Native Nanoelectrospray Analysis of VLPs

In order to enable native nES-based analysis of VLPs, employed storage buffers (often including additional salt components or stabilizing agents) had to be replaced by a volatile electrolyte solution. Else, these additional sample components were shown to lead to an increased peak heterogeneity of the analytes of interest and, in nES GEMMA, an elevated baseline resulting from clustering of small, non-volatile molecules during the nES process [55 (link)]. As in previous studies, we opted for ammonium acetate and carried out removal of small, buffer-associated sample components via spin filtration [42 (link)] employing 10 kDa M_W cutoff filters (Vivaspin-polyethersulfone membrane, Vivacon-regenerated cellulose membrane-both from Sartorius or centrifugal filters-polyethersulfone membrane, VWR, Vienna, Austria). Between 3 and 5 filtration steps were necessary to remove non-volatile additional sample components. Sample concentration for measurements was typically well below 1 mg/mL protein content (based on originally determined values and sample dilution).