This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.

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.

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.

Dextran is widely exploited in medical products and as a component of drug-delivering nanoparticles (NPs). Here, we tested whether dextran can serve as the main substrate of NPs and form a stable backbone. We tested dextrans with several molecular masses under several synthesis conditions to optimize NP stability. The analysis of the obtained nanoparticles showed that dextran NPs that were synthesized from 70 kDa dextran with a 5% degree of oxidation of the polysaccharide chain and 50% substitution with dodecylamine formed a NP backbone composed of modified dextran subunits, the mean diameter of which in an aqueous environment was around 100 nm. Dextran NPs could be stored in a dry state and reassembled in water. Moreover, we found that different chemical moieties (e.g., drugs such as doxorubicin) can be attached to the dextran NPs via a pH-dependent bond that allows release of the drug with lowering pH. We conclude that dextran NPs are a promising nano drug carrier.

We present a modelled approach of scattering contribution to the radiation transmission through a scattering medium, such as an aerosol, yielding a correction term to the Lambert-Beer law. The correction is essential because a certain amount of the forward scattered light flux is always overlaid on the transmitted radiation. Hence it enters together with the attenuated beam into the finite aperture of any detector system and therefore constitutes a potential problem in the inversion of measured data. This correction depends not only on the geometry of the measuring system but also substantially on the optical depth of the medium. We discuss the numerical analysis of the magnitude and functional behaviour of the scattering correction for a number of important measuring parameters and we give a simple approximation for the determination of the range of applicability of the scattering correction for single scattering conditions. Finally, we show that the derived expressions yield useful values of optical depths at which non-negligible multiple scattering effects occur.

Differential mobility analysis (DMA) is a technique suited for size analysis as well as preparative collection of airborne nanosized airborne particles. In the recent decade, the analysis of intact viruses, proteins, DNA fragments, polymers, and inorganic nanoparticles was possible when combining this method with a nano-electrospray charge-reduction source for producing aerosols from a sample solution/suspensions. Mass analysis of high molecular weight noncovalent complexes is also possible with this methodology due to the linear correlation of the electrophoretic mobility diameter and the molecular mass. In this work, we present the analysis (size and molecular mass) of high molecular weight multimers (noncovalent functional homocomplex) of Jack bean urease in a mass range from 275 kDa up to 2.5 MDa, with mainly present tri-and hexamers but also higher oligomers of the 91 kDa monomer subunit. In a second experiment, the size analysis of intact very-low-density (ϳ35 nm), low-density (ϳ22 nm) and high-density lipoparticles (ϳ10 nm), which are heterocomplexes consisting of cholesterol, lipids, and proteins in different ratios, is presented. Results from mobility analysis were in excellent agreement with particle diameters found in literature. The last presented experiment demonstrates size analysis of a rod-like virus and selective sampling of a selected size fraction of electrosprayed, singly-charged tobacco mosaic virus particles. Sampling and subsequent transmission electron microscopic investigations of a specific size fraction (40 nm electrophoretic mobility diameter) revealed the folding of virus particles during the electrospray and charge reduction (electrical stress) as well as solvent evaporation (mechanical stress) process, leading to an observed geometry of 150 (length) ϫ 35 (width) nm (average cylindrical geometry of unsprayed intact virus 300 ϫ 18 nm). and viruses. Analysis of the formation process, stoichiometry, and molecular weight of those complexes can be very challenging as noncovalent interactions can easily be destroyed or biased when leaving specific native conditions [5], and as their molecular mass can easily exceed the working range of modern mass spectrometry. For these reasons, a demand for new analytical techniques, which preserve noncovalent interactions, deliver molecular mass and/or size related information (in contrast to capillary electrophoresis and native gel electrophoresis) and which have a working range that exceed conventional mass spectrometry, exists.Differential mobility analysis (DMA) is a technique developed to classify charged aerosolized particles under ambient pressure according to their electrophoretic mobility diameter [6]. In the recent decade, its working range was extended from m down to the nm size range [7], or in terms of molecular mass, into the kDa to GDa molecular mass range, thus closing the gap between classic aerosol particle technology (low m into mm range) and mass spectrometry (sub-nm to 10 nm). The size range of 10 to 200 nm is therefore of great in...

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The performance of a narrow-angle and a wide-angle, forward scattering laser aerosol spectrometer has been studied as a function of particle size and refractive index. The results have been compared with theoretical calculations based on light scattering theory. The results indicate that for the narrow-angle instrument, the scattered-lighl intensity is not a monotonic function of particle size for transparent particles (a monotonic relationship is required for unambiguous particle size measurement) above 0.7 pm. The instrument is therefore limited in its useful range to size distribution measurement between 0.2 pmits lower particle size limitand 0.7 pm for transparent particles. In the case of the wide-angle instrument, the instrument output is a monotonic function of particle size for transparent particles, but the output is severely attenuated for light absorbing particles above 0.3 pm. The instrument, therefore, cannot be used for accurate size measurements above 0.3 pm for light absorbing particles.