Nano-electrospray Gas-phase Electrophoretic Mobility Research Articles

Exploring the manganese-dependent interaction between a transcription factor and its corresponding DNA: insights from gas-phase electrophoresis on a nES GEMMA instrument

Manganese ion homeostasis is vital for bacteria and is achieved via manganese-dependent transcription factors. Manganese mediation of transcription factor attachment to the corresponding oligonucleotide sequences can be investigated, e.g. via electrophoretic mobility shift assays (EMSA). Formation of specific biocomplexes leads to differences in the migration pattern upon gel electrophoresis. Focusing on electrophoresis in the gas-phase, applying a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) also known as nES differential mobility analyzer (nES DMA), and on transcription factors (MntR proteins) from Bacillus subtilis and Mycobacterium tuberculosis, we took interest in the gas-phase electrophoresis of the corresponding biospecific complexes. We compared nES GEMMA, separating analytes in the nanometer regime (a few to several hundred nm in diameter) in the gas-phase in their native state according to particle size, to EMSA data. Indeed we were able to demonstrate manganese-mediated attachment of MntR to target genomic sequences with both analytical techniques. Despite some inherent pitfalls of the nES GEMMA method like analyte/instrument surface interactions, we were able to detect the target complexes. Moreover, we were able to calculate the molecular weight (MW) of the obtained species by application of a correlation function based on nES GEMMA obtained data. As gas-phase electrophoresis also offers the possibility of offline hyphenation to orthogonal analysis techniques, we are confident that nES GEMMA measurements are not just complementary to EMSA, but will offer the possibility of further in-depth characterization of biocomplexes in the future.Graphical

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.

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.Graphical abstractPlatelet-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.

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.

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

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.

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.

NES GEMMA Analysis of Lectins and Their Interactions with Glycoproteins \u2013 Separation, Detection, and Sampling of Noncovalent Biospecific Complexes

In order to better understand biological events, lectin–glycoprotein interactions are of interest. The possibility to gather more information than the mere positive or negative response for interactions brought mass spectrometry into the center of many research fields. The presented work shows the potential of a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) to detect weak, noncovalent, biospecific interactions besides still unbound glycoproteins and unreacted lectins without prior liquid phase separation. First results for Sambucus nigra agglutinin, concanavalin A, and wheat germ agglutinin and their retained noncovalent interactions with glycoproteins in the gas phase are presented. Electrophoretic mobility diameters (EMDs) were obtained by nES GEMMA for all interaction partners correlating very well with molecular masses determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the individual molecules. Moreover, EMDs measured for the lectin–glycoprotein complexes were in good accordance with theoretically calculated mass values. Special focus was laid on complex formation for different lectin concentrations and binding specificities to evaluate the method with respect to results obtained in the liquid phase. The latter was addressed by capillary electrophoresis on-a-chip (CE-on-a-chip). Of exceptional interest was the fact that the formed complexes could be sampled according to their size onto nitrocellulose membranes after gas-phase separation. Subsequent immunological investigation further proved that the collected complex actually retained its native structure throughout nES GEMMA analysis and sampling.Graphical ᅟ

Combining gas-phase electrophoretic mobility molecular analysis (GEMMA), light scattering, field flow fractionation and cryo electron microscopy in a multidimensional approach to characterize liposomal carrier vesicles

For drug delivery, characterization of liposomes regarding size, particle number concentrations, occurrence of low-sized liposome artefacts and drug encapsulation are of importance to understand their pharmacodynamic properties. In our study, we aimed to demonstrate the applicability of nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyser (nES GEMMA) as a suitable technique for analyzing these parameters. We measured number-based particle concentrations, identified differences in size between nominally identical liposomal samples, and detected the presence of low-diameter material which yielded bimodal particle size distributions. Subsequently, we compared these findings to dynamic light scattering (DLS) data and results from light scattering experiments coupled to Asymmetric Flow-Field Flow Fractionation (AF4), the latter improving the detectability of smaller particles in polydisperse samples due to a size separation step prior detection. However, the bimodal size distribution could not be detected due to method inherent limitations. In contrast, cryo transmission electron microscopy corroborated nES GEMMA results. Hence, gas-phase electrophoresis proved to be a versatile tool for liposome characterization as it could analyze both vesicle size and size distribution. Finally, a correlation of nES GEMMA results with cell viability experiments was carried out to demonstrate the importance of liposome batch-to-batch control as low-sized sample components possibly impact cell viability.

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.

Comparative method evaluation for size and size‐distribution analysis of gold nanoparticles

Gold nanoparticles (GNPs) are popular colloidal substrates in various sensor, imaging, and nanomedicine applications. In separation science, they have raised some interest as a support for sample preparation. Reasons for their popularity are their low cost, ability for size-controlled synthesis with well-defined narrow nanoparticle size distributions, as well as straightforward surface functionalization by self-assembling (thiol-containing) molecules on the surface, which allows flexible introduction of functionalities for the selective capture of analytes. Most commonly, the method of first choice for size determination is dynamic light scattering (DLS). However, DLS has some serious shortcomings, and results from DLS may be misleading. For this reason, in this contribution several distinct complementary nanoparticle sizing methodologies were utilized and compared to characterize citrate-capped GNPs of different diameters in the range of 13-26 nm. Weaknesses and strengths of DLS, transmission electron microscopy, asymmetrical-flow field-flow fractionation and nanoelectrospray gas-phase electrophoretic mobility molecular analysis are discussed and the results comparatively assessed. Furthermore, the distinct GNPs were characterized by measuring their zeta-potential and surface plasmon resonance spectra. Overall, the combination of methods for GNP characterization gives a more realistic and comprehensive picture of their real physicochemical properties, (hydrodynamic) diameter, and size distribution.

PEGylated recombinant von Willebrand factor analyzed by means of MALDI-TOF-MS, CGE-on-a-chip and nES-GEMMA

Recombinant VWF (rVWF) is a candidate for therapy of von Willebrand disease and the largest known multimeric glycoprotein. In this study rVWF was covalently linked with a 20 kDa branched polyethylene glycol (PEG) to obtain PEGylated rVWF (PEGrVWF). This conjugation results in a further increase of heterogeneity besides glycoheterogeneity and a challenge in analyzing of such a bioconjugate, particular when investigated on the intact molecule level. Four different techniques including SDS-PAGE, MALDI-TOF-MS, capillary-gel-electrophoresis-on-a-chip (CGE-on-a-chip) and nano electrospray gas-phase electrophoretic mobility molecular analysis (nES-GEMMA) were applied to determine the molecular weight (MW) and the PEGylation degree of the monomeric rVWF. The degree and distribution of PEGylation of rVWF obtained by CGE-on-a-chip were in good agreement with results obtained by MALDI-TOF-MS with a special high mass detector. An average PEGylation degree of 3.1 PEG chains coupled to the monomeric glycoforms was found. MW determination by MALDI-TOF-MS (317.4 ± 1.0 kDa; 3 PEG chains attached) showed in comparison to CGE-on-a-chip (413.4 ± 2.1 kDa) the highest precision. Furthermore the orthogonal method, nES-GEMMA provided first information on the globular size (11.3 ± 0.1 nm) and based on that the MW (251 ± 7.2 kDa for the average PEGylation) of the PEGrVWF.

A comparison of nano-electrospray gas-phase electrophoretic mobility macromolecular analysis and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry for the characterization of the recombinant coagulation glycoprotein von W

Von Willebrand factor (VWF), an adhesive glycoprotein with an approximate molecular weight (MW) of the monomer of 260 kDa, circulates in human blood plasma as a series of multimers ranging in size up to 20.000 kDa; thus the determination of the accurate MW of the monomer is of great importance and due to its high MW quite challenging. In this study accurate MW determination of intact recombinant VWF monomer (rVWF) was performed with GEMMA (gas-phase electrophoretic mobility macromolecular analysis) and MALDI TOF MS (matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry). Three rVWF preparations with differing buffer systems and glycoprotein concentrations were analyzed. First investigations directed towards heterogeneity determination by means of capillary gel electrophoresis (CGE)-on-the-chip with a laser-induced fluorescence detector revealed two compounds (MW of 277 kDa (migration time 44.3 s) and 341 kDa (migration time 49.5 s)) present in each sample to varying extents, namely mature and pro-rVWF. MALDI MS analysis in the linear positive ion mode allowed the detection of mature rVWF with an exact MW of 256.1 kDa (+/-0.8%) and pro-rVWF with a MW of 349.8 kDa (+/-0.8%). Two samples containing pro-rVWF in very minor concentration resulted in GEMMA detection of the mature rVWF with a MW of 227.4 kDa (+/-2.5%), derived from the measured globular size of 10.9 nm. For one sample containing both rVWF species in almost equal concentrations no differentiation of the two species was possible with GEMMA. Due to its lower resolution only a peak representing a mixture of both species at 11.8 nm could be observed, yielding a MW of 298.8 kDa (+/-1.6%).

Determination of Molecular Weight, Particle Size, and Density of High Number Generation PAMAM Dendrimers Using MALDI−TOF−MS and nES−GEMMA

In this work we present the characterization of PAMAM dendrimers from generation two (G2) up to ten (G10) with a focus on the G5−G10 dendrimers with matrix-assisted laser desorption/ionization linear mass spectrometry (MALDI−MS) and nanoelectrospray gas-phase electrophoretic mobility molecular analysis (nES−GEMMA). For the first time the molecular masses of high-mass dendrimers G8−G10 were determined by MALDI−MS and nES−GEMMA, techniques which are based on different physicochemical principles. Obtained experimental data allows the determination of the molecular mass (up to 580 kDa with a precision below ±0.9%), of the spherical size (from 3.3 to 14.0 nm with a precision of ±0.2 nm) and the calculation of their densities. Amounts in the nanogramm range were sufficient for an analysis that could be performed within several minutes. The results based on these methods for high-generation dendrimers exhibited an excellent correlation and were compared with published data using techniques based on different principles.