Analytical and Bioanalytical Chemistry

, Volume 405, Issue 15, pp 5267–5278

Mass spectrometry based phospholipidomics of mammalian thymus and leukemia patients: implication for function of iNKT cells

  • Xiukun Xu
  • Yunhui Yu
  • Zheng Wang
  • Tingting Zhu
  • Yanping Wang
  • Jian Zhu
  • Zijun Chen
  • Yun He
  • Linling Ju
  • Yunsen Li
Research Paper


In previous studies phospholipids have been proved to be involved in biochemical, physiological, and pathological processes. As a special class of phospholipids, peroxisome-derived lipids (PDLs) have been proved to be potential ligands of invariant natural killer T (iNKT) cells in recent studies. Here, on the basis of phospholipidomics, we focused on the relative quantity of PDLs extracted from mammalian thymus or bone marrow using electrospray ionization mass spectrometry (MS). In phospholipid analysis, we identified 12 classes of phospholipids and accounted for their relative quantities by comparing their relative abundances in the MS1 map. Our results show that PDLs are present in mammalian thymus as well as mouse spleen and liver. Interestingly, the relative quantity of PDLs extracted from human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) bone marrows is higher than that extracted from bone marrow of healthy donors. Our results may help to explain the close correlation between PDLs and iNKT cell function in thymus, spleen, liver, and especially in leukemia patients. We think that our phospholipidomics work may reveal a function of iNKT cells.


Phospholipids Natural killer T cells Lipidomics Peroxisome-derived lipids Function 





Acute myeloid leukemia


Acute lymphoblastic leukemia


Coefficient of variation


Electrospray ionization


High-performance liquid chromatography




Invariant natural killer T


Liquid chromatography


Lysophosphatidic acid




Mass spectrometry


Natural killer T


Peroxisome-derived lipid







Supplementary material

216_2013_6923_MOESM1_ESM.pdf (292 kb)
ESM 1(PDF 292 kb)


  1. 1.
    Han X, Holtzman DM, McKeel DW Jr (2001) Plasmalogen deficiency in early Alzheimer's disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry. J Neurochem 77(4):1168–1180CrossRefGoogle Scholar
  2. 2.
    Schumann J, Facciotti F, Panza L, Michieletti M, Compostella F, Collmann A, Mori L, De Libero G (2007) Differential alteration of lipid antigen presentation to NKT cells due to imbalances in lipid metabolism. Eur J Immunol 37(6):1431–1441. doi:10.1002/eji.200737160 CrossRefGoogle Scholar
  3. 3.
    Facciotti F, Ramanjaneyulu GS, Lepore M, Sansano S, Cavallari M, Kistowska M, Forss-Petter S, Ni G, Colone A, Singhal A, Berger J, Xia C, Mori L, De Libero G (2012) Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus. Nat Immunol 13(5):474–480. doi:10.1038/ni.2245 CrossRefGoogle Scholar
  4. 4.
    Gumperz JE, Roy C, Makowska A, Lum D, Sugita M, Podrebarac T, Koezuka Y, Porcelli SA, Cardell S, Brenner MB, Behar SM (2000) Murine CD1d-restricted T cell recognition of cellular lipids. Immunity 12(2):211–221CrossRefGoogle Scholar
  5. 5.
    Zhou D, Mattner J, Cantu C 3rd, Schrantz N, Yin N, Gao Y, Sagiv Y, Hudspeth K, Wu YP, Yamashita T, Teneberg S, Wang D, Proia RL, Levery SB, Savage PB, Teyton L, Bendelac A (2004) Lysosomal glycosphingolipid recognition by NKT cells. Science 306(5702):1786–1789. doi:10.1126/science.1103440 CrossRefGoogle Scholar
  6. 6.
    Mattner J, Debord KL, Ismail N, Goff RD, Cantu C 3rd, Zhou D, Saint-Mezard P, Wang V, Gao Y, Yin N, Hoebe K, Schneewind O, Walker D, Beutler B, Teyton L, Savage PB, Bendelac A (2005) Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434(7032):525–529. doi:10.1038/nature03408 CrossRefGoogle Scholar
  7. 7.
    Speak AO, Salio M, Neville DC, Fontaine J, Priestman DA, Platt N, Heare T, Butters TD, Dwek RA, Trottein F, Exley MA, Cerundolo V, Platt FM (2007) Implications for invariant natural killer T cell ligands due to the restricted presence of isoglobotrihexosylceramide in mammals. Proc Natl Acad Sci USA 104(14):5971–5976. doi:10.1073/pnas.0607285104 CrossRefGoogle Scholar
  8. 8.
    Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC, Feuerstein IM, Horowitz ME, Magrath IT, Shad AT, Steinberg SM et al (1995) Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. New Engl J Med 332(3):143–149. doi:10.1056/NEJM199501193320303 CrossRefGoogle Scholar
  9. 9.
    Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC, Feuerstein IM, Magrath IT, Wexler LH, Dimitrov DS, Gress RE (1997) Distinctions between CD8+ and CD4+ T-cell regenerative pathways result in prolonged T-cell subset imbalance after intensive chemotherapy. Blood 89(10):3700–3707Google Scholar
  10. 10.
    Metelitsa LS, Naidenko OV, Kant A, Wu HW, Loza MJ, Perussia B, Kronenberg M, Seeger RC (2001) Human NKT cells mediate antitumor cytotoxicity directly by recognizing target cell CD1d with bound ligand or indirectly by producing IL-2 to activate NK cells. J Immunol 167(6):3114–3122Google Scholar
  11. 11.
    Nicol A, Nieda M, Koezuka Y, Porcelli S, Suzuki K, Tadokoro K, Durrant S, Juji T (2000) Human invariant valpha24+ natural killer T cells activated by alpha-galactosylceramide (KRN7000) have cytotoxic anti-tumour activity through mechanisms distinct from T cells and natural killer cells. Immunology 99(2):229–234CrossRefGoogle Scholar
  12. 12.
    Gansuvd B, Hagihara M, Yu Y, Inoue H, Ueda Y, Tsuchiya T, Masui A, Ando K, Nakamura Y, Munkhtuvshin N, Kato S, Thomas JM, Hotta T (2002) Human umbilical cord blood NK T cells kill tumors by multiple cytotoxic mechanisms. Hum Immunol 63(3):164–175CrossRefGoogle Scholar
  13. 13.
    Wang Z, Wen L, Ma X, Chen Z, Yu Y, Zhu J, Wang Y, Liu Z, Liu H, Wu D, Zhou D, Li Y (2012) High expression of lactotriaosylceramide, a differentiation-associated glycosphingolipid, in the bone marrow of acute myeloid leukemia patients. Glycobiology 22(7):930–938. doi:10.1093/glycob/cws061 CrossRefGoogle Scholar
  14. 14.
    Olsnes AM, Motorin D, Ryningen A, Zaritskey AY, Bruserud O (2006) T lymphocyte chemotactic chemokines in acute myelogenous leukemia (AML): local release by native human AML blasts and systemic levels of CXCL10 (IP-10), CCL5 (RANTES) and CCL17 (TARC). Cancer Immunol Immunother 55(7):830–840. doi:10.1007/s00262-005-0080-z CrossRefGoogle Scholar
  15. 15.
    Bruserud O, Ryningen A, Olsnes AM, Stordrange L, Oyan AM, Kalland KH, Gjertsen BT (2007) Subclassification of patients with acute myelogenous leukemia based on chemokine responsiveness and constitutive chemokine release by their leukemic cells. Haematologica 92(3):332–341CrossRefGoogle Scholar
  16. 16.
    Behl D, Porrata LF, Markovic SN, Letendre L, Pruthi RK, Hook CC, Tefferi A, Elliot MA, Kaufmann SH, Mesa RA, Litzow MR (2006) Absolute lymphocyte count recovery after induction chemotherapy predicts superior survival in acute myelogenous leukemia. Leukemia 20(1):29–34. doi:10.1038/sj.leu.2404032 CrossRefGoogle Scholar
  17. 17.
    Metelitsa LS, Weinberg KI, Emanuel PD, Seeger RC (2003) Expression of CD1d by myelomonocytic leukemias provides a target for cytotoxic NKT cells. Leukemia 17(6):1068–1077. doi:10.1038/sj.leu.2402943 CrossRefGoogle Scholar
  18. 18.
    Ekroos K, Chernushevich IV, Simons K, Shevchenko A (2002) Quantitative profiling of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer. Anal Chem 74(5):941–949CrossRefGoogle Scholar
  19. 19.
    Touchstone JC (1995) Thin-layer chromatographic procedures for lipid separation. J Chromatogr B Biomed Appl 671(1–2):169–195CrossRefGoogle Scholar
  20. 20.
    Xia YQ, Jemal M (2009) Phospholipids in liquid chromatography/mass spectrometry bioanalysis: comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects. Rapid Commun Mass Spectrom 23(14):2125–2138. doi:10.1002/rcm.4121 CrossRefGoogle Scholar
  21. 21.
    Kim HY, Wang TC, Ma YC (1994) Liquid chromatography/mass spectrometry of phospholipids using electrospray ionization. Anal Chem 66(22):3977–3982CrossRefGoogle Scholar
  22. 22.
    Kim HY, Salem N Jr (1987) Application of thermospray high-performance liquid chromatography/mass spectrometry for the determination of phospholipids and related compounds. Anal Chem 59(5):722–726CrossRefGoogle Scholar
  23. 23.
    Robins SJ, Patton GM (1986) Separation of phospholipid molecular species by high performance liquid chromatography: potentials for use in metabolic studies. J Lipid Res 27(2):131–139Google Scholar
  24. 24.
    Weintraub ST, Pinckard RN, Hail M (1991) Electrospray ionization for analysis of platelet-activating factor. Rapid Commun Mass Spectrom 5(7):309–311. doi:10.1002/rcm.1290050702 CrossRefGoogle Scholar
  25. 25.
    Schwudke D, Oegema J, Burton L, Entchev E, Hannich JT, Ejsing CS, Kurzchalia T, Shevchenko A (2006) Lipid profiling by multiple precursor and neutral loss scanning driven by the data-dependent acquisition. Anal Chem 78(2):585–595. doi:10.1021/ac051605m CrossRefGoogle Scholar
  26. 26.
    Han X, Gross RW (2003) Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J Lipid Res 44(6):1071–1079. doi:10.1194/jlr.R300004-JLR200 CrossRefGoogle Scholar
  27. 27.
    Lim S, Byeon SK, Lee JY, Moon MH (2012) Computational approach to structural identification of phospholipids using raw mass spectra from nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry. J Mass Spectrom 47(8):1004–1014. doi:10.1002/jms.3033 CrossRefGoogle Scholar
  28. 28.
    Taguchi R, Hayakawa J, Takeuchi Y, Ishida M (2000) Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry. J Mass Spectrom 35(8):953–966. doi:10.1002/1096-9888(200008)35:8<953::AID-JMS23>3.0.CO;2-4 CrossRefGoogle Scholar
  29. 29.
    Isaac G, Bylund D, Mansson JE, Markides KE, Bergquist J (2003) Analysis of phosphatidylcholine and sphingomyelin molecular species from brain extracts using capillary liquid chromatography electrospray ionization mass spectrometry. J Neurosci Methods 128(1–2):111–119CrossRefGoogle Scholar
  30. 30.
    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 CrossRefGoogle Scholar
  31. 31.
    Han X, Gross RW (1994) Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids. Proc Natl Acad Sci USA 91(22):10635–10639CrossRefGoogle Scholar
  32. 32.
    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 CrossRefGoogle Scholar
  33. 33.
    Blanksby SJ, Mitchell TW (2010) Advances in mass spectrometry for lipidomics. Annu Rev Anal Chem 3:433–465. doi:10.1146/annurev.anchem.111808.073705 CrossRefGoogle Scholar
  34. 34.
    Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226(1):497–509Google Scholar
  35. 35.
    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8):911–917CrossRefGoogle Scholar
  36. 36.
    Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246(4926):64–71CrossRefGoogle Scholar
  37. 37.
    Ejsing CS, Duchoslav E, Sampaio J, Simons K, Bonner R, Thiele C, Ekroos K, Shevchenko A (2006) Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning. Anal Chem 78(17):6202–6214. doi:10.1021/ac060545x CrossRefGoogle Scholar
  38. 38.
    Alberici RM, Simas RC, Sanvido GB, Romao W, Lalli PM, Benassi M, Cunha IB, Eberlin MN (2010) Ambient mass spectrometry: bringing MS into the "real world". Anal Bioanal Chem 398(1):265–294. doi:10.1007/s00216-010-3808-3 CrossRefGoogle Scholar
  39. 39.
    Han X, Gross RW (2001) Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry. Anal Biochem 295(1):88–100. doi:10.1006/abio.2001.5178 CrossRefGoogle Scholar
  40. 40.
    Han X (2002) Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry. Anal Biochem 302(2):199–212. doi:10.1006/abio.2001.5536 CrossRefGoogle Scholar
  41. 41.
    Eberl G, Lees R, Smiley ST, Taniguchi M, Grusby MJ, MacDonald HR (1999) Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells. J Immunol 162(11):6410–6419Google Scholar
  42. 42.
    Hammond KJ, Pelikan SB, Crowe NY, Randle-Barrett E, Nakayama T, Taniguchi M, Smyth MJ, van Driel IR, Scollay R, Baxter AG, Godfrey DI (1999) NKT cells are phenotypically and functionally diverse. Eur J Immunol 29(11):3768–3781. doi:10.1002/(SICI)1521-4141(199911)29:11<3768::AID-IMMU3768>3.0.CO;2-G CrossRefGoogle Scholar
  43. 43.
    Hammond KJ, Pellicci DG, Poulton LD, Naidenko OV, Scalzo AA, Baxter AG, Godfrey DI (2001) CD1d-restricted NKT cells: an interstrain comparison. J Immunol 167(3):1164–1173Google Scholar
  44. 44.
    Kamani N, Kattamis A, Carroll A, Campbell D, Bunin N (2000) Immune reconstitution after autologous purged bone marrow transplantation in children. J Pediatr Hematol Oncol 22(1):13–19CrossRefGoogle Scholar
  45. 45.
    Barrett AJ, Savani BN (2009) Does chemotherapy modify the immune surveillance of hematological malignancies? Leukemia 23(1):53–58. doi:10.1038/leu.2008.273 CrossRefGoogle Scholar
  46. 46.
    Najera Chuc AE, Cervantes LA, Retiguin FP, Ojeda JV, Maldonado ER (2012) Low number of invariant NKT cells is associated with poor survival in acute myeloid leukemia. J Cancer Res Clin Oncol 138(8):1427–1432. doi:10.1007/s00432-012-1251-x CrossRefGoogle Scholar
  47. 47.
    Ohnishi K, Yamanishi H, Naito K, Utsumi M, Yokomaku S, Hirabayashi N, Ohno R (1998) Reconstitution of peripheral blood lymphocyte subsets in the long-term disease-free survivors of patients with acute myeloblastic leukemia. Leukemia 12(1):52–58CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Xiukun Xu
    • 1
  • Yunhui Yu
    • 1
  • Zheng Wang
    • 1
  • Tingting Zhu
    • 1
  • Yanping Wang
    • 1
  • Jian Zhu
    • 1
  • Zijun Chen
    • 2
  • Yun He
    • 1
  • Linling Ju
    • 1
  • Yunsen Li
    • 1
  1. 1.Laboratory of Cellular and Molecular Tumor Immunology, Institutes of Biology and Medical Sciences, Jiangsu Laboratory of Infection ImmunitySoochow UniversitySuzhouChina
  2. 2.College of Chinese Materia MedicaShanghai University of Traditional Chinese MedicineShanghaiChina

Personalised recommendations