Analytical and Bioanalytical Chemistry

, Volume 411, Issue 19, pp 4739–4749 | Cite as

In-depth structural characterization of phospholipids by pairing solution photochemical reaction with charge inversion ion/ion chemistry

  • Elissia T. Franklin
  • Stella K. Betancourt
  • Caitlin E. Randolph
  • Scott A. McLuckeyEmail author
  • Yu XiaEmail author
Research Paper
Part of the following topical collections:
  1. Young Investigators in (Bio-)Analytical Chemistry


Shotgun lipid analysis based on electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in lipidomic studies. One challenge for the shotgun approach is the discrimination of lipid isomers and isobars. Gas-phase charge inversion via ion/ion reactions has been used as an effective method to identify multiple isomeric/isobaric components in a single MS peak by exploiting the distinctive functionality of different lipid classes. In doing so, fatty acyl chain information can be obtained without recourse to condensed-phase separations or derivatization. This method alone, however, cannot provide carbon–carbon double bond (C=C) location information from fatty acyl chains. Herein, we provide an enhanced method pairing photochemical derivatization of C=C via the Paternò–Büchi reaction with charge inversion ion/ion tandem mass spectrometry. This method was able to provide gas-phase separation of phosphatidylcholines and phosphatidylethanolamines, the fatty acyl compositions, and the C=C location within each fatty acyl chain. We have successfully applied this method to bovine liver lipid extracts and identified 40 molecular species of glycerophospholipids with detailed structural information including head group, fatty acyl composition, and C=C location.

Graphical Abstract


Lipidomics The Paternò–Büchi reaction Unsaturated lipid Charge inversion Mass spectrometry 


Funding information

This research was supported by the National Institutes of Health (R01GM118484 to Y. X. and GM R37-45372 to S. A. M.) and Sciex.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1537_MOESM1_ESM.pdf (758 kb)
ESM 1 (PDF 758 kb)


  1. 1.
    Han X, Gross RW. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom Rev. 2005;24(3):367–412.Google Scholar
  2. 2.
    Armstrong D, editor. Lipidomics [Internet]. Totowa, NJ: Humana Press; 2009. (Methods in Molecular Biology; vol. 579). Available from: Accessed 9 Oct 2017.
  3. 3.
    Han X, Gross RW. Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes. Expert Rev Proteomics. 2005;2(2):253–64.Google Scholar
  4. 4.
    Fhaner CJ, Liu S, Ji H, Simpson RJ, Reid GE. Comprehensive lipidome profiling of isogenic primary and metastatic colon adenocarcinoma cell lines. Anal Chem. 2012;84(21):8917–26.Google Scholar
  5. 5.
    van Meer G, Voelker DR, Feigenson GW. Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Bio. 2008;9(2):112–24.Google Scholar
  6. 6.
    Eberlin LS, Dill AL, Golby AJ, Ligon KL, Wiseman JM, Cooks RG, et al. Discrimination of human astrocytoma subtypes by lipid analysis using desorption electrospray ionization imaging mass spectrometry. Angew Chem Int Ed. 2010;49(34):5953–6.Google Scholar
  7. 7.
    Gross RW, Han X. Lipidomics at the interface of structure and function in systems biology. Chem Biol. 2011;18(3):284–91.Google Scholar
  8. 8.
    Ejsing CS, Sampaio JL, Surendranath V, Duchoslav E, Ekroos K, Klemm RW, et al. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc Natl Acad Sci U S A. 2009;106(7):2136–41.Google Scholar
  9. 9.
    Loizides-Mangold U. On the future of mass spectrometry-based lipidomics. FEBS J. 2013;280(12):2817–29.Google Scholar
  10. 10.
    Fahy E, Subramaniam S, Murphy RC, Nishijima M, Raetz CRH, Shimizu T, et al. Update of the LIPID MAPS comprehensive classification system for lipids. J Lipid Res. 2009;50(Supplement):S9–14.Google Scholar
  11. 11.
    Whiley L, Sen A, Heaton J, Proitsi P, García-Gómez D, Leung R, et al. Evidence of altered phosphatidylcholine metabolism in Alzheimer’s disease. Neurobiol Aging. 2014;35(2):271–8.Google Scholar
  12. 12.
    Phoenix DA, Harris F, Mura M, Dennison SR. The increasing role of phosphatidylethanolamine as a lipid receptor in the action of host defense peptides. Prog Lipid Res. 2015;59:26–37.Google Scholar
  13. 13.
    Guan Z-Z, Wang Y-N, Xiao K-Q, Hu P-S, Liu J-L. Activity of phosphatidylethanolamine-N-methyltransferase in brain affected by Alzheimers disease. Neurochem Int. 1999;34(1):41–7.Google Scholar
  14. 14.
    Brouwers JF. Liquid chromatographic–mass spectrometric analysis of phospholipids. Chromatography, ionization and quantification. BBA-Mol Cell Bio Lipids. 2011;1811(11):763–75.Google Scholar
  15. 15.
    Wang C, Kong H, Guan Y, Yang J, Gu J, Yang S, et al. Plasma phospholipid metabolic profiling and biomarkers of type 2 diabetes mellitus based on high-performance liquid chromatography/electrospray mass spectrometry and multivariate statistical analysis. Anal Chem. 2005;77(13):4108–16.Google Scholar
  16. 16.
    Bird SS, Marur VR, Sniatynski MJ, Greenberg HK, Kristal BS. Lipidomics profiling by high-resolution LC–MS and high-energy collisional dissociation fragmentation: focus on characterization of mitochondrial cardiolipins and monolysocardiolipins. Anal Chem. 2011;83(3):940–9.Google Scholar
  17. 17.
    Nygren H, Seppänen-Laakso T, Castillo S, Hyötyläinen T, Orešič M. Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics for studies of body fluids and tissues. In: Metz TO, editor. Metabolic profiling [Internet]. Totowa, NJ: Humana Press; 2011. p. 247–57. Available from: Accessed 15 Nov 2017.
  18. 18.
    Brügger B, Erben G, Sandhoff R, Wieland FT, Lehmann WD. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc Natl Acad Sci U S A. 1997;94(6):2339.Google Scholar
  19. 19.
    Han X, Gross RW. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids. Proc Natl Acad Sci U S A. 1994;91(22):10635–9.Google Scholar
  20. 20.
    Han X, Gross RW. Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J Lipid Res. 2003;44(6):1071–9.Google Scholar
  21. 21.
    Han X, Yang K, Gross RW. Multi-dimensional mass spectrometry-based shotgun lipidomics and novel strategies for lipidomic analyses. Mass Spectrom Rev. 2012;31(1):134–78.Google Scholar
  22. 22.
    Lintonen TPI, Baker PRS, Suoniemi M, Ubhi BK, Koistinen KM, Duchoslav E, et al. Differential mobility spectrometry-driven shotgun lipidomics. Anal Chem. 2014;86(19):9662–9.Google Scholar
  23. 23.
    Buré C, Ayciriex S, Testet E, Schmitter J-M. A single run LC-MS/MS method for phospholipidomics. Anal Bioanal Chem. 2013;405(1):203–13.Google Scholar
  24. 24.
    Hsu F-F, Turk J. Electrospray ionization with low-energy collisionally activated dissociation tandem mass spectrometry of glycerophospholipids: mechanisms of fragmentation and structural characterization. J Chromatogr B. 2009;877(26):2673–95.Google Scholar
  25. 25.
    Wasslen KV, Canez CR, Lee H, Manthorpe JM, Smith JC. Trimethylation enhancement using diazomethane (TrEnDi) II: rapid in-solution concomitant quaternization of glycerophospholipid amino groups and methylation of phosphate groups via reaction with diazomethane significantly enhances sensitivity in mass spectrometry analyses via a fixed, permanent positive charge. Anal Chem. 2014;86(19):9523–32.Google Scholar
  26. 26.
    Canez CR, Shields SWJ, Bugno M, Wasslen KV, Weinert HP, Willmore WG, et al. Trimethylation enhancement using 13 C-diazomethane (13C-TrEnDi): increased sensitivity and selectivity of phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine lipids derived from complex biological samples. Anal Chem. 2016;88(14):6996–7004.Google Scholar
  27. 27.
    Wang M, Palavicini JP, Cseresznye A, Han X. Strategy for quantitative analysis of isomeric bis (monoacylglycero) phosphate and phosphatidylglycerol species by shotgun lipidomics after one-step methylation. Anal Chem. 2017;89(16):8490–5.Google Scholar
  28. 28.
    Stutzman JR, Blanksby SJ, McLuckey SA. Gas-phase transformation of phosphatidylcholine cations to structurally informative anions via ion/ion chemistry. Anal Chem. 2013;85(7):3752–7.Google Scholar
  29. 29.
    Rojas-Betancourt S, Stutzman JR, Londry FA, Blanksby SJ, McLuckey SA. Gas-phase chemical separation of phosphatidylcholine and phosphatidylethanolamine cations via charge inversion ion/ion chemistry. Anal Chem. 2015;87(22):11255–62.Google Scholar
  30. 30.
    Pulfer M, Murphy RC. Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev. 2003;22(5):332–64.Google Scholar
  31. 31.
    Tomer KB, Crow FW, Gross ML. Location of double-bond position in unsaturated fatty acids by negative ion MS/MS. J Am Chem Soc. 1983;105(16):5487–8.Google Scholar
  32. 32.
    Thomas MC, Mitchell TW, Harman DG, Deeley JM, Nealon JR, Blanksby SJ. Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions. Anal Chem. 2008;80(1):303–11.Google Scholar
  33. 33.
    Brown SHJ, Mitchell TW, Blanksby SJ. Analysis of unsaturated lipids by ozone-induced dissociation. BBA-Mol Cell Bio Lipids. 2011;1811(11):807–17.Google Scholar
  34. 34.
    Pham HT, Maccarone AT, Thomas MC, Campbell JL, Mitchell TW, Blanksby SJ. Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: the next step towards “top-down” lipidomics. Analyst. 2014;139(1):204–14.Google Scholar
  35. 35.
    Klein DR, Brodbelt JS. Structural characterization of phosphatidylcholines using 193 nm ultraviolet photodissociation mass spectrometry. Anal Chem. 2017;89(3):1516–22.Google Scholar
  36. 36.
    Pham HT, Trevitt AJ, Mitchell TW, Blanksby SJ. Rapid differentiation of isomeric lipids by photodissociation mass spectrometry of fatty acid derivatives: photodissociation of derivatized fatty acids. Rapid Commun Mass Spectrom. 2013;27(7):805–15.Google Scholar
  37. 37.
    Pham HT, Julian RR. Radical delivery and fragmentation for structural analysis of glycerophospholipids. Int J Mass Spectrom. 2014;370:58–65.Google Scholar
  38. 38.
    Deimler RE, Sander M, Jackson GP. Radical-induced fragmentation of phospholipid cations using metastable atom-activated dissociation mass spectrometry (MAD-MS). Int J Mass Spectrom. 2015;390:178–86.Google Scholar
  39. 39.
    Li P, Hoffmann WD, Jackson GP. Multistage mass spectrometry of phospholipids using collision-induced dissociation (CID) and metastable atom-activated dissociation (MAD). Int J Mass Spectrom. 2016;403:1–7.Google Scholar
  40. 40.
    Campbell JL, Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics. Anal Chem. 2015;87(11):5837–45.Google Scholar
  41. 41.
    Baba T, Campbell JL, Le Blanc JCY, Baker PRS. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics (EIEIO). J Lipid Res. 2016;57(11):2015–27.Google Scholar
  42. 42.
    Stinson CA, Zhang W, Xia Y. UV lamp as a facile ozone source for structural analysis of unsaturated lipids via electrospray ionization-mass spectrometry. J Am Soc Mass Spectr. 2018;29(3):481–9.Google Scholar
  43. 43.
    Harris RA, May JC, Stinson CA, Xia Y, McLean JA. Determining double bond position in lipids using online ozonolysis coupled to liquid chromatography and ion mobility-mass spectrometry. Anal Chem. 2018;90(3):1915–24.Google Scholar
  44. 44.
    Ma X, Xia Y. Pinpointing double bonds in lipids by Paternò-Büchi reactions and mass spectrometry. Angew Chem Int Ed. 2014;53(10):2592–6.Google Scholar
  45. 45.
    Stinson CA, Xia Y. A method of coupling the Paternò–Büchi reaction with direct infusion ESI-MS/MS for locating the C=C bond in glycerophospholipids. Analyst. 2016;141(12):3696–704.Google Scholar
  46. 46.
    Ma X, Chong L, Tian R, Shi R, Hu TY, Ouyang Z, et al. Identification and quantitation of lipid C=C location isomers: a shotgun lipidomics approach enabled by photochemical reaction. Proc Natl Acad Sci U S A. 2016;113(10):2573–8.Google Scholar
  47. 47.
    Ren J, Franklin ET, Xia Y. Uncovering structural diversity of unsaturated fatty acyls in cholesteryl esters via photochemical reaction and tandem mass spectrometry. J Am Soc Mass Spectrom. 2017;28(7):1432–41.Google Scholar
  48. 48.
    Murphy RC, Okuno T, Johnson CA, Barkley RM. Determination of double bond positions in polyunsaturated fatty acids using the photochemical Paternò-Büchi reaction with acetone and tandem mass spectrometry. Anal Chem. 2017;89(16):8545–53.Google Scholar
  49. 49.
    Liebisch G, Vizcaíno JA, Köfeler H, Trötzmüller M, Griffiths WJ, Schmitz G, et al. Shorthand notation for lipid structures derived from mass spectrometry. J Lipid Res. 2013;54(6):1523–30.Google Scholar
  50. 50.
    Xia Y, Chrisman PA, Erickson DE, Liu J, Liang X, Londry FA, et al. Implementation of ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer. Anal Chem. 2006;78(12):4146–54.Google Scholar
  51. 51.
    Cao W, Ma X, Li Z, Zhou X, Ouyang Z. Locating carbon–carbon double bonds in unsaturated phospholipids by epoxidation reaction and tandem mass spectrometry. Anal Chem. 2018;90(17):10286–92.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ChemistryPurdue UniversityWest LafayetteUSA
  2. 2.Department of ChemistryTsinghua UniversityBeijingChina

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