Spesyvyi Anatolii, Cebecauer Marek, Žabka Ján, Olżyńska Agnieszka, Malečková Michaela, Johanovská Zuzana, Polášek Miroslav, Charvat Ales, Abel Bernd
J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Prague 18223, Czechia.
Faculty of Mathematics and Physics, Charles University, Prague 12116, Czechia.
Anal Chem. 2025 May 6;97(17):9131-9138. doi: 10.1021/acs.analchem.4c05730. Epub 2025 Apr 22.
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 / 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 /-range frequency-controlled quadrupoles. The /, charges, and masses of individual vesicles were obtained by the nondestructive single-pass charge detector. The resolving mode of the second quadrupole with / RSD < 10% allowed to separate size selected distributions of vesicles with modal diameters of 88, 112, 130, 162, and 190 nm at corresponding quadrupole / settings of 2.5 × 10, 5 × 10, 8 × 10, 1.5 × 10, and 2.5 × 10, respectively with a rate of 20-100 counts per minute. The distributions of bioparticles with masses between 10 and 10 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.
细胞外囊泡（EVs）是细胞释放的膜性颗粒，被认为是各种疾病生物标志物的有前景来源。对EVs进行质谱（MS）分析需要纯化且经去污剂裂解的EVs样本。基于大小、密度或表面性质对EVs进行纯化很费力，且需要大量的源材料（如血液、脑脊液）。我们使用合成产生的大单层脂质囊泡（LUVs）作为EVs的类似物，以展示一种用于囊泡分离的替代方法，以便随后对其组成进行质谱分析。采用质荷比/通过频率控制四极杆进行分离，从水样中过滤出窄尺寸分布的LUVs。脂质囊泡通过纳米电喷雾带正电荷，并使用两个宽范围频率控制四极杆转移到真空中。通过无损单通道电荷探测器获得单个囊泡的质荷比、电荷和质量。第二个四极杆的分辨率模式（相对标准偏差<RSD<10%）允许在相应的四极杆设置分别为2.5×10、5×10、8×10、1.5×10和2.5×10时，以每分钟20 - 100次计数的速率分离模态直径为88、112、130、162和190 nm的囊泡的大小选择分布。在初步实验中，从人血清中分离出了质量在10到10 Da之间的生物颗粒分布。所提出的脂质囊泡分离方法促进了用于在真空中对MS分离的EVs中的生物标志物进行直接质谱分析的新技术的开发。
