Automated on-line isolation and fractionation system for nanosized biomacromolecules from human plasma.

Multia, Evgen; Liangsupree, Thanaporn; Jussila, Matti; Ruiz-Jimenez, Jose; Kemell, Marianna; Riekkola, Marja-Liisa

An automated on-line isolation and fractionation system including controlling software was developed for selected nanosized biomacromolecules from human plasma by on-line coupled immunoaffinity chromatography - asymmetric flow field-flow fractionation (IAC - AsFlFFF). The on-line system was versatile, only different monoclonal antibodies, anti-apolipoprotein B-100, anti-CD9, or anti-CD61, were immobilized on monolithic disk columns for isolation of lipoproteins and extracellular vesicles (EVs). The platelet-derived CD61- positive EVs and CD9- positive EVs, isolated by IAC, were further fractionated by AsFlFFF to their size-based subpopulations (e.g. exomeres and exosomes) for further analysis. Field-emission scanning electron microscope elucidated the morphology of the subpopulations, and 20 free amino acids and glucose in EV subpopulations were identified and quantified in ng/mL range using hydrophilic interaction liquid chromatography - tandem mass spectrometry (HILIC-MS/MS). The study revealed that there were significant differences between EV origin and size based subpopulations. The on-line coupled IAC-AsFlFFF system was successfully programmed for reliable execution of 10 sequential isolation and fractionation cycles (37-80 min per cycle) with minimal operator involvement, minimal sample losses, and contamination. The relative standard deviations (RSD) between the cycles for human plasma samples were 0.84-6.6%.