Asymmetrical flow field-flow fractionation for improved characterization of human plasma lipoproteins.

High- and low-density lipoproteins (HDL and LDL) are attractive targets for biomarker discovery. However, ultracentrifugation (UC), the current methodology of choice for isolating HDL and LDL, is tedious, requires large sample volume, results in sample loss, and does not readily provide information on particle size. In this work, human plasma HDL and LDL are separated and collected using semi-preparative asymmetrical flow field-flow fractionation (SP-AF4) and UC. The SP-AF4 and UC separation conditions, sample throughput, and liquid chromatography/mass spectrometry (LC/MS) lipidomic results are compared. Over 600 μg of total proteins is recovered in a single SP-AF4 run, and Western blot results confirm apoA1 pure and apoB100 pure fractions, consistent with HDL and LDL, respectively. The SP-AF4 separation requires ~ 60 min per sample, thus providing a marked improvement over UC which can span hours to days. Lipidome analysis of SP-AF4-prepared HDL and LDL fractions is compared to UC-prepared HDL and LDL samples. Over 270 lipids in positive MS mode and over 140 lipids in negative MS mode are identified by both sample preparation techniques with over 98% overlap between the lipidome. Additionally, lipoprotein size distributions are determined using analytical scale AF4 coupled with multiangle light scattering (MALS) and dynamic light scattering (DLS) detectors. These developments position SP-AF4 as a sample preparation method of choice for lipoprotein biomarker characterization and identification. Graphical abstract ᅟ.