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

Abstract

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 (SPAF4) 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 are 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 minutes 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 are 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.

Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery

“…Still, most of smaller particles were not included in particle radius calculations in our study, as they co-eluted with other contaminants that absorb UV light. Our 31 and other studies namely showed that UV detector at 280 nm measures proteins 64 and lipoproteins 65 in the sample. To exclude the effect of impurities on EV quantification, we effectively included in the AF4-UV-MALS analysis only particles with 2* R geom larger than ≥ 120 nm.…”

Asymmetric flow field-flow fractionation as a multifunctional technique for the characterization of polymeric nanocarriers

“…6, AF4 can be particularly useful in these situations, being an ideal technique when it comes to fractionating complex mixtures. Numerous methods have been developed for the analysis of complete plasma or serum and can be easily applied to study drug transfer to plasma proteins [163][164][165][166][167]. As a common rule, plasma samples are fractionated in physiological buffers to preserve the structure and properties of the proteins.…”

“…AF4 presents several limitations, though: peak broadening is more accentuated than chromatography, and baseline separation is often unfeasible. Although deconvolution methods have been developed to improve peak resolution [170], more often than not it may not be possible to identify exactly the protein to which the drug is transferred: consider, for example, that albumin and high-density lipoproteins are both acceptors of lipophilic drugs, and being of similar size their peaks have a high chance of overlapping [164,167]. Once acknowledged its limitations, AF4 is a powerful tool in the study of drug release to serum proteins.…”

“…Detergents [ 163 ] or other suitable reagents such as imidazole [ 165 ] can be added to prevent non-specific interactions with the channel walls or the membrane. The identity of the proteins can be guessed by studying their size and shape factor as determined by online MALS [ 165 ] or by running protein standards [ 163 , 164 ] or, for greater accuracy, by adding a selective stain [ 168 ] or by performing HPLC-MS analyses offline [ 167 ]. The proteins with which the drug interacts can be identified by downstream analyses of the collected fractions.…”

Characterization of human plasma lipoproteins using anion exchange fast protein liquid chromatography and targeted mass spectrometry assay

“…Most proteomic studies of plasma lipoproteins rely on ultracentrifugation techniques that are time consuming (e.g., 5.7 h), demand large sample volumes (e.g., 640 μL), and damage the integrity and loss of apolipoprotein content of lipoproteins due to its high shear force [ 9 ] . Recently, there have been advancement of methods for lipoprotein fractionation such as flow field‐flow fractionation [ 10 ] . HDL subclass proteomics analysis also has revealed dynamic protein change during HDL maturation [ 11 ] .…”