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

Abstract

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.

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

“…Hence, based on a protein EMD value, its M W can be calculated in good approximation. Similar approaches have been demonstrated, e.g., for intact viruses [4] and polysaccharides [32]. It is of note that for the latter two, the obtained EMD/M W correlations deviated from the protein case.…”

“…3). Notably, this deviates from a correlation for proteins as was the case for filled virions [32]. Hence, a basic knowledge concerning the analyte class prior EMD-based M W calculation (but not for gas-phase electrophoresis itself) is necessary.

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“…Simply because VLPs are analyzed in an EMD/M W range, in which pure protein complexes (in a non-aggregated or structured form) rarely exist, the extrapolation of the protein EMD/M W correlation to larger EMD and M W values has to be taken with extreme caution. It is of note that the largest protein analyzed in [32] was the octamer of β-galactosidase with an EMD of 16.83 nm and a M W of 931.28 kDa.…”

“…At least N = 3 measurements were considered per EMD value. M W values and data for instrument A either taken from [32] or Table 1. M monomer, D dimer; errors provided are standard deviations…”

A New Role of Biological Macromolecules as Reference Materials for the Sub-10 nm Calibration for the Real-time Electrophoretic ES-SMPS

“…The correlation between mobility size and molecular weight of biomolecules has been reported. [6,13], and the aggregate of proteins can accordingly characterized [14,15]. Another advantage of the nanoscale ES-SMPS is the real time scanning of sample, which make the technique fascinating for biomanufacturing process, nanomedicine and pharmaceutical development.…”