Separation and detection of charged unilamellar vesicles in vacuum by a frequency-scanned quadrupole mass sensor

Extracellular vesicles (EVs) are membranous particles released by cells considered as a promising source of biomarkers for various diseases. Mass spectrometry (MS) analysis of EVs requires a sample of purified and disintegrated 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 a simplified method of vesicle separation for mass spectrometry analysis. Mass-to-charge ratio m/z separation by frequency-scanned quadrupole was employed to filter narrow size distributions of LUVs from a water sample. Lipid vesicles were charged with nano-electrospray and transferred into a vacuum using two wide m/z-range frequency-scanned quadrupoles. The m/z, charges and masses of individual vesicles were obtained by the nondestructive single-pass charge detector. The resolving regime of the second quadrupole with m/z RSD < 10% allowed to separate size selected distributions of vesicles with modal diameters 87, 111, 130, 160, 181 nm at corresponding quadrupole m/z settings 2.5×105, 5×105, 8×105, 1.5×106, 2.5×106 with detection frequencies 20-100 per minute. The presented approach for lipid vesicles separation encourages the development of new techniques for direct mass-spectrometric analysis of biomarkers in MS-separated EVs in a vacuum.