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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
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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?
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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
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Shotgun lipidomics for analyses of cardiolipin
molecular species
Identification and Quantitation
(Applications)J. Lipid Res. 47 (2006), 864
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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
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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.
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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)
• ……
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Identification
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• Doubly charged chemical nature
• ESI mass spectrometers with modest to high mass resolution (both QqQ- and QqTOF type)
Identification: The basics of the technique
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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)
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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)
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Identification: Negative-ion ESI/MS analysis of a lipid extract of mouse heart by direct infusion utilizing a QqTOF-type
instrument
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Identification: Product-ion analysis of cardiolipin molecular species in mouse heart lipid extract utilizing a QqQ-type instrument (FWHM 0.3 Th)
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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)
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Quantitation
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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
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Quantitation: Quantitative analyses of equimolar mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqTOF-type instrument
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Quantitation: Quantitative analyses of equimolar mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqTOF-type instrument
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Quantitation: Quantitative analyses of equimolar mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqTOF-type instrument
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Quantitation: De-isotoping of cardiolipin molecular species based on [M+1]2- isotopomer intensity (area)
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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)
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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)
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Quantitation: The effects of “ion suppression” on cardiolipin quantitation by shotgun lipidomics
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Applications
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Application: Shotgun lipidomics analyses of mouse skeletal muscle cardiolipin molecular species
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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