Advertisement

Analytical and Bioanalytical Chemistry

, Volume 411, Issue 3, pp 777–786 | Cite as

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

  • Carmen R. M. Bria
  • Farsad Afshinnia
  • Patrick W. Skelly
  • Thekkelnaycke M. Rajendiran
  • Pradeep Kayampilly
  • Thommey P. Thomas
  • Victor P. Andreev
  • Subramaniam Pennathur
  • S. Kim Ratanathanawongs WilliamsEmail author
Research Paper

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 (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

Keywords

Asymmetrical flow field-flow fractionation Ultracentrifugation Lipoproteins Lipidomics Mass spectrometry 

Notes

Author’s contribution

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.

Funding information

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.

Supplementary material

216_2018_1499_MOESM1_ESM.pdf (287 kb)
ESM 1 (PDF 286 kb)

References

  1. 1.
    Austin MA, Rodriguez BL, McKnight B, McNeely MJ, Edwards KL, Curb JD, et al. Low-density lipoprotein particle size, triglycerides, and high-density lipoprotein cholesterol as risk factors for coronary heart disease in older Japanese-American men. Am J Cardiol. 2000;86:412–6.  https://doi.org/10.1016/S0002-9149(00)00956-5.CrossRefGoogle Scholar
  2. 2.
    Teerlink T, Scheffer PG. LDL particles are nonspherical: consequences for size determination and phenotypic classification. Clin Chem. 2007;53:361–2.  https://doi.org/10.1373/clinchem.2006.079871.CrossRefGoogle Scholar
  3. 3.
    Gerszten RE, Wang TJ. The search for new cardiovascular biomarkers. Nature. 2008;451:949–52.  https://doi.org/10.1038/nature06802.CrossRefGoogle Scholar
  4. 4.
    Davidson WS, Heink A, Sexmith H, Melchior JT, Gordon SM, Kuklenyik Z, et al. The effects of apolipoprotein B depletion on HDL subspecies composition and function. J Lipid Res. 2016;57:674–86.  https://doi.org/10.1194/jlr.M066613.CrossRefGoogle Scholar
  5. 5.
    Larner CD, Henriquez RR, Johnson JD, Macfarlane RD. Developing high performance lipoprotein density profiling for use in clinical studies relating to cardiovascular disease. Anal Chem. 2011;83:8524–30.  https://doi.org/10.1021/ac2018124.CrossRefGoogle Scholar
  6. 6.
    Munroe WH, Phillips ML, Schumaker VN. Excessive centrifugal fields damage high density lipoprotein. J Lipid Res. 2015;56:1172–81.  https://doi.org/10.1194/jlr.M058735.CrossRefGoogle Scholar
  7. 7.
    Kunitake ST, Kane JP. Factors affecting the integrity of high density lipoproteins in the ultracentrifuge. J Lipid Res. 1982;23:936–40.Google Scholar
  8. 8.
    Ståhlman M, Davidsson P, Kanmert I, Rosengren B, Borén J, Fagerberg B, et al. Proteomics and lipids of lipoproteins isolated at low salt concentrations in D2O/sucrose or in KBr. J Lipid Res. 2008;49:481–90.  https://doi.org/10.1194/jlr.D700025-JLR200.CrossRefGoogle Scholar
  9. 9.
    Jones J, Toth C, Kuklenyik Z, Schieltz D, Williamson Y, Andrews M, et al. Effects of ultracentrifugation on HDL and LDL size distribution. GA: Pittcon Atlanta; 2016.Google Scholar
  10. 10.
    Lu M, Gursky O. Aggregation and fusion of low-density lipoproteins in vivo and in vitro. Biomol Concepts. 2013;4:501–18.  https://doi.org/10.1515/bmc-2013-0016.CrossRefGoogle Scholar
  11. 11.
    Hsieh J, Chang C, Huang MT, Chang C, Chen C, Shen M-Y, et al. Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults. Anal Chem. 2013;85:11440–8.  https://doi.org/10.1021/ac402516u.CrossRefGoogle Scholar
  12. 12.
    Scheffer PG, Bakker SJL, Heine RJ, Teerlink T. Measurement of low-density lipoprotein particle size by high-performance gel-filtration chromatography. Clin Chem. 1997;43:1904–12.Google Scholar
  13. 13.
    Bobály B, Beck A, Fekete J, Guillarme D, Fekete S. Systematic evaluation of mobile phase additives for the LC-MS characterization of therapeutic proteins. Talanta. 2015;136:60–7.  https://doi.org/10.1016/j.talanta.2014.12.006.CrossRefGoogle Scholar
  14. 14.
    Williams SKR, Runyon JR, Ashames AA. Field-flow fractionation: addressing the nano challenge. Anal Chem. 2011;83:634–42.  https://doi.org/10.1021/ac101759z.CrossRefGoogle Scholar
  15. 15.
    Li P, Giddings JC. Isolation and measurement of colloids in human plasma by membrane-selective flow field-flow fractionation: lipoproteins and pharmaceutical colloids. J Pharm Sci. 1996;85:895–8.  https://doi.org/10.1021/js950335s.CrossRefGoogle Scholar
  16. 16.
    Wahlund K. Flow field-flow fractionation: critical overview. J Chromatogr A. 2013;1287:97–112.  https://doi.org/10.1016/j.chroma.2013.02.028.CrossRefGoogle Scholar
  17. 17.
    Johann C, Elsenberg S, Roesch U, Rambaldi DC, Zattoni A, Reschiglian P. A novel approach to improve operation and performance in flow field-flow fractionation. J Chromatogr A. 2011;1218:4126–31.  https://doi.org/10.1016/j.chroma.2010.12.077.CrossRefGoogle Scholar
  18. 18.
    Park I, Paeng K-J, Kang D, Moon MH. Performance of hollow-fiber flow field-flow fractionation in protein separation. J Sep Sci. 2005;28:2043–9.  https://doi.org/10.1002/jssc.200500125.CrossRefGoogle Scholar
  19. 19.
    Kim KH, Moon MH. Chip-type asymmetrical flow field-flow fractionation channel coupled with mass spectrometry for top-down protein identification. Anal Chem. 2011;83:8652–8.  https://doi.org/10.1021/ac202098b.CrossRefGoogle Scholar
  20. 20.
    Kuklenyik Z, Gardner M, Parks B, Schieltz D, Rees J, McWilliams L, et al. Multivariate DoE optimization of asymmetric flow field flow fractionation coupled to quantitative LC-MS/MS for analysis of lipoprotein subclasses. Chromatography. 2015;2:96–117.  https://doi.org/10.3390/chromatography2010096.CrossRefGoogle Scholar
  21. 21.
    Lee JY, Kim KH, Moon MH. Evaluation of multiplexed hollow fiber flow field-flow fractionation for semi-preparative purposes. J Chromatogr A. 2009;1216:6539–42.  https://doi.org/10.1016/j.chroma.2009.07.044.CrossRefGoogle Scholar
  22. 22.
    Lee JY, Min HK, Choi D, Moon MH. Profiling of phospholipids in lipoproteins by multiplexed hollow fiber flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2010;1217:1660–6.  https://doi.org/10.1016/j.chroma.2010.01.006.CrossRefGoogle Scholar
  23. 23.
    Byeon SK, Lee JY, Lim S, Choi D, Moon MH, Kee S, et al. Discovery of candidate phospholipid biomarkers in human lipoproteins with coronary artery disease by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2012;1270:246–53.  https://doi.org/10.1016/j.chroma.2012.11.012.CrossRefGoogle Scholar
  24. 24.
    Byeon SK, Kim JY, Lee JY, Chung BC, Seo HS, Moon MH. Top-down and bottom-up lipidomic analysis of rabbit lipoproteins under different metabolic conditions using flow field-flow fractionation, nanoflow liquid chromatography and mass spectrometry. J Chromatogr A. 2015;1405:140–8.  https://doi.org/10.1016/j.chroma.2015.05.059.CrossRefGoogle Scholar
  25. 25.
    Lee JY, Byeon SK, Moon MH. Profiling of oxidized phospholipids in lipoproteins from patients with coronary artery disease by hollow fiber flow field-flow fractionation and nanoflow liquid chromatography–tandem mass spectrometry. Anal Chem. 2015;87:1266–73.  https://doi.org/10.1021/ac503973p.CrossRefGoogle Scholar
  26. 26.
    Kuklenyik Z, Jones JI, Gardner MS, Schieltz DM, Parks BA, Toth CA, et al. Core lipid, surface lipid and apolipoprotein composition analysis of lipoprotein particles as a function of particle size in one workflow integrating asymmetric flow field-flow fractionation and liquid chromatography-tandem mass spectrometry. PLoS One. 2018;13:e0194797.  https://doi.org/10.1371/journal.pone.0194797.CrossRefGoogle Scholar
  27. 27.
    Bria CRM, Skelly PW, Morse JR, Schaak RE, Williams SKR. Semi-preparative asymmetrical flow field-flow fractionation: a closer look at channel dimensions and separation performance. J Chromatogr A. 2017;1499:149–57.  https://doi.org/10.1016/j.chroma.2017.03.017.CrossRefGoogle Scholar
  28. 28.
    Kim SH, Yang JS, Lee JC, Lee J-Y, Lee J-Y, Kim E, et al. Lipidomic alterations in lipoproteins of patients with mild cognitive impairment and Alzheimer’s disease by asymmetrical flow field-flow fractionation and nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2018;1568:91–100.  https://doi.org/10.1016/J.CHROMA.2018.07.018.CrossRefGoogle Scholar
  29. 29.
    McPherson PAC, Young IS, McKibben B, McEneny J. High density lipoprotein subfractions: isolation, composition, and their duplicitous role in oxidation. J Lipid Res. 2007;48:86–95.  https://doi.org/10.1194/jlr.M600094-JLR200.CrossRefGoogle Scholar
  30. 30.
    Vivekanandan-Giri A, Slocum JL, Byun J, Tang C, Sands RL, Gillespie BW, et al. High density lipoprotein is targeted for oxidation by myeloperoxidase in rheumatoid arthritis. Ann Rheum Dis. 2013;72:1725–31.  https://doi.org/10.1136/annrheumdis-2012-202033.CrossRefGoogle Scholar
  31. 31.
    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.  https://doi.org/10.1016/0003-2697(76)90527-3.CrossRefGoogle Scholar
  32. 32.
    Afshinnia F, Rajendiran TM, Karnovsky A, Soni T, Wang X, Xie D, et al. Lipidomic signature of progression of chronic kidney disease in the chronic renal insufficiency cohort. Kidney Int Reports. 2016;1:256–68.  https://doi.org/10.1016/j.ekir.2016.08.007.CrossRefGoogle Scholar
  33. 33.
    Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–7.  https://doi.org/10.1139/o59-099.CrossRefGoogle Scholar
  34. 34.
  35. 35.
    Mackness MI, Hallam SD, Peard T, Warner S, Walker CH. The separation of sheep and human serum “A”-esterase activity into the lipoprotein fraction by ultracentrifugation. Comp Biochem Physiol - Part B Biochem Mol Biol. 1985;82:675–7.CrossRefGoogle Scholar
  36. 36.
    Williams PT, Zhao X-Q, Marcovina SM, Otvos JD, Brown BG, Krauss RM. Comparison of four methods of analysis of lipoprotein particle subfractions for their association with angiographic progression of coronary artery disease. Atherosclerosis. 2014;233:713–20.  https://doi.org/10.1016/j.atherosclerosis.2014.01.034.CrossRefGoogle Scholar
  37. 37.
    Leeuwenburgh C, Rasmussen JE, Hsu FF, Mueller DM, Pennathur S, Heinecke JW. Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J Biol Chem. 1997;272:3520–6.  https://doi.org/10.1074/JBC.272.6.3520.CrossRefGoogle Scholar
  38. 38.
    Pennathur S, Wagner JD, Leeuwenburgh C, Litwak KN, Heinecke JW. A hydroxyl radical-like species oxidizes cynomolgus monkey artery wall proteins in early diabetic vascular disease. J Clin Invest. 2001;107:853–60.  https://doi.org/10.1172/JCI11194.CrossRefGoogle Scholar
  39. 39.
    Pennathur S, Ido Y, Heller JI, Byun J, Danda R, Pergola P, et al. Reactive carbonyls and polyunsaturated fatty acids produce a hydroxyl radical-like species: a potential pathway for oxidative damage of retinal proteins in diabetes. J Biol Chem. 2005;280:22706–14.  https://doi.org/10.1074/jbc.M500839200.CrossRefGoogle Scholar
  40. 40.
    German JB, Smilowitz JT, Zivkovic AM. Lipoproteins: when size really matters. Curr Opin Colloid Interface Sci. 2006;11:171–83.  https://doi.org/10.1016/j.cocis.2005.11.006.CrossRefGoogle Scholar
  41. 41.
    Afshinnia F, Rajendiran TM, Soni T, Byun J, Wernisch S, Sas KM, et al. Impaired β-oxidation and altered complex lipid fatty acid partitioning with advancing CKD. J Am Soc Nephrol. 2017;29:295–306.  https://doi.org/10.1681/ASN.2017030350.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Carmen R. M. Bria
    • 1
  • Farsad Afshinnia
    • 2
  • Patrick W. Skelly
    • 1
  • Thekkelnaycke M. Rajendiran
    • 3
    • 4
  • Pradeep Kayampilly
    • 2
  • Thommey P. Thomas
    • 2
  • Victor P. Andreev
    • 5
  • Subramaniam Pennathur
    • 2
    • 6
  • S. Kim Ratanathanawongs Williams
    • 1
    Email author
  1. 1.Laboratory for Advanced Separations Technologies, Department of ChemistryColorado School of MinesGoldenUSA
  2. 2.Department of Internal Medicine – NephrologyUniversity of MichiganAnn ArborUSA
  3. 3.Department of PathologyUniversity of MichiganAnn ArborUSA
  4. 4.Michigan Regional Comprehensive Metabolomics Resource CoreAnn ArborUSA
  5. 5.Arbor Research Collaborative for HealthAnn ArborUSA
  6. 6.Molecular and Integrative PhysiologyUniversity of MichiganAnn ArborUSA

Personalised recommendations