Skip to main content

Development of an Automated Multi-Injection Shotgun Lipidomics Approach Using a Triple Quadrupole Mass Spectrometer

Abstract

Shotgun lipidomics is a well-suited approach to monitor lipid alterations due to its ability to scan for varying lipid types on a global, class and individual species level. However, the ability to perform high-throughput shotgun lipidomics has remained challenging due to time-consuming data processing and hardware limitations. To increase the throughput nature of shotgun lipidomics, an automated shotgun lipidomics approach is described utilizing conventional low flow gradient liquid chromatography (LC) analysis (post-injection) coupled with multiple sample injections per sample (on a lipid scan per injection basis). The proposed automated multi-injection approach resulted in a reproducible lipid scanning period of 2.5 min (in a 4.5 min total data acquisition period), thereby providing a sufficient scanning period for performing either mass spectrometric or tandem mass spectrometric analyses. In addition to being simple, robust and reproducible, this approach was also constructed to be cost-effective by using common LC instrumentation and customizable as the data acquisition period can be tailored to perform different scan types, period lengths and scan numbers. Combined with a strategy to create multiple lipid-specific aliquots per sample, the overall approach provides a simple and efficient platform to perform high-throughput lipid profiling.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Abbreviations

Ptd2Gro:

Cardiolipins

Cer:

Ceramides

CE:

Cholesteryl esters

DAG:

Diacylglycerols

ESI:

Electrospray ionization

FFA:

Free fatty acids

FIA:

Flow-injection analysis

FS:

Full scan

CerGal:

Galactosylceramides

LC:

Liquid chromatography

LysoPtdCho:

Lysophosphatidylcholines

LysoPtdEtn:

Lysophosphatidylethanolamines

MS:

Mass spectrometry

MRM:

Multiple-reaction Monitoring

NL:

Neutral loss

PtdOH:

Phosphatidic acid

PtdCho:

Phosphatidylcholines

PtdEtn:

Phosphatidylethanolamines

PtdGro:

Phosphatidylglycerols

PtdIns:

Phosphatidylinositols

PtdSer:

Phosphatidylserines

PI:

Precursor ion

CerPCho:

Sphingomyelins

ST:

Sulfatides

MS/MS:

Tandem mass spectrometry

TAG:

Triacylglycerols

References

  1. Gross RW, Han X (2011) Lipidomics at the interface of structure and function in systems biology. Chem Biol 18:284–291

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Watson AD (2006) Thematic review series: systems biology approaches to metabolic and cardiovascular disorders. Lipidomics: a global approach to lipid analysis in biological systems. J Lipid Res 47:2101–2111

    Article  CAS  PubMed  Google Scholar 

  3. Wenk MR (2005) The emerging field of lipidomics. Nat Rev Drug Discov 4:594–610

    Article  CAS  PubMed  Google Scholar 

  4. Shevchenko A, Simons K (2010) Lipidomics: coming to grips with lipid diversity. Nat Rev Mol Cell Biol 11:593–598

    Article  CAS  PubMed  Google Scholar 

  5. Han X, Rozen S, Boyle SH, Hellegers C, Cheng H, Burke JR, Welsh-Bohmer KA, Doraiswamy PM, Kaddurah-Daouk R (2011) Metabolomics in early Alzheimer’s disease: identification of altered plasma sphingolipidome using shotgun lipidomics. PLoS ONE 6:e21643

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. 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 (BBA) Mol Cell Biol Lipids 1801:774–783

    Article  CAS  Google Scholar 

  7. Han X (2007) Potential mechanisms contributing to sulfatide depletion at the earliest clinically recognizable stage of Alzheimer’s disease: a tale of shotgun lipidomics. J Neurochem 103:171–179

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Hu C, van der Heijden R, Wang M, van der Greef J, Hankemeier T, Xu G (2009) Analytical strategies in lipidomics and applications in disease biomarker discovery. J Chromatogr B Analyt Technol Biomed Life Sci 877:2836–2846

    Article  CAS  PubMed  Google Scholar 

  9. Gross RW, Han X (2007) Lipidomics in diabetes and the metabolic syndrome. Methods Enzymol 433:73–90

    Article  CAS  PubMed  Google Scholar 

  10. Aguilera CM, Gil-Campos M, Canete R, Gil A (2008) Alterations in plasma and tissue lipids associated with obesity and metabolic syndrome. Clin Sci 114:183–193

    Article  CAS  PubMed  Google Scholar 

  11. Pietiläinen KH, Sysi-Aho M, Rissanen A, Seppänen-Laakso T, Yki-Järvinen H, Kaprio J, Oresic M (2007) Acquired obesity is associated with changes in the serum lipidomic profile independent of genetic effects-a monozygotic twin study. PLoS ONE 2:e218

    Article  PubMed Central  PubMed  Google Scholar 

  12. Stegemann C, Drozdov I, Shalhoub J, Humphries J, Ladroue C, Didangelos A, Baumert M, Allen M, Davies AH, Monaco C, Smith A, Xu Q, Mayr M (2011) Comparative lipidomics profiling of human atherosclerotic plaques. Circ Cardiovasc Genet 4:232–242

    Article  CAS  PubMed  Google Scholar 

  13. Ekroos K, Jänis M, Tarasov K, Hurme R, Laaksonen R (2010) Lipidomics: a tool for studies of atherosclerosis. Curr Atheroscler Rep 12:273–281

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Min H, Lim S, Chung B, Moon M (2011) Shotgun lipidomics for candidate biomarkers of urinary phospholipids in prostate cancer. Anal Bioanal Chem 399:823–830

    Article  CAS  PubMed  Google Scholar 

  15. Sewell GW, Hannun YA, Han X, Koster G, Bielawski J, Goss V, Smith PJ, Rahman FZ, Vega R, Bloom SL, Walker AP, Postle AD, Segal AW (2012) Lipidomic profiling in Crohn’s disease: abnormalities in phosphatidylinositols, with preservation of ceramide, phosphatidylcholine and phosphatidylserine composition. Int J Biochem Cell Biol 44:1839–1846

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Gross RW, Han X (2009) Shotgun lipidomics of neutral lipids as an enabling technology for elucidation of lipid-related diseases. Am J Phys Endocrinol Metab 297:E297–E303

    Article  CAS  Google Scholar 

  17. Han X, Yang K, Cheng H, Fikes KN, Gross RW (2005) Shotgun lipidomics of phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization. J Lipid Res 46:1548–1560

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Jiang X, Cheng H, Yang K, Gross RW, Han X (2007) Alkaline methanolysis of lipid extracts extends shotgun lipidomics analyses to the low-abundance regime of cellular sphingolipids. Anal Biochem 371:135–145

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Schwudke D, Schuhmann K, Herzog R, Bornstein SR, Shevchenko A (2011) Shotgun lipidomics on high resolution mass spectrometers. Cold Spring Harb Perspect Biol 3:a004614

    Article  PubMed Central  PubMed  Google Scholar 

  20. Song H, Ladenson J, Turk J (2009) Algorithms for automatic processing of data from mass spectrometric analyses of lipids. J Chromatogr B 877:2847–2854

    Article  CAS  Google Scholar 

  21. Herzog R, Schuhmann K, Schwudke D, Sampaio JL, Bornstein SR, Schroeder M, Shevchenko A (2012) LipidXplorer: a software for consensual cross-platform lipidomics. PLoS ONE 7:e29851

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Ståhlman M, Ejsing CS, Tarasov K, Perman J, Borén J, Ekroos K (2009) High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry. J Chromatogr B 877:2664–2672

    Article  Google Scholar 

  24. Jung HR, Sylvänne T, Koistinen KM, Tarasov K, Kauhanen D, Ekroos K (2011) High throughput quantitative molecular lipidomics. Biochim Biophys Acta Mol Cell Biol Lipids 1811:925–934

    Article  CAS  Google Scholar 

  25. Han X, Yang K, Gross RW (2008) Microfluidics-based electrospray ionization enhances the intrasource separation of lipid classes and extends identification of individual molecular species through multi-dimensional mass spectrometry: development of an automated high-throughput platform for shotgun lipidomics. Rapid Commun Mass Spectrom 22:2115–2124

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Hermansson M, Uphoff A, Käkelä R, Somerharju P (2005) Automated quantitative analysis of complex lipidomes by liquid chromatography/mass spectrometry. Anal Chem 77:2166–2175

    Article  CAS  PubMed  Google Scholar 

  27. Scherer M, Leuthäuser-Jaschinski K, Ecker J, Schmitz G, Liebisch G (2010) A rapid and quantitative LC-MS/MS method to profile sphingolipids. J Lipid Res 51:2001–2011

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Myers DS, Ivanova PT, Milne SB, Brown HA (2011) Quantitative analysis of glycerophospholipids by LC/MS: acquisition, data handling, and interpretation. Biochim Biophys Acta Mol Cell Biol Lipids 1811:748–757

    Article  CAS  Google Scholar 

  29. Sandra K, Pereira AS, Vanhoenacker G, David F, Sandra P (2010) Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry. J Chromatogr A 1217:4087–4099

    Article  CAS  PubMed  Google Scholar 

  30. Han X, Gross RW (1994) Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids. PNAS 91:10635–10639

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. 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 24:367–412

    Article  CAS  PubMed  Google Scholar 

  32. Han X, Yang J, Cheng H, Ye H, Gross RW (2004) Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry. Anal Biochem 330:317–331

    Article  CAS  PubMed  Google Scholar 

  33. Han X, Yang K, Gross RW (2012) Multi-dimensional mass spectrometry-based shotgun lipidomics and novel strategies for lipidomic analyses. Mass Spectrom Rev 31:134–178

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Schuhmann K, Almeida R, Baumert M, Herzog R, Bornstein SR, Shevchenko A (2011) Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes. J Mass Spectrom 47:96–104

    Article  Google Scholar 

  35. Wiesner P, Leidl K, Boettcher A, Schmitz G, Liebisch G (2009) Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry. J Lipid Res 50:574–585

    Article  CAS  PubMed  Google Scholar 

  36. Liebisch G, Lieser B, Rathenberg J, Drobnik W, Schmitz G (2004) High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm. Biochim Biophys Acta Mol Cell Biol Lipids 1686:108–117

    Article  CAS  Google Scholar 

  37. Liebisch G, Drobnik W, Lieser B, Schmitz G (2002) High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry. Clin Chem 48:2217–2224

    CAS  PubMed  Google Scholar 

  38. Köfeler HC, Fauland A, Rechberger GN, Trötzmüller M (2012) Mass spectrometry based lipidomics: an overview of technological platforms. Metabolites 2:19–38

    Article  Google Scholar 

  39. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Phys 37:911–917

    Article  CAS  Google Scholar 

  40. Bowden JA, Albert CJ, Barnaby OS, Ford DA (2011) Analysis of cholesteryl esters and diacylglycerols using lithiated adducts and electrospray ionization-tandem mass spectrometry. Anal Biochem 417:202–210

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. 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 295:88–100

    Article  CAS  PubMed  Google Scholar 

  42. Hsu FF, Bohrer A, Turk J (1998) Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry. J Am Soc Mass Spectrom 9:516–526

    Article  CAS  PubMed  Google Scholar 

  43. Hsu FF, Turk J (1999) Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisionally activated dissociation on a triple stage quadrupole instrument. J Am Soc Mass Spectrom 10:587–599

    Article  CAS  PubMed  Google Scholar 

  44. 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 44:1071–1079

    Article  CAS  PubMed  Google Scholar 

  45. Liebisch G, Binder M, Schifferer R, Langmann T, Schulz B, Schmitz G (2006) High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Biochim Biophys Acta Mol Cell Biol Lipids 1761:121–128

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to John A. Bowden.

Additional information

Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology; nor does it imply that the materials or equipment identified are necessarily the best for the purpose.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 324 kb)

About this article

Cite this article

Bowden, J.A., Bangma, J.T. & Kucklick, J.R. Development of an Automated Multi-Injection Shotgun Lipidomics Approach Using a Triple Quadrupole Mass Spectrometer. Lipids 49, 609–619 (2014). https://doi.org/10.1007/s11745-014-3903-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-014-3903-x

Keywords

  • Shotgun lipidomics
  • Lipid profiling
  • Triple quadrupole mass spectrometry
  • Flow-injection analysis
  • Multi-injection shotgun lipidomics
  • Tandem mass spectrometry
  • Direct-infusion