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A reversed-phase capillary ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) method for comprehensive top-down/bottom-up lipid profiling

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Abstract

Lipidomics is a critical part of metabolomics and aims to study all the lipids within a living system. We present here the development and evaluation of a sensitive capillary UPLC-MS method for comprehensive top-down/bottom-up lipid profiling. Three different stationary phases were evaluated in terms of peak capacity, linearity, reproducibility, and limit of quantification (LOQ) using a mixture of lipid standards representative of the lipidome. The relative standard deviations of the retention times and peak abundances of the lipid standards were 0.29% and 7.7%, respectively, when using the optimized method. The linearity was acceptable at >0.99 over 3 orders of magnitude, and the LOQs were sub-fmol. To demonstrate the performance of the method in the analysis of complex samples, we analyzed lipids extracted from a human cell line, rat plasma, and a model human skin tissue, identifying 446, 444, and 370 unique lipids, respectively. Overall, the method provided either higher coverage of the lipidome, greater measurement sensitivity, or both, when compared to other approaches of global, untargeted lipid profiling based on chromatography coupled with MS.

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References

  1. van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9(2):112–124. doi:10.1038/nrm2330

    Article  Google Scholar 

  2. Astrup A, Dyerberg J, Selleck M, Stender S (2008) Nutrition transition and its relationship to the development of obesity and related chronic diseases. Obes Rev 9(Suppl 1):48–52. doi:10.1111/j.1467-789X.2007.00438.x

    Article  Google Scholar 

  3. Russo GL (2009) Dietary n-6 and n-3 polyunsaturated fatty acids: from biochemistry to clinical implications in cardiovascular prevention. Biochem Pharmacol 77(6):937–946. doi:10.1016/j.bcp.2008.10.020

    Article  CAS  Google Scholar 

  4. Brasaemle DL (2007) Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis. J Lipid Res 48(12):2547–2559. doi:10.1194/jlr.R700014-JLR200

    Article  CAS  Google Scholar 

  5. Wenk MR (2005) The emerging field of lipidomics. Nat Rev Drug Discov 4(7):594–610. doi:10.1038/nrd1776

    Article  CAS  Google Scholar 

  6. Oresic M, Simell S, Sysi-Aho M, Nanto-Salonen K, Seppanen-Laakso T, Parikka V, Katajamaa M, Hekkala A, Mattila I, Keskinen P, Yetukuri L, Reinikainen A, Lahde J, Suortti T, Hakalax J, Simell T, Hyoty H, Veijola R, Ilonen J, Lahesmaa R, Knip M, Simell O (2008) Dysregulation of lipid and amino acid metabolism precedes islet autoimmunity in children who later progress to type 1 diabetes. J Exp Med 205(13):2975–2984. doi:10.1084/jem.20081800

    Article  CAS  Google Scholar 

  7. Sorensen CM, Ding J, Zhang Q, Alquier T, Zhao R, Mueller PW, Smith RD, Metz TO (2010) Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: lipidomics analysis of a Diabetes Antibody Standardization Program sample subset. Clin Biochem 43(12):948–956. doi:10.1016/j.clinbiochem.2010.04.075

    Article  CAS  Google Scholar 

  8. Gross RW, Han X (2007) Lipidomics in diabetes and the metabolic syndrome. Methods Enzymol 433:73–90. doi:10.1016/S0076-6879(07)33004-8

    Article  CAS  Google Scholar 

  9. Han X (2010) Multi-dimensional mass spectrometry-based shotgun lipidomics and the altered lipids at the mild cognitive impairment stage of Alzheimer’s disease. Biochim Biophys Acta 1801(8):774–783. doi:10.1016/j.bbalip.2010.01.010

    CAS  Google Scholar 

  10. Zhao L, Spassieva SD, Jucius TJ, Shultz LD, Shick HE, Macklin WB, Hannun YA, Obeid LM, Ackerman SL (2011) A deficiency of ceramide biosynthesis causes cerebellar Purkinje cell neurodegeneration and lipofuscin accumulation. PLoS Genet 7(5):e1002063. doi:10.1371/journal.pgen.1002063

    Article  CAS  Google Scholar 

  11. Cutler RG, Kelly J, Storie K, Pedersen WA, Tammara A, Hatanpaa K, Troncoso JC, Mattson MP (2004) Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer’s disease. Proc Natl Acad Sci USA 101(7):2070–2075. doi:10.1073/pnas.0305799101

    Article  CAS  Google Scholar 

  12. Fernandis AZ, Wenk MR (2009) Lipid-based biomarkers for cancer. J Chromatogr B Analyt Technol Biomed Life Sci 877(26):2830–2835. doi:10.1016/j.jchromb.2009.06.015

    Article  CAS  Google Scholar 

  13. Aboagye EO, Bhujwalla ZM (1999) Malignant transformation alters membrane choline phospholipid metabolism of human mammary epithelial cells. Cancer Res 59(1):80–84

    CAS  Google Scholar 

  14. Diamond DL, Syder AJ, Jacobs JM, Sorensen CM, Walters KA, Proll SC, McDermott JE, Gritsenko MA, Zhang Q, Zhao R, Metz TO, Camp DG 2nd, Waters KM, Smith RD, Rice CM, Katze MG (2010) Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics. PLoS Pathog 6(1):e1000719. doi:10.1371/journal.ppat.1000719

    Article  Google Scholar 

  15. Jain M, Petzold CJ, Schelle MW, Leavell MD, Mougous JD, Bertozzi CR, Leary JA, Cox JS (2007) Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling. Proc Natl Acad Sci USA 104(12):5133–5138. doi:10.1073/pnas.0610634104

    Article  CAS  Google Scholar 

  16. Wenk MR (2006) Lipidomics of host–pathogen interactions. FEBS Lett 580(23):5541–5551. doi:10.1016/j.febslet.2006.07.007

    Article  CAS  Google Scholar 

  17. Fahy E, Subramaniam S, Brown HA, Glass CK, Merrill AH Jr, Murphy RC, Raetz CR, Russell DW, Seyama Y, Shaw W, Shimizu T, Spener F, van Meer G, VanNieuwenhze MS, White SH, Witztum JL, Dennis EA (2005) A comprehensive classification system for lipids. J Lipid Res 46(5):839–861. doi:10.1194/jlr.E400004-JLR200

    Article  CAS  Google Scholar 

  18. Fahy E, Subramaniam S, Murphy RC, Nishijima M, Raetz CR, Shimizu T, Spener F, van Meer G, Wakelam MJ, Dennis EA (2009) Update of the LIPID MAPS comprehensive classification system for lipids. J Lipid Res 50(Suppl):S9–14. doi:10.1194/jlr.R800095-JLR200

    Article  Google Scholar 

  19. Harkewicz R, Dennis EA (2011) Applications of mass spectrometry to lipids and membranes. Annu Rev Biochem 80:301–325. doi:10.1146/annurev-biochem-060409-092612

    Article  CAS  Google Scholar 

  20. Han X, Gross RW (2005) Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom Rev 24(3):367–412. doi:10.1002/mas.20023

    Article  CAS  Google Scholar 

  21. Duffin KL, Henion JD, Shieh JJ (1991) Electrospray and tandem mass spectrometric characterization of acylglycerol mixtures that are dissolved in nonpolar solvents. Anal Chem 63(17):1781–1788

    Article  CAS  Google Scholar 

  22. Dennis EA (2009) Lipidomics joins the omics evolution. Proc Natl Acad Sci USA 106(7):2089–2090. doi:10.1073/pnas.0812636106

    Article  CAS  Google Scholar 

  23. Mitchell TW, Pham H, Thomas MC, Blanksby SJ (2009) Identification of double bond position in lipids: from GC to OzID. J Chromatogr B Analyt Technol Biomed Life Sci 877(26):2722–2735. doi:10.1016/j.jchromb.2009.01.017

    Article  CAS  Google Scholar 

  24. Li M, Zhou Z, Nie H, Bai Y, Liu H (2011) Recent advances of chromatography and mass spectrometry in lipidomics. Anal Bioanal Chem 399(1):243–249. doi:10.1007/s00216-010-4327-y

    Article  CAS  Google Scholar 

  25. Hutchins PM, Barkley RM, Murphy RC (2008) Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry. J Lipid Res 49(4):804–813. doi:10.1194/jlr.M700521-JLR200

    Article  CAS  Google Scholar 

  26. Nie H, Liu R, Yang Y, Bai Y, Guan Y, Qian D, Wang T, Liu H (2010) Lipid profiling of rat peritoneal surface layers by online normal- and reversed-phase 2D LC QToF-MS. J Lipid Res 51(9):2833–2844. doi:10.1194/jlr.D007567

    Article  CAS  Google Scholar 

  27. Ding J, Sorensen CM, Jaitly N, Jiang H, Orton DJ, Monroe ME, Moore RJ, Smith RD, Metz TO (2008) Application of the accurate mass and time tag approach in studies of the human blood lipidome. J Chromatogr B Analyt Technol Biomed Life Sci 871(2):243–252. doi:10.1016/j.jchromb.2008.04.040

    Article  CAS  Google Scholar 

  28. Sandra K, Pereira Ados S, Vanhoenacker G, David F, Sandra P (2010) Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry. J Chromatogr A 1217(25):4087–4099. doi:10.1016/j.chroma.2010.02.039

    Article  CAS  Google Scholar 

  29. Metz TO, Zhang Q, Page JS, Shen Y, Callister SJ, Jacobs JM, Smith RD (2007) The future of liquid chromatography-mass spectrometry (LC-MS) in metabolic profiling and metabolomic studies for biomarker discovery. Biomark Med 1(1):159–185. doi:10.2217/17520363.1.1.159

    Article  CAS  Google Scholar 

  30. Nygren H, Seppanen-Laakso T, Castillo S, Hyotylainen T, Oresic M (2011) Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics for studies of body fluids and tissues. Methods Mol Biol 708:247–257. doi:10.1007/978-1-61737-985-7_15

    Article  CAS  Google Scholar 

  31. Masoodi M, Eiden M, Koulman A, Spaner D, Volmer DA (2010) Comprehensive lipidomics analysis of bioactive lipids in complex regulatory networks. Anal Chem 82(19):8176–8185. doi:10.1021/ac1015563

    Article  CAS  Google Scholar 

  32. Rainville PD, Stumpf CL, Shockcor JP, Plumb RS, Nicholson JK (2007) Novel application of reversed-phase UPLC-oaTOF-MS for lipid analysis in complex biological mixtures: a new tool for lipidomics. J Proteome Res 6(2):552–558. doi:10.1021/pr060611b

    Article  CAS  Google Scholar 

  33. Shockcor J, Crowe H, Yu K, Shion H (2010) Analysis of intact lipids from biologics matrices by UPLC/high definition MS. Application Note. Waters Corporation, Milford, MA

  34. Schuhmann K, Herzog R, Schwudke D, Metelmann-Strupat W, Bornstein SR, Shevchenko A (2011) Bottom-up shotgun lipidomics by higher energy collisional dissociation (HCD) on LTQ orbitrap mass spectrometers. Anal Chem. doi:10.1021/ac102505f

  35. Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 75(13):3019–3030

    Article  CAS  Google Scholar 

  36. Kelly RT, Page JS, Luo Q, Moore RJ, Orton DJ, Tang K, Smith RD (2006) Chemically etched open tubular and monolithic emitters for nanoelectrospray ionization mass spectrometry. Anal Chem 78(22):7796–7801. doi:10.1021/ac061133r

    Article  CAS  Google Scholar 

  37. Kiebel GR, Auberry KJ, Jaitly N, Clark DA, Monroe ME, Peterson ES, Tolic N, Anderson GA, Smith RD (2006) PRISM: a data management system for high-throughput proteomics. Proteomics 6(6):1783–1790. doi:10.1002/pmic.200500500

    Article  CAS  Google Scholar 

  38. Jaitly N, Mayampurath A, Littlefield K, Adkins JN, Anderson GA, Smith RD (2009) Decon2LS: an open-source software package for automated processing and visualization of high resolution mass spectrometry data. BMC Bioinformatics 10:87. doi:10.1186/1471-2105-10-87

    Article  Google Scholar 

  39. Monroe ME, Tolic N, Jaitly N, Shaw JL, Adkins JN, Smith RD (2007) VIPER: an advanced software package to support high-throughput LC-MS peptide identification. Bioinformatics 23(15):2021–2023. doi:10.1093/bioinformatics/btm281

    Article  CAS  Google Scholar 

  40. Jaitly N, Monroe ME, Petyuk VA, Clauss TR, Adkins JN, Smith RD (2006) Robust algorithm for alignment of liquid chromatography-mass spectrometry analyses in an accurate mass and time tag data analysis pipeline. Anal Chem 78(21):7397–7409. doi:10.1021/ac052197p

    Article  CAS  Google Scholar 

  41. Quehenberger O, Armando AM, Brown AH, Milne SB, Myers DS, Merrill AH, Bandyopadhyay S, Jones KN, Kelly S, Shaner RL, Sullards CM, Wang E, Murphy RC, Barkley RM, Leiker TJ, Raetz CR, Guan Z, Laird GM, Six DA, Russell DW, McDonald JG, Subramaniam S, Fahy E, Dennis EA (2010) Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res 51(11):3299–3305. doi:10.1194/jlr.M009449

    Article  CAS  Google Scholar 

  42. Anderson NL, Anderson NG (2002) The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 1(11):845–867

    Article  CAS  Google Scholar 

  43. Schmidt A, Karas M, Dulcks T (2003) Effect of different solution flow rates on analyte ion signals in nano-ESI MS, or: when does ESI turn into nano-ESI? J Am Soc Mass Spectrom 14(5):492–500. doi:10.1016/S1044-0305(03)00128-4

    Article  CAS  Google Scholar 

  44. Smith RD, Tang KQ, Page JS (2004) Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry. J Am Soc Mass Spectrom 15(10):1416–1423. doi:10.1016/j.jasms.2004.04.034

    Article  Google Scholar 

  45. Shen Y, Jacobs JM, Camp DG 2nd, Fang R, Moore RJ, Smith RD, Xiao W, Davis RW, Tompkins RG (2004) Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome. Anal Chem 76(4):1134–1144. doi:10.1021/ac034869m

    Article  CAS  Google Scholar 

  46. Wilm MS, Mann M (1994) Electrospray and Taylor-Cone Theory, Doles Beam of macromolecules at last. Int J Mass Spectrom 136(2–3):167–180

    Article  CAS  Google Scholar 

  47. Delamora JF, Loscertales IG (1994) The current emitted by highly conducting Taylor Cones. J Fluid Mech 260:155–184

    Article  Google Scholar 

  48. Plumb R, Castro-Perez J, Granger J, Beattie I, Joncour K, Wright A (2004) Ultra-performance liquid chromatography coupled to quadrupole-orthogonal time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 18(19):2331–2337. doi:10.1002/rcm.1627

    Article  CAS  Google Scholar 

  49. Vandeemter JJ, Zuiderweg FJ, Klinkenberg A (1956) Longitudinal diffusion and resistance to mass transfer as causes of nonideality in chromatography. Chem Eng Sci 5(6):271–289

    Article  CAS  Google Scholar 

  50. Castro-Perez JM, Kamphorst J, DeGroot J, Lafeber F, Goshawk J, Yu K, Shockcor JP, Vreeken RJ, Hankemeier T (2010) Comprehensive LC-MS E lipidomic analysis using a shotgun approach and its application to biomarker detection and identification in osteoarthritis patients. J Proteome Res 9(5):2377–2389. doi:10.1021/pr901094j

    Article  CAS  Google Scholar 

  51. Shen Y, Zhao R, Berger SJ, Anderson GA, Rodriguez N, Smith RD (2002) High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics. Anal Chem 74(16):4235–4249

    Article  CAS  Google Scholar 

  52. Giddings JC (1991) United separation science. Wiley, New York

    Google Scholar 

  53. Shen Y, Zhang R, Moore RJ, Kim J, Metz TO, Hixson KK, Zhao R, Livesay EA, Udseth HR, Smith RD (2005) Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000–1500 and capabilities in proteomics and metabolomics. Anal Chem 77(10):3090–3100. doi:10.1021/ac0483062

    Article  CAS  Google Scholar 

  54. Graessler J, Schwudke D, Schwarz PE, Herzog R, Shevchenko A, Bornstein SR (2009) Top-down lipidomics reveals ether lipid deficiency in blood plasma of hypertensive patients. PLoS One 4(7):e6261. doi:10.1371/journal.pone.0006261

    Article  Google Scholar 

  55. Schwudke D, Hannich JT, Surendranath V, Grimard V, Moehring T, Burton L, Kurzchalia T, Shevchenko A (2007) Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra. Anal Chem 79(11):4083–4093. doi:10.1021/ac062455y

    Article  CAS  Google Scholar 

  56. Pulfer M, Murphy RC (2003) Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev 22(5):332–364. doi:10.1002/mas.10061

    Article  CAS  Google Scholar 

  57. Yang K, Cheng H, Gross RW, Han X (2009) Automated lipid identification and quantification by multidimensional mass spectrometry-based shotgun lipidomics. Anal Chem 81(11):4356–4368. doi:10.1021/ac900241u

    Article  CAS  Google Scholar 

  58. Fang JS, Barcelona MJ (1998) Structural determination and quantitative analysis of bacterial phospholipids using liquid chromatography electrospray ionization mass spectrometry. J Microbiol Methods 33(1):23–35

    Article  CAS  Google Scholar 

  59. Bushee JL, Argikar UA (2011) An experimental approach to enhance precursor ion fragmentation for metabolite identification studies: application of dual collision cells in an orbital trap. Rapid Commun Mass Spectrom 25(10):1356–1362. doi:10.1002/rcm.4996

    Article  CAS  Google Scholar 

  60. Quehenberger O, Armando A, Dumlao D, Stephens DL, Dennis EA (2008) Lipidomics analysis of essential fatty acids in macrophages. Prostaglandins Leukot Essent Fatty Acids 79(3–5):123–129. doi:10.1016/j.plefa.2008.09.021

    Article  CAS  Google Scholar 

  61. Thomas A, Deglon J, Lenglet S, Mach F, Mangin P, Wolfender JL, Steffens S, Staub C (2010) High-throughput phospholipidic fingerprinting by online desorption of dried spots and quadrupole-linear ion trap mass spectrometry: evaluation of atherosclerosis biomarkers in mouse plasma. Anal Chem 82(15):6687–6694. doi:10.1021/ac101421b

    Article  CAS  Google Scholar 

  62. Ahn EJ, Kim H, Chung BC, Kong G, Moon MH (2008) Quantitative profiling of phosphatidylcholine and phosphatidylethanolamine in a steatosis/fibrosis model of rat liver by nanoflow liquid chromatography/tandem mass spectrometry. J Chromatogr A 1194(1):96–102. doi:10.1016/j.chroma.2008.04.031

    Article  CAS  Google Scholar 

  63. Kim H, Ahn E, Moon MH (2008) Profiling of human urinary phospholipids by nanoflow liquid chromatography/tandem mass spectrometry. Analyst 133(12):1656–1663. doi:10.1039/b804715d

    Article  CAS  Google Scholar 

  64. Kim H, Min HK, Kong G, Moon MH (2009) Quantitative analysis of phosphatidylcholines and phosphatidylethanolamines in urine of patients with breast cancer by nanoflow liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem 393(6–7):1649–1656. doi:10.1007/s00216-009-2621-3

    Article  CAS  Google Scholar 

  65. Lee JY, Min HK, Moon MH (2011) Simultaneous profiling of lysophospholipids and phospholipids from human plasma by nanoflow liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 400(9):2953–2961. doi:10.1007/s00216-011-4958-7

    Article  CAS  Google Scholar 

  66. Taguchi R, Houjou T, Nakanishi H, Yamazaki T, Ishida M, Imagawa M, Shimizu T (2005) Focused lipidomics by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 823(1):26–36. doi:10.1016/j.jchromb.2005.06.005

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Department of Health and Human Services, under Contract Number HHSN272200800060C. Additional support was provided by the NIAID under Award Number U54AI081680 and by the Office of Science, U.S. Department of Energy (DOE), under the Low Dose Radiation Research Program. Work was performed at the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is a multi-program national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RLO 1830.

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Correspondence to Thomas O. Metz.

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Xiaoli Gao and Qibin Zhang contributed equally to this work.

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Gao, X., Zhang, Q., Meng, D. et al. A reversed-phase capillary ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) method for comprehensive top-down/bottom-up lipid profiling. Anal Bioanal Chem 402, 2923–2933 (2012). https://doi.org/10.1007/s00216-012-5773-5

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