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Shotgun Lipidomics of Cardiolipin Molecular Species. Xianlin Han, Kui Yang, Jingyue Yang, Hua Cheng, and Richard W. Gross. Division of Bioorganic Chemistry and Molecular Pharmacology Department of Medicine Washington University School of Medicine - St. Louis. What is shotgun lipidomics?.

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slide1

Shotgun Lipidomics of Cardiolipin Molecular Species

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

Division of Bioorganic Chemistry

and Molecular Pharmacology

Department of Medicine

Washington University School of Medicine - St. Louis

slide2

What is shotgun lipidomics?

Individual

Molecular

Species of Lipids

Shotgun Lipidomics

ESI/MS

Cellular Lipid

Extract(s) of a

Biological Sample

slide3

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

slide4

Shotgun lipidomics for analyses of cardiolipin molecular species

Identification and Quantitation

(Applications)

J. Lipid Res. 47 (2006), 864

slide5

Shotgun lipidomics for analyses of cardiolipin molecular species

Identification and Quantitation

(Applications)

J. Lipid Res. 47 (2006), 864

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.

slide6

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.
slide7

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)
  • ……
slide9

Identification: The basics of the technique

  • Doubly charged chemical nature
  • ESI mass spectrometers with modest to high mass resolution (both QqQ- and QqTOF type)
slide10

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)

slide11

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)

slide12

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

slide13

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

slide14

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)

slide16

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

Linear correlation

Dynamic range of quantitation

slide17

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

slide18

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

slide19

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

slide20

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

slide21

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/n

M+1 0.01082n I1

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

…… …… ……

slide22

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/n

M+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) + ……]

slide23

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

slide25

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

slide26

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