Division of Bioorganic Chemistry

and Molecular Pharmacology

Department of Medicine

Washington University School of Medicine - St. Louis

Shotgun Lipidomics of Cardiolipin Molecular Species

Xianlin Han, Kui Yang, Jingyue Yang, Hua Cheng, and Richard W. Gross

Cellular Lipid Extract(s) of a

Biological Sample

Individual Molecular

Species of Lipids

ESI/MSShotgun Lipidomic

s

What is shotgun lipidomics?What is shotgun lipidomics?

Shotgun lipidomics includes:

• Multiplexed extractions

• Intrasource separation and selective ionization

• Multi-dimensional MS identification

• Quantitation using a two-step procedure

• Bioinformatics and data processing

J. Lipid Res. 44 (2003), 1071-1079;Mass Spectrom. Rev. 24 (2005), 367-412;Expert Rev. Proteomics 2 (2005) 253-264

Shotgun lipidomics for analyses of cardiolipin

molecular species

Identification and Quantitation

(Applications)J. Lipid Res. 47 (2006), 864

Shotgun lipidomics for analyses of cardiolipin

molecular species

Identification and Quantitation

(Applications)

Hsu, F.F., et al. (2005) J. Am. Soc. Mass Spectrom. 16, 491;Valianpour, F. (2002) Clin. Chem. 48, 1390;Sparagna, G.C. (2005) J. Lipid Res. 46, 1196.

J. Lipid Res. 47 (2006), 864

What is cardiolipin?

• One minor phospholipid class, almost exclusively present in mitochondria;

• Two phosphates;

• Three glycerols;

• Four identical/almost identical acyl chains predominant in most of mammalian tissues.

Cardiolipin is a class of very important phospholipids:

• Highly anionic: interact with different cations and proteins, and influence ion channel function

• Large aliphatic chain to polar head group volume: promote membrane fusion and fission

• Specific binding with proteins in ETC;

• Anchoring cytochrome c (apoptosis)

• Barth’s syndrome (altered cardiolipin profile)

• ……

Identification

• Doubly charged chemical nature

• ESI mass spectrometers with modest to high mass resolution (both QqQ- and QqTOF type)

Identification: The basics of the technique

Identification: Negative-ion ESI/MS analysis of a lipid extract of mouse heart by direct infusion utilizing a QqQ-type instrument (FWHM 0.3 Th)

Identification: Negative-ion ESI/MS analysis of a lipid extract of mouse heart by direct infusion utilizing a QqQ-type instrument (FWHM 0.3 Th)

Identification: Negative-ion ESI/MS analysis of a lipid extract of mouse heart by direct infusion utilizing a QqTOF-type

instrument

Identification: Product-ion analysis of cardiolipin molecular species in mouse heart lipid extract utilizing a QqQ-type instrument (FWHM 0.3 Th)

Identification: 2D MS analysis of some representative building blocks of mouse heart cardiolipin molecular species after direct infusion utilizing a QqQ-type instrument (FWHM 0.3 Th)

Quantitation

Quantitation: Quantitative analyses of different molar ratio mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqQ-type instrument

Dynamic range of quantitation

Linear correlation

Quantitation: Quantitative analyses of equimolar mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqTOF-type instrument

Quantitation: Quantitative analyses of equimolar mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqTOF-type instrument

Quantitation: Quantitative analyses of equimolar mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqTOF-type instrument

Quantitation: De-isotoping of cardiolipin molecular species based on [M+1]2- isotopomer intensity (area)

Isotopomers Isotope Ratio Peak Intensity (area)

M 1 92.42 I1/nM+1 0.01082n I1

M+2 0.010822n(n-1)/2 0.00541(n-1)I1

…… …… ……

Quantitation: De-isotoping of cardiolipin molecular species based on [M+1]2- isotopomer intensity (area)

Isotopomers Isotope Ratio Peak Intensity (area)

M 1 92.42 I1/nM+1 0.01082n I1

M+2 0.010822n(n-1)/2 0.00541(n-1)I1

…… …… ……

De-isotoping based on [M+1]2- peak intensity (area):

Itotal=I1 x [92.42/n +1 + 0.00541(n - 1) + 1.95x10-5(n -1)(n - 2) + ……]

Quantitation: De-isotoping of cardiolipin molecular species based on [M+1]2- isotopomer intensity (area)

Quantitation: The effects of “ion suppression” on cardiolipin quantitation by shotgun lipidomics

Applications

Application: Shotgun lipidomics analyses of mouse skeletal muscle cardiolipin molecular species

Conclusion:

• Cardiolipin molecular species can be “recognized” by searching for the doubly-charged ions

• Cardiolipin molecular species can be identified by the specific neutral loss of ketenes from the doubly-charged molecular ions

• Cardiolipin molecular species can be quantitated after de-isotoping based on [M+1]2- isotopomer intensity (area)

• Quantitation of cardiolipin can be performed using both QqQ- and QqTOF-type instruments with care

• “Ion suppression” does not affect quantitation of cardiolipin at the low concentration region using shotgun lipidomics