillary gel electrophoresis.

Glycosylations severely influence a protein's biological and physicochemical properties. Five exemplary proteins with varying glycan moieties were chosen to establish molecular weight (MW) determination (sizing), quantitation, and sensitivity of detection for microchip capillary gel electrophoresis (MCGE). Although sizing showed increasing deviations from literature values (SDS-PAGE or MALDI-MS) with a concomitant higher degree of analyte glycosylation, the reproducibility of MW determination and accuracy of quantitation with high sensitivity and reliability were demonstrated. Additionally, speed of analysis together with the low level of analyte consumption render MCGE attractive as an alternative to conventional SDS-PAGE.

Chip electrophoretic separation of highly homologous ammodytoxin isoforms: three neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom.

Ammodytoxins (Atxs), a group of Ca(2+) -dependent neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom, are mainly responsible for venom toxicity. Within the Atx group, LD50 values between three isoforms, A, B, and C are differing with AtxA exhibiting an LD50 value by an order of magnitude lower (more toxic) than the other two isoforms. This difference in toxicity justifies the necessity to prepare suitable antibodies and thus isoform separation to characterize the Atx content of Vipera ammodytes ammodytes venom is of importance. However, a high homology between the three Atx isoforms (differences in only two, respectively, three residues within the last 18 amino acids at the C-terminus, total length 122 residues) hindered the successful separation of isoforms to date. As the investigated phospholipases A2 were reported to exhibit differences in pI values, we concentrate with the current work on the separation of Atx isoforms after fluorescence labeling via chip electrophoresis on a commercially available instrument to build the basis for a fast and easy to handle screening method. In the course of our work, we were able to show that samples of AtxA, AtxB, and AtxC declared to be homogenous by standard analytical techniques consisted indeed of more than one isoform of which the relative amounts were calculated by using the newly developed method.

Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids.

The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions ('spikes') on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the 'α-determinant') or in the 'floor' regions between them (the 'ß-determinant'). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the ß-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes.

Chip electrophoresis of gelatin-based nanoparticles.

Recently, biodegradable nanoparticles received increasing attention for pharmaceutical applications as well as applications in the food industry. With the current investigation we demonstrate chip electrophoresis of fluorescently (FL) labeled gelatin nanoparticles (gelatin NPs) on a commercially available instrument. FL labeling included a step for the removal of low molecular mass material (especially excess dye molecules). Nevertheless, for the investigated gelatin NP preparation two analyte peaks, one very homogeneous with an electrophoretic net mobility of µ = -24.6 ± 0.3 × 10(-9) m(2) /Vs at the peak apex (n = 17) and another more heterogeneous peak with µ between approximately -27.2 ± 0.2 × 10(-9) m(2) /Vs and -36.6 ± 0.2 × 10(-9) m(2) /Vs at the peak beginning and end point (n = 11, respectively) were recorded. Filtration allowed enrichment of particles in the size range of approximately 35 nm (pore size employed for concentration of gelatin NPs) to 200 nm (pore size employed during FL labeling). This corresponded to the very homogeneous peak linking it to gelatin NPs, whereas the more heterogeneous peak probably corresponds to gelatin not cross-linked to such a high degree (NP building blocks). Several further gelatin NP preparations were analyzed according to the same protocol yielding peaks with electrophoretic net mobilities between -23.3 ± 0.3 × 10(-9) m(2) /Vs and -28.9 ± 0.2 × 10(-9) m(2) /Vs at peak apexes (n = 15 and 6). Chip electrophoresis allows analyte separation in less than two minutes (including electrophoretic sample injection). Together with the high sensitivity of the FL detection - the LOD as derived for the first main peak of the applied dye from the threefold standard deviation of the background noise values 80 pM for determined separation conditions - this leads to a very promising high throughput separation technique especially for the analysis of bionanoparticles. For gelatin NP preparations, chip electrophoresis allows for example the comparison of preparation batches concerning the amount of NPs and gelatin building blocks as well as the indirect assessment of the degree of gelatin cross-linking (from obtained FL signals).

Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas phase electrophoretic mobility molecular analysis: part II.

Human rhinoviruses (HRVs) are valuable tools in the investigation of early viral infection steps due to their far reaching (although still incomplete) characterization. During endocytosis, native virions first loose one of the four capsid proteins (VP4); corresponding particles sediment at 135S and were termed subviral A particles. Subsequently, the viral RNA genome leaves the viral shell giving rise to empty capsids. In continuation of our previous work with HRV serotype 2 (HRV2) intermediate subviral particles, in which we were able to discriminate by CE even between two intermediates (AI and AII) of virus uncoating, we further concentrated on the characterization of AI particles with the electrophoretic mobility of around -17.2 × 10(-9) m(2) /Vs at 20°C. In the course of our present work we related these particles to virions as previously described at the subviral A stage of uncoating (and as such sedimenting at 135S) by determination of their protein and RNA content--in comparison to native virions AI particles did not include VP4, however, still 93% of their initial RNA content. Binding of an mAb specific for subviral particles demonstrated antigenic rearrangements on the capsid surface at the AI stage. Furthermore, we investigated possible factors stabilizing intermediates of virus uncoating. We could exclude the influence of the previously suspected so-called contaminant of virus preparation on HRV2 subviral particle formation. Instead, we regarded other factors being part of the virus preparation system and found a dependence of AI particle formation on the presence of divalent cations.

Characterization of cross-linked gelatin nanoparticles by electrophoretic techniques in the liquid and the gas phase.

Biodegradable nanoparticles (NPs) and hence e.g. NPs prepared from glutaraldehyde crosslinked gelatin (gelatin NPs) are lately receiving increased attention in various fields like pharmaceutical technology and nutraceutics as biocompatible carriers for hardly water soluble drugs, molecules intended for sustained release or targeted transport. However, in vivo application of such materials requires a thoroughly characterization of corresponding particles. In a previous manuscript we demonstrated the applicability of chip electrophoresis for the separation of gelatin NPs from NP building blocks. Following our previous results we intensified our efforts in the characterization of gelatin NPs by electrophoresis in the liquid (capillary and chip format) and the gas phase (gas phase electrophoretic mobility molecular analysis, GEMMA). In doing so, we demonstrated differences between nominally comparable (from the concentration of initially employed material for crosslinking) gelatin NP preparation batches concerning (i) the amount of obtained NPs, (ii) the degree of NP crosslinking, (iii) the amount of NP building blocks present within samples and (iv) the electrophoretic mobility diameter of NPs. Differences were even more pronounced when NP preparations from batches with different content of initially employed gelatin were compared. Additionally, we compared three setups for the removal of low molecular weight components from samples after fluorescence labeling of NPs. In overall, the combination of the three employed analytical methods for gelatin NP characterization - CE in the capillary and the chip format as well as GEMMA - allows a more thoroughly characterization of NP containing samples.

In situ assembly of macromolecular complexes triggered by light.

Chemical biology aims for a perfect control of protein complexes in time and space by their site-specific labeling, manipulation, and structured organization. Here we developed a self-inactivated, lock-and-key recognition element whose binding to His-tagged proteins can be triggered by light from zero to nanomolar affinity. Activation is achieved by photocleavage of a tethered intramolecular ligand arming a multivalent chelator head for high-affinity protein interaction. We demonstrate site-specific, stable, and reversible binding in solution as well as at interfaces controlled by light with high temporal and spatial resolution. Multiplexed organization of protein complexes is realized by an iterative in situ writing and binding process via laser scanning microscopy. This light-triggered molecular recognition should allow for a spatiotemporal control of protein-protein interactions and cellular processes by light-triggered protein clustering.