260 likes | 410 Views
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?.
E N D
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? Individual Molecular Species of Lipids Shotgun Lipidomics ESI/MS Cellular Lipid Extract(s) of a Biological Sample
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) 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.
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: The basics of the technique • Doubly charged chemical nature • ESI mass spectrometers with modest to high mass resolution (both QqQ- and QqTOF type)
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: 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
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)
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 …… …… ……
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) + ……]
Quantitation: The effects of “ion suppression” on cardiolipin quantitation by shotgun lipidomics
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