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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

The Bottom Up Solution to the Triacylglycerol Lipidome

William Craig Byrdwell

Florida Atlantic University

Dept. of Chemistry & Biochem.

Boca Raton, FL 33432


Overview
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


Atmospheric pressure chemical ionization
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


Apci ms of tag
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]+


Apci ms mass spectra of aaa tag

[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


The mh s dag varies with sites
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.


Apci ms mass spectra of aba aab tag

[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


Apci ms mass spectra of aba aab tag1

[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


Apci ms mass spectra of abc tag

[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


Apci ms mass spectra of abc tag1

[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.


Apci ms mass spectra of aba aab tag2

[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.


Quantification of regioisomers

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


Quantification of regioisomers1

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


Quantification of regioisomers2

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.


Quantification of regioisomers3

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:


Quantification of regioisomers4

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:


The critical ratios for aba aab tag
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.


The critical ratios for aba aab tag1
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.


The critical ratios for abc tag
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.


The critical ratios for abc tag1
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.


The shape of the aaa tag lipidome

[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



The critical value for the aba tag lipidome

[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


The critical limit for the aba tag lipidome

[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


Quantification of regioisomers5

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


The shape of the type ii tag lipidome
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


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]+


The shape of the type iii tag lipidome
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



Critical ratios and mass spectra for a type ii tag
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†


Critical ratios and mass spectra for a type ii tag1
Critical Ratios and Mass Spectra for a Type II TAG

Case 2

Case 2.4

Case 2.3

Case 1.3

Case 1




Conclusions
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.


Conclusions1
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.


Acknowledgements
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


Other web links
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