Extracellular vesicles (EVs) are membranous particles released by cells and are considered to be promising sources of biomarkers for various diseases. Mass spectrometry (MS) analysis of EVs requires a sample of purified and detergent-lysed EVs. Purification of EVs is laborious, based on size, density, or surface nature, and requires large amounts of the source material (e.g., blood, spinal fluid). We have employed synthetically produced large unilamellar lipid vesicles (LUVs) as analogs of EVs to demonstrate an alternative approach to vesicle separation for subsequent mass spectrometry analysis of their composition. Mass-to-charge ratio m/z separation by frequency-controlled quadrupole was employed to filter narrow-size distributions of LUVs from a water sample. Lipid vesicles were positively charged with nanoelectrospray and transferred into a vacuum using two wide m/z-range frequency-controlled quadrupoles. The m/z, charges, and masses of individual vesicles were obtained by the nondestructive single-pass charge detector. The resolving mode of the second quadrupole with m/z RSD < 10% allowed to separate size selected distributions of vesicles with modal diameters of 88, 112, 130, 162, and 190 nm at corresponding quadrupole m/z settings of 2.5 × 105, 5 × 105, 8 × 105, 1.5 × 106, and 2.5 × 106, respectively with a rate of 20-100 counts per minute. The distributions of bioparticles with masses between 108 and 1010 Da were separated from human blood serum in the pilot experiment. The presented approach for lipid vesicle separation encourages the development of new techniques for the direct mass-spectrometric analysis of biomarkers in MS-separated EVs in a vacuum.

Faculty of Mathematics and Physics Charles University Prague 12116 Czechia

Institute of Chemical Technology Leipzig University Leipzig 04103 Germany

J Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences Prague 18223 Czechia

Leibniz Institute of Surface Engineering Leipzig 04318 Germany

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