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The Bottom Up Solution to the Triacylglycerol Lipidome

The Bottom Up Solution to the Triacylglycerol Lipidome. William Craig Byrdwell Florida Atlantic University Dept. of Chemistry & Biochem. Boca Raton, FL 33432. Overview. What is Atmospheric Pressure Chemical Ionization (APCI) mass spectrometry (MS)?

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The Bottom Up Solution to the Triacylglycerol Lipidome

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  1. The Bottom Up Solution to the Triacylglycerol Lipidome William Craig Byrdwell Florida Atlantic University Dept. of Chemistry & Biochem. Boca Raton, FL 33432

  2. Overview • What is Atmospheric Pressure Chemical Ionization (APCI) mass spectrometry (MS)? • What does an APCI-MS mass spectrum look like? • What Information does an APCI-MS mass spectrum contain? • What Information is desired from MS of TAG? • Critical Ratios provide structural information • Critical Values and Critical Limits define Cases • The Complete Bottom Up Solution • The Shape of the Triacylglycerol Lipidome

  3. Atmospheric Pressure Chemical Ionization • APCI-MS involves spraying a liquid flow down a heated nebulizer cylinder past a high voltage needle that has a corona discharge plasma formed at the tip • Ions from atmospheric gases and moisture act as chemical ionization reagents to give mostly protonated molecules • APCI-MS is a soft ionization technique

  4. APCI-MS of TAG • Triacylglycerols (TAG) give simple mass spectra by APCI-MS which contain : • A protonated molecule ion, [M+H]+ • Diacylglycerol-like fragment ions, [DAG]+ • For Example, [OL]+ C57H103O6+ Exact Mass: 883.78 [M+H]+ OLO C39H69O4 + Exact Mass: 601.52 [OL]+ C39H71O4 + Exact Mass: 603.54 [OO]+ • and, [OO]+

  5. [AA]+ [MH]+ 1 1 [MH]+ [AA]+ [MH]+ 1 [AA]+ [AA]+ 1 Polyunsaturated TAG [MH]+ Saturated TAG APCI-MS Mass Spectra of AAA TAG Type I TAG • Type I (AAA) TAG give two ions: • Protonated Molecule, [M+H]+ • One Diacylglycerol ion, [DAG]+ The proportion of the Protonated Molecule, [M+H]+, to the Diacylglycerol ion, [DAG]+, depends on the amount of unsaturation in the TAG. e.g. LnLnLn e.g. SSS

  6. The MH/SDAG varies with # Sites From: Byrdwell, W.C., Neff, W.E., “Qualitative and Quantitative Analysis of Triacylglycerols Using Atmospheric Pressure Chemical Ionization Mass Spectrometry”, in New Techniques and Applications In Lipid Analysis, R.E. McDonald and M.M. Mossoba, Ed., AOCS Press, Champaign, IL 1997.

  7. [AB]+ [MH]+ [MH]+ 1 1 1 [AB]+ [AB]+ [AA]+ [AA]+ [AA]+ [MH]+ [AB]+ 1 [AA]+ [MH]+ Polyunsaturated TAG Saturated TAG APCI-MS Mass Spectra of ABA/AAB TAG Type II TAG • Type II (ABA/AAB) TAG give three ions: • Protonated Molecule, [M+H]+ • Two Diacylglycerol ions, [DAG]+ The proportion of the Protonated Molecule, [M+H]+, to the Diacylglycerol ions, [DAG]+, depends on the amount of unsaturation in the TAG. e.g. LnOLn/LnLnO e.g. SSP

  8. [MH]+ [AB]+ [MH]+ 1 1 1 [AB]+ [AB]+ [AA]+ [AA]+ [MH]+ [AA]+ ABA TAG [MH]+ [AB]+ 1 1 [AB]+ [AA]+ [AA]+ [MH]+ AAB TAG Saturated TAG APCI-MS Mass Spectra of ABA/AAB TAG Type II TAG • Type II (ABA/AAB) TAG give three ions: • Protonated Molecule, [M+H]+ • Two Diacylglycerol ions, [DAG]+ The proportion of the Diacylglycerol fragment ions, [DAG]+, depends on the location of the Fatty Acid (FA) in the TAG. The proportion of the Protonated Molecule, [M+H]+, to the Diacylglycerol ion, [DAG]+, depends on the amount of unsaturation in the TAG. e.g. LnLnO e.g. LnOLn

  9. [AB]+ [MH]+ [MH]+ 1 1 1 [BC]+ [AB]+ [AB]+ [AC]+ [BC]+ [BC]+ [AC]+ [AC]+ [MH]+ [AB]+ 1 [BC]+ [AC]+ e.g. LnLS [MH]+ Polyunsaturated TAG Saturated TAG APCI-MS Mass Spectra of ABC TAG Type III TAG • Type III (ABC) TAG give three ions: • Protonated Molecule, [M+H]+ • Three Diacylglycerol ions, [DAG]+ The proportion of the Protonated Molecule, [M+H]+, to the Diacylglycerol ions, [DAG]+, depends on the amount of unsaturation in the TAG. e.g. SPM

  10. [AB]+ [MH]+ 1 1 [BC]+ [AC]+ [AB]+ 1 1 [AC]+ [BC]+ [AC]+ [AB]+ [MH]+ [BC]+ [MH]+ CAB TAG [MH]+ 1 1 [AB]+ [BC]+ [AC]+ ABC TAG APCI-MS Mass Spectra of ABC TAG Type III TAG • Type III (ABC) TAG give three ions: • Protonated Molecule, [M+H]+ • Three Diacylglycerol ions, [DAG]+ The proportion of the Diacylglycerol fragment ions, [DAG]+, depends on the location of the Fatty Acid (FA) in the TAG. The [DAG]+ from the sn-1,3 regioisomer is energetically disfavored. It is expected to be the smallest [DAG]+ ion.

  11. [AB]+ [AB]+ 1 1 [AA]+ [AA]+ [MH]+ [MH]+ [MH]+ 1 [AA]+ [AB]+ Should be no AA > AB APCI-MS Mass Spectra of ABA/AAB TAG Type II TAG For Example: PPO = 0.87 For Example: POP = 0.29 The proportion of the Diacylglycerol fragment ions, [DAG]+, depends on the location of the Fatty Acid (FA) in the TAG.

  12. They used the ratio of [LL]+ [OL]+ versus the percentage of regioisomers Quantification of Regioisomers • Jakab, Jablonkai and Forgacs showed a linear calibration line for LOL/LLO isomers LLO LOL

  13. 75 70 65 60 55 50 [LL]+/[LO]+ (%) 45 We could make the ratio of [LL]+ [OL]+ versus the percentage of LLO go from Low to High 40 35 30 25 20 15 0 10 20 30 40 50 60 70 80 90 100 LLO concentration (% v/v, 100*LLO/(LOL+LLO)) Quantification of Regioisomers • The calibration line could be converted to go from low to high. LOL LLO

  14. 75 70 65 60 55 50 [LL]+/[LO]+ (%) 45 40 35 30 25 20 15 0 10 20 30 40 50 60 70 80 90 100 LLO concentration (% v/v, 100*LLO/(LOL+LLO)) Quantification of Regioisomers • When the plot is converted to go from low to high, any observed ratio should fall on the line.

  15. 75 70 65 60 55 50 [LL]+/[LO]+ (%) 45 40 35 30 25 20 15 0 10 20 30 40 50 60 70 80 90 100 LLO concentration (% v/v, 100*LLO/(LOL+LLO)) Quantification of Regioisomers • The equation for the calibration line can be found in terms of %LLO:

  16. 75 70 65 60 55 50 [LL]+/[LO]+ (%) 45 40 35 30 25 20 15 0 10 20 30 40 50 60 70 80 90 100 LLO concentration (% v/v, 100*LLO/(LOL+LLO)) Quantification of Regioisomers • Or, the equation for the calibration line can be found in terms of % LOL:

  17. The Critical Ratios for ABA/AAB TAG • The [AA]+/[AB]+ ratio provides information to be able to quantify the amounts of regioisomers. • The [MH]+/S[DAG]+ ratio provides information about the amount of unsaturation in a TAG. • These ratios act a ‘Critical Ratios’ to provide structural information about a Type II TAG.

  18. The Critical Ratios for ABA/AAB TAG • The Critical Ratios also constitute a ‘Reduced Data Set’ from which the mass spectrum can be reproduced • These two ratios contain all of the information necessary to reproduce the protonated molecule, [M+H]+, and two [DAG]+ fragment ions.

  19. The Critical Ratios for ABC TAG • The [AC]+/([AB]++[BC]+) ratio provides information to be able to quantify the amounts of regioisomers. • The [MH]+/S[DAG]+ ratio provides information about the amount of unsaturation in a TAG. • These ratios act as ‘Critical Ratios’ to provide structural information about a Type III TAG.

  20. The Critical Ratios for ABC TAG • The Critical Ratios also constitute a ‘Reduced Data Set’ from which the mass spectrum can be reproduced • These three ratios contain all of the information necessary to reproduce the protonated molecule, [M+H]+, and three [DAG]+ fragment ions.

  21. [AA]+ [MH]+ 1 1 [MH]+ [AA]+ [AA]+ [MH]+ 1 The Shape of the AAA TAG Lipidome [MH]+ Base Peak Case 2 Critical Value Case 1 [AA]+ Base Peak

  22. The Bottom Up Solution foran AAA TAG

  23. [AA]+ [AB]+ [MH]+ [MH]+ 1 1 [AB]+ [AA]+ [AB]+ 1 [AA]+ [MH]+ The Critical Value for the ABA TAG Lipidome [MH]+ Base Peak Case 2 Critical Value Case 1 [DAG]+ Base Peak

  24. [AB]+ [MH]+ [MH]+ 1 1 [AB]+ [AA]+ [AA]+ [AB]+ 1 [AA]+ [MH]+ The Critical Limit for the ABA TAG Lipidome [MH]+ Base Peak Case 2 Critical Limit Case 1 [DAG]+ Base Peak

  25. 75 70 65 60 55 50 [AA]+/[AB]+ (%) 45 40 35 30 25 20 15 0 10 20 30 40 50 60 70 80 90 100 AAB concentration (% v/v, 100*AAB/(ABA+AAB)) Quantification of Regioisomers • A calibration line for % ABA can be found

  26. The Shape of the Type II TAG Lipidome Case 2 1 1 Case 2.4 Case 2.3 Case 1.4 0 0 1 Case 1.3 0 Case 1

  27. N Y Case 2.0 Y Y % [M+H]+ = 100 % (base peak) N N Case 1.0 a [DAG]+ = 100 % (base peak) Case n.3 Y Case 1.3 †Implies strong [AA]+ preference or large difference in FA unsat. Case n.4 % [AB]+ = 100 % (base peak) Case 2.0 % [M+H]+ = 100 % (base peak) N† Y [AA]+ < [AB]+ Case 2.3 Same for Both Cases: Case 1.4† N† % [AA]+ = 100 % (base peak) Case 2.4† [AA]+³ [AB]+

  28. The Shape of the Type III TAG Lipidome Case 2 [M+H]+ Base Peak Case 2.4 CL2 CV 2 Case 2.3 Case 1.4 Case 1.3 [DAG]+ base peak Case 1

  29. Critical Ratios and Mass Spectra for Type I TAG

  30. Critical Ratios and Mass Spectra for a Type II TAG N Y Case 2.0 Y Y % [M+H]+ = 100 % N N Case 1.0 a [DAG]+ = 100 % Case n.3 Y Case 1.3 Case n.4 % [AB]+ = 100 % Case 2.0 % [M+H]+ = 100 % N Y Case 2.3 Same for Both Cases: Case 1.4† N % [AA]+ = 100 % Case 2.4†

  31. Critical Ratios and Mass Spectra for a Type II TAG Case 2 Case 2.4 Case 2.3 Case 1.3 Case 1

  32. Mass Spectra from Critical Ratiosfor a Type II TAG

  33. Mass Spectra from Critical Ratiosfor a Type III TAG

  34. Conclusions • Critical Ratios provide structural information, such as the percentage of regioisomers. • The Bottom Up Solution allows data to be expressed as Critical Ratios, to provide information at face value. • The mass spectrum of any TAG can be reproduced from its Critical Ratios.

  35. Conclusions • Critical Ratios constitute a ‘reduced data set’. • It takes fewer Critical Ratios to express the data than the raw abundances. • The Critical Ratios provide more information in fewer values than raw abundances alone.

  36. Acknowledgements • Thanks to William E. Neff for his years of collaboration and friendship • Thanks to Kathleen Warner, Sevim Erhan, and the National Center for Agricultural Utilization Research (NCAUR) in Peoria, IL for collaboration and loan of a triple-stage quadrupole (TSQ) instrument • For more information see: http://Byrdwell.com or http://TheBottomUpSolution.com

  37. Other Web Links • Mass Lists for fragments from APCI-MS of triacylglycerols (TAG) can be found at: • http://www.triacylglycerols.com • Mass Lists for fragments from APCI-MS of sphingolipids can be found at: • http://www.sphingomyelin.com • More information regarding LC/MS analysis of TAG can be found at: • http://www.hplc-ms.com

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