The Bottom Up Solution to the Triacylglycerol Lipidome

William Craig Byrdwell

Florida Atlantic UniversityDept. of Chemistry & Biochem.

Boca Raton, FL 33432

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

MS

MMM

MM

MS

S SS

SS

SSM

M

H3O+ H2O

(M+H)+

H3O+

H2O

N4+N2

e-

++ ++ ++ ++

N2+

e- N4+

H2O

N2

O2

H2ON2

N 2+

N2+N2

e-

N4+

H2O+

H2O

(H2O)nH+

CoronaDischargeRegion

CoronaDischargeElectrode

Positive High Voltage

SampleIntroductionTube (Inner)

AuxiliaryGas

SheathGas Tube(Outer)

Vaporizer Heater

Sample (M)andSolvent (S)Vapor

HeatedCapillary

N2

H3O+

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• Triacylglycerols (TAG)

give simple mass spectra by APCI-MS which contain :

H2C

CH

H2C

O O

OC

C

C+HO

O

O

– A protonated molecule ion, [M+H]+

– Diacylglycerol-like fragment ions, [DAG]+

C57H103O6+

Exact Mass: 883.78[M+H]+

OLO

C39H69O4 +

Exact Mass: 601.52[OL]+

C39H71O4 +

Exact Mass: 603.54[OO]+

H2C

CH

H2C

O O

C CO +HO

H2C

CH

H2C

O

OC C

+HO

O

• For Example, [OL]+

• and, [OO]+

Saturated TAG

1

[MH]+

[AA]+

e.g. LnLnLn

1[MH]+

[AA]+

Polyunsaturated TAG

APCI-MS Mass Spectra of AAA TAG1

[MH]+

[AA]+

1

[MH]+

[AA]+

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 unsaturationin the TAG.

Type I TAG

e.g. SSS

The MH/DAG varies with # SitesTAG Quotient vs. # Sites of Unsaturation

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.8000

0.9000

0 1 2 3 4 5 6 7 8 9

# Sites

TAG

Quo

tient

From: Byrdwell, W.C., Neff, W.E., “Qualitative and Quantitative Analysis of Triacylglycerols UsingAtmospheric Pressure Chemical Ionization Mass Spectrometry”, in New Techniques and ApplicationsIn Lipid Analysis, R.E. McDonald and M.M. Mossoba, Ed., AOCS Press, Champaign, IL 1997.

e.g. LnOLn/LnLnO

1

[MH]+

[AB]+

[AA]+

Saturated TAG

APCI-MS Mass Spectra of ABA/AAB TAG

The proportion of the Protonated Molecule, [M+H]+, to the Diacylglycerol ions, [DAG]+, depends on the amount of unsaturationin the TAG.

Type II TAG

1[MH]+

[AB]+

[AA]+

1

[MH]+

[AB]+

[AA]+

Polyunsaturated TAG

1[MH]+

[AB]+

[AA]+

Type II (ABA/AAB) TAG give three ions: Protonated Molecule, [M+H]+

Two Diacylglycerol ions, [DAG]+

e.g. SSP

The proportion of the Protonated Molecule, [M+H]+, to the Diacylglycerol ion, [DAG]+, depends on the amount of unsaturationin the TAG.

e.g. LnLnO

The proportion of the Diacylglycerol fragment ions, [DAG]+, depends on the location of the Fatty Acid (FA) in the TAG.

AAB TAG

1[MH]+

[AB]+

[AA]+

APCI-MS Mass Spectra of ABA/AAB TAG

Type II TAG

1[MH]+

[AB]+

[AA]+

1

[MH]+

[AB]+

[AA]+

Type II (ABA/AAB) TAG give three ions: Protonated Molecule, [M+H]+

Two Diacylglycerol ions, [DAG]+

e.g. LnOLn

1

[MH]+

[AB]+

[AA]+

Saturated TAGABA TAG

1[MH]+

[AB]+

[AA]+

APCI-MS Mass Spectra of ABC TAG

The proportion of the Protonated Molecule, [M+H]+, to the Diacylglycerol ions, [DAG]+, depends on the amount of unsaturation in the TAG.

Type III TAG

1[MH]+

[AB]+

[AC]+[BC]+

1

[MH]+

[AB]+

[AC]+

[BC]+

Type III (ABC) TAG give three ions: Protonated Molecule, [M+H]+

Three Diacylglycerol ions, [DAG]+

e.g. SPM

Polyunsaturated TAG

1[MH]+

[AB]+

[AC]+[BC]+

e.g. LnLS

Saturated TAG

[MH]+

[AB]+

[AC]+

[BC]+1

The [DAG]+ from the sn-1,3 regioisomer is energetically disfavored. It is expected to be the smallest [DAG]+ ion.

The proportion of the Diacylglycerol fragment ions, [DAG]+, depends on the location of the Fatty Acid (FA)in the TAG.

1

ABC TAG

1[MH]+

[AB]+

[AC]+

[BC]+

11

CAB TAG

[MH]+

[AB]+

[AC]+

[BC]+

APCI-MS Mass Spectra of ABC TAG

Type III TAG

1[MH]+

[AB]+

[AC]+[BC]+

1

[MH]+

[AB]+

[AC]+

[BC]+

Type III (ABC) TAG give three ions: Protonated Molecule, [M+H]+

Three Diacylglycerol ions, [DAG]+

1[MH]+

[AB]+

[AA]+

Should be no AA > AB

The proportion of the Diacylglycerol fragment ions,

[DAG]+, depends on the location of the Fatty Acid (FA)

in the TAG.

APCI-MS Mass Spectra of ABA/AAB TAG

Type II TAG

1

[MH]+

[AB]+

[AA]+1

[MH]+

[AB]+

[AA]+

For Example:PPO = 0.87

For Example:POP = 0.29

H2C

CH

H2C

O O

OC

C

C+HO

O

O

Quantification of Regioisomers• Jakab, Jablonkai and Forgacs showed a linear

calibration line for LOL/LLO isomersH2C

CH

H2C

O O

OC

C

C+HO

O

O

LOL

They used the ratio of

[LL]+

[OL]+

versus the

percentageof

regioisomers

LLO

H2C

CH

H2C

O O

OC

C

C+HO

O

O

We could make the ratio of

[LL]+

[OL]+

versus the

percentageof LLO go from

Low to High

Quantification of Regioisomers• The calibration line could be converted to go

from low to high.H2C

CH

H2C

O O

OC

C

C+HO

O

O

LLO100 20 30 40 50 60 70 80 90 100

1520253035

4045505560657075

LLO concentration (% v/v, 100*LLO/(LOL+LLO))

[LL]

+ /[LO

]+ (%

)

LOL

Quantification of Regioisomers• When the plot is converted to go from low to

high, any observed ratio should fall on the line.

100 20 30 40 50 60 70 80 90 1001520253035

4045505560657075

LLO concentration (% v/v, 100*LLO/(LOL+LLO))

[LL]

+ /[LO

]+ (%

)

ABA

[LO][LL]

AAB

[LO][LL]

Obs

[LO][LL]

Quantification of Regioisomers

• The equation for the calibration line can be found in terms of %LLO:

100 20 30 40 50 60 70 80 90 1001520253035

4045505560657075

LLO concentration (% v/v, 100*LLO/(LOL+LLO))

[LL]

+ /[LO

]+ (%

)

ABA

[LO][LL]

AAB

[LO][LL]

Obs

[LO][LL]

100

[AB][AA]

[AB][AA]

[AB][AA]

[AB][AA]

LLO %

ABAAAB

ABA xObs

Quantification of Regioisomers

• Or, the equation for the calibration line can be found in terms of % LOL:

100 20 30 40 50 60 70 80 90 1001520253035

4045505560657075

LLO concentration (% v/v, 100*LLO/(LOL+LLO))

[LL]

+ /[LO

]+ (%

)

ABA

[LO][LL]

AAB

[LO][LL]

Obs

[LO][LL]

100

[AB][AA]

[AB][AA]

[AB][AA]

[AB][AA]

LOL %

ABAAAB

AAB xObs

The Critical Ratios for ABA/AAB TAG

• The [AA]+/[AB]+ ratio provides information to be able to quantify the amounts of regioisomers.

• The [MH]+/[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.

[AB][AA]

[DAG][MH]

The Critical Ratios for ABA/AAB TAG

• The Critical Ratios also constitute a ‘Reduced Data Set’ from which the mass spectrum can be reproduced

[AB][AA]

[DAG][MH]

• These two ratios contain all of the information necessary to reproduce the protonated molecule, [M+H]+, and two [DAG]+ fragment ions.

• The [AC]+/([AB]++[BC]+) ratio provides information to be able to quantify the amounts of regioisomers.

• The [MH]+/[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.

)]BC[([AB][AC]

[DAG][MH]

]AB[[BC]

The Critical Ratios for ABC TAG

The Critical Ratios for ABC TAG• The Critical Ratios also constitute a ‘Reduced

Data Set’ from which the mass spectrum can be reproduced

[DAG][MH]

• These three ratios contain all of the information necessary to reproduce the protonated molecule, [M+H]+, and three [DAG]+ fragment ions.

]AB[[BC]

)[BC]([AB]

[AC]

The Shape of the AAA TAG Lipidome

Case 2

1 [DAG][MH]

1[MH]+

[AA]+

1

[MH]+

[AA]+

1[AA]+ [MH]+

Critical Value

[AA]+ Base Peak

[MH]+ Base Peak

Case 1

1 [DAG][MH]

1 [DAG][MH]

The Bottom Up Solution foran AAA TAG

[MH]+

[AA]+ 1

[MH]+

[AA]+

1

1 ?

[DAG][MH]

I) ‘AAA’ TAG Critical Value = 1

100

[DAG][MH]

1[DAG] % x

Y

% [M+H]+ = 100 % (base peak)

N

the [DAG]+ = 100 % (base peak)

100[DAG]

[MH]H][M % x

Case 2

Case 1

)

[DAG][MH]

1 1 ( )[DAG]

[MH] 1 ( )[DAG] ([MH] )(I

*

*11

or

Case 1 Case 2

The Critical Value for the ABA TAG Lipidome

Case 2

0.521

[DAG][MH]

Critical Value

[DAG]+ Base Peak

[MH]+ Base Peak

Case 1

Limit Critical [DAG][MH]

0.5 [DAG][MH]

1[AB]+ [MH]+[AA]+

1

[AB]+

[MH]+

[AA]+

1[AB]+

[MH]+

[AA]+

The Critical Limit for the ABA TAG Lipidome

Case 2

0.521

[DAG][MH]

Critical Limit

[DAG]+ Base Peak

[MH]+ Base Peak

Case 1

Limit Critical [DAG][MH]

C.L. [DAG][MH]

1[AB]+ [MH]+

[AA]+

1

[AB]+

[MH]+

[AA]+

1[AB]+

[MH]+

[AA]+

[AB][AA]

11

1

or

[AB][AA]1

1 [DAG][MH]for Limit Critical

Quantification of Regioisomers

• A calibration line for % ABA can be found

100 20 30 40 50 60 70 80 90 1001520253035

4045505560657075

AAB concentration (% v/v, 100*AAB/(ABA+AAB))

[AA

]+ /[A

B]+ (

%)

ABA

[AB][AA]

AAB

[AB][AA]

Obs

[AB][AA]

100

[AB][AA]

[AB][AA]

[AB][AA]

[AB][AA]

ABA %

ABAAAB

AAB xObs

0

The Shape of the Type II TAG Lipidome

Case 2.4

Case 1.4

Case 1.3

Case 2.3

0.521

[DAG][MH]

C.L.

[DAG][MH]

Case 1

Case 2

1 [AB][AA]

1 [AB][AA]

AAB[AB][AA]

ABA[AB][AA]

AAB[AB][AA]

ABA[AB][AA]

1

0

0

1

1

[DAG][MH]

[AB][AA]

Limit Critical?

[DAG][MH]

Value Critical?

[DAG][MH]

% [M+H]+ = 100 % (base peak)Case 2.0

1 ?

[DAG][MH]

YN

Y

N

Y

N

(usual) 1.0 ?

[AB][AA]

Y

N†

100[AB][AA]1

[DAG][MH]H][M % x

100[AB][AA] [AA] % x

% [AB]+ = 100 % (base peak)

100

[AB][AA]

1 [AB] % x

% [AA]+ = 100 % (base peak)

Case 1.3

Case 1.4†

a [DAG]+ = 100 % (base peak)Case 1.0

100

[AB][AA]

11[DAG]

[MH]H][M % x

[AB][AA]1

1

: (usual) 0.1 [AB][AA] if

Limit

†Implies strong [AA]+ preference or large difference in FA unsat.

[AB][AA]

11

1

: (unusual) 1.0 [AB][AA] if †

Limit

Case n.3

Case n.4

(usual) 1.0 ?

[AB][AA]

N†100

[AB][AA]1

[DAG][MH]

1 [AB] % x

100

[AB][AA]

11[DAG]

[MH]

1 [AA] % x

Case 2.4†

% [M+H]+ = 100 % (base peak)

YCase 2.3

Same for Both Cases:

[AA]+ < [AB]+

[AA]+ [AB]+

Case 2.0

CV 2

Case 2.4

0[BC][AB]

[AC]

1[BC][AB]

[AC]

2[BC][AB]

[AC]

LimitCritical

2 Limit CriticalObs

[BC][AB][AC]

0[DAG][MH]

1[DAG][MH]

Obs

[DAG][MH]

LimitCritical

[DAG][MH]

33.0[DAG][MH]

ValueCritical

Case 2 [M+H]+ Base Peak

[DAG]+ base peak Case 1

Case 1.3

1.3.6 Case , 1[AB][BC]

1.3.5 Case , 1[AB][BC]

1[AB][BC] 1.4.6, Case

1[AB][BC] 1.4.5, Case

2.4.6 Case , 1[AB][BC]

2.4.5 Case , 1[AB][BC]

Case 1.4CL2Obs

[BC][AB]

[AC]

1[BC][AB]

[AC]

5.0[BC][AB]

[AC]

2 ValueCritical

CL2

2 Limit CriticalObs

[BC][AB]

[AC]

Case 2.3

2.3.6 Case , 1[AB][BC]

2.3.5 Case , 1[AB][BC]

The Shape of the Type III TAG Lipidome

Critical Ratios and Mass Spectra for Type I TAG

TAG MH/DAG

PPP 0.00

SSS 0.00

OOO 3.24

LLL 120.07

LnLnLn 222.80

[MH]+ [AA]+ Case

PPP 0.00 100.00 1

SSS 0.00 100.00 1

OOO 3.24 100.00 1

LLL 100.00 83.28 2

LnLnLn 100.00 44.88 2

1 ?

[DAG][MH]

100

[DAG][MH]

1[DAG] % x

Y

% [M+H]+ = 100 % (base peak)

N

the [DAG]+ = 100 % (base peak)

100[DAG]

[MH]H][M % x

Case 2

Case 1

Critical Ratios and Mass Spectra for a Type II TAG

TAG MH/DAG AA/ABPPS/PSP 0.00 49.26

PPO/POP 0.55 65.28

PPL/PLP 0.40 84.11

PPLn/PLnP 12.81 69.94

SSP/SPS 0.00 40.91

SSO/SOS 0.45 75.14

SSL/SLS 0.42 82.89

SSLn/SLnS 9.59 60.98

OOP/POP 1.89 36.12

OOS/SOS 1.12 39.82

OOL/OLO 11.93 50.10

OOLn/OLnO 36.94 41.62

LLP/LPL 27.64 88.84

LLS/LSL 43.47 76.07

LLO/LOL 44.44 74.62

LLLn/LLnL 101.35 51.97

LnLnP/LnPLn 101.33 74.69

LnLnS/LnSLn 61.31 95.10

LnLnO/LnOLn 101.34 83.80

LnLnL/LnLLn 128.01 64.08

Limit Critical?

[DAG][MH]

Value Critical?

[DAG][MH]

% [M+H]+ = 100 %Case 2.0

1 ?

[DAG][MH]

YN

Y

N

Y

N

(usual) 1.0 ?

[AB][AA]

Y

N100

[AB][AA]1

[DAG][MH]H][M % x

100[AB][AA] [AA] % x

% [AB]+ = 100 %

100

[AB][AA]

1 [AB] % x

% [AA]+ = 100 %

Case 1.3

Case 1.4†

a [DAG]+ = 100 %Case 1.0

100

[AB][AA]

11[DAG]

[MH]H][M % x

[AB][AA]1

1

: (usual) 0.1 [AB][AA] if

Limit

[AB][AA]

11

1

: (unusual) 1.0 [AB][AA] if †

LimitCase n.3

Case n.4

(usual) 1.0 ?

[AB][AA]

N 100

[AB][AA]1

[DAG][MH]

1 [AB] % x

100

[AB][AA]

11[DAG]

[MH]

1 [AA] % x

Case 2.4†

% [M+H]+ = 100 %Y Case 2.3

Same for Both Cases:

Case 2.0

Critical Ratios and Mass Spectra for a Type II TAG

TAG MH/DAG AA/ABPPS/PSP 0.00 49.26

PPO/POP 0.55 65.28

PPL/PLP 0.40 84.11

PPLn/PLnP 12.81 69.94

SSP/SPS 0.00 40.91

SSO/SOS 0.45 75.14

SSL/SLS 0.42 82.89

SSLn/SLnS 9.59 60.98

OOP/POP 1.89 36.12

OOS/SOS 1.12 39.82

OOL/OLO 11.93 50.10

OOLn/OLnO 36.94 41.62

LLP/LPL 27.64 88.84

LLS/LSL 43.47 76.07

LLO/LOL 44.44 74.62

LLLn/LLnL 101.35 51.97

LnLnP/LnPLn 101.33 74.69

LnLnS/LnSLn 61.31 95.10

LnLnO/LnOLn 101.34 83.80

LnLnL/LnLLn 128.01 64.08

Case 2.4

Case 1.3

Case 2.3

Case 1

Case 2

1 [AB][AA]

1 [AB][AA]

AAB[AB][AA]

AAB[AB][AA]

AAB[AB][AA]

AAB[AB][AA]

Mass Spectra from Critical Ratiosfor a Type II TAG

TAG MH/DAG AA/ABPPS/PSP 0.00 49.26PPO/POP 0.55 65.28PPL/PLP 0.40 84.11

PPLn/PLnP 12.81 69.94SSP/SPS 0.00 40.91SSO/SOS 0.45 75.14SSL/SLS 0.42 82.89

SSLn/SLnS 9.59 60.98OOP/POP 1.89 36.12OOS/SOS 1.12 39.82OOL/OLO 11.93 50.10

OOLn/OLnO 36.94 41.62LLP/LPL 27.64 88.84LLS/LSL 43.47 76.07LLO/LOL 44.44 74.62

LLLn/LLnL 101.35 51.97LnLnP/LnPLn 101.33 74.69LnLnS/LnSLn 61.31 95.10LnLnO/LnOLn 101.34 83.80LnLnL/LnLLn 128.01 64.08

TAG MH AA ABPPS/PSP 0.00 49.26 100.00PPO/POP 0.91 65.28 100.00PPL/PLP 0.74 84.11 100.00

PPLn/PLnP 21.77 69.94 100.00SSP/SPS 0.00 40.91 100.00SSO/SOS 0.79 75.14 100.00SSL/SLS 0.77 82.89 100.00

SSLn/SLnS 15.44 60.98 100.00OOP/POP 2.57 36.12 100.00OOS/SOS 1.57 39.82 100.00OOL/OLO 17.91 50.10 100.00

OOLn/OLnO 52.31 41.62 100.00LLP/LPL 52.20 88.84 100.00LLS/LSL 76.54 76.07 100.00LLO/LOL 77.60 74.62 100.00

LLLn/LLnL 100.00 33.74 64.93LnLnP/LnPLn 100.00 42.19 56.49LnLnS/LnSLn 100.00 79.50 83.60LnLnO/LnOLn 100.00 44.99 53.69LnLnL/LnLLn 100.00 30.51 47.61

Case1.31.31.31.31.31.31.31.31.31.31.31.31.31.31.32.32.32.32.32.3

Case

1 3 5

1 3 5

1 3 5

1 3 5

1 3 5

2 3 5

1 3 5

1 3 5

2 3 5

2 3 5

Mass Spectra from Critical Ratiosfor a Type III TAG

TAG MH/DAG

AC/(AB+BC) BC/AB

SPO 0.47 37.64 69.79

SPL 0.46 35.39 59.43

SPLn 9.28 39.78 76.70

LOP 6.44 40.39 98.27

LnOP 27.18 42.00 91.76

LnLP 48.86 36.71 73.38

LOS 7.45 43.86 85.70

OLnS 19.95 42.47 89.53

LnLS 45.35 38.65 78.57

LLnO 56.61 40.18 77.66

TAG MH AC AB BCSPO 1.10 63.91 100.00 69.79

SPL 0.99 56.42 100.00 59.43

SPLn 22.92 70.29 100.00 76.70

LOP 17.93 80.08 100.00 98.27

LnOP 74.01 80.54 100.00 91.76

LnLP 100.00 54.96 86.35 63.36

LOS 19.90 81.45 100.00 85.70

OLnS 53.87 80.49 100.00 89.53

LnLS 100.00 61.47 89.06 69.98

LLnO 100.00 50.63 70.93 55.08

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.

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• 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

orhttp://TheBottomUpSolution.com

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