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.
KeywordsAsymmetrical flow field-flow fractionation Ultracentrifugation Lipoproteins Lipidomics Mass spectrometry
Carmen R. M. Bria and Farsad Afshinnia contributed equally to this work. Carmen R. M. Bria performed semi-preparative and analytical scale asymmetrical flow field-flow fraction and light scattering experiments and was involved in experimental design, data interpretation, and manuscript preparation. Patrick W. Skelly performed semi-preparative asymmetrical flow field-flow fractionation experiments. Farsad Afshinnia contributed to the experimental design, preparation of LDL and HDL lipoprotein standards, protein quantification, Western blot, mass spectrometry run and data acquisition, analysis, interpretation, and drafting of the manuscript. Thekkelnaycke M. Rajendiran contributed to mass spectrometry analysis, interpretation of the lipidomic results, and drafting of corresponding analytical aspects of the manuscript. Pradeep Kayampilly contributed to lipoprotein Western blot, interpretation of the corresponding results, and writing of the manuscript. Thommey P. Thomas contributed to developing methodology for lipoprotein isolation, purity analysis and protein quantification and data interpretation, and drafting of manuscript. Kim Ratanathanawongs Williams, Victor P. Andreev, and Subramaniam Pennathur conceptualized the study, supported funding for the work, and were actively involved with discussions about experimental design, data interpretation, and manuscript writing.
CRMB and SKRW and the SP-AF4 and analytical AF4 work are supported by NSF-CHE1508827. FA’s time and salary is supported by the grant K08DK106523. Lipoprotein lipidomic studies, metabolomic core support, and clinical sample procurement was funded by NIH grants P30DK081943, P30DK0829503, U24DK097153, and R24DK082841 (all to SP).
Compliance with ethical standards
This research did not involve human participants or animals, and therefore, obtaining informed consent was not required.
Human plasma was obtained from the American Red Cross which was a pool of over 1000 de-identified plasma donors which were to be discarded due to expiration of safe window for transfusion. The study is therefore exempt from Institutional Review Board (IRB) approval.
Conflict of interest
The authors declare that they have no conflict of interest.
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