Mass-Spectrometric Analysis of Lipids (Lipidomics) 1. Identification 2. Quantification 3. Metabolism
Why to do lipidomics? Biology: Functions of different lipids? Medicine: Diagnostics and Therapy Industry: ealthier food, Quality control
GlyceroPhospholipids P 2 C C C 2 N >10 classes (PC, PE, PS, PI, PA etc) - Each class consists of numerous species with different fatty acid combinations (>20 different fatty acids) => Thousands of different molecular species possible Phosphatidylcholine (PC)
Neutral Glycerolipids - Triacylglycerols (TG) - Diacylglycerols (DG) - Monoacylglycerols (MG) -Each class consists of numerous species due to different fatty acid combinations => undreads of different molecular species TG DG
Sphingolipids - Ceramides - Neutral Glycosphingolipids - Acidic Glycosphingolipids -Each class consists of numerous species due to different fatty acid => undreads of different molecular species Lactosylceramide Ganglioside Sulfatide
The complete lipidome of no cell or tissue has ever been determined...because of technical limitations
Advantages of MS analysis Sensitivity >1000-fold higher than with conventional methods Resolution - Allows quantification of hundreds of lipid species Speed -100 times faster Can be automated - igh troughput possible
Ionization methods used in lipid MS Electrospray (ESI) Does not cause fragmentation Can be easily automated Compatible with on-line LC Matrix-assisted laser desorption (MALDI) Less used thus far Suppression by PC/SM > All lipids not detected n-line LC separation not feasible
Electrospray ionization Competition for charge => Suppression effects!
MS spectrum of cellular lipid extract = a Mess! Intens. PE 18:0/18:2 6000 PS 18:0/18:1 PE 18:0/20:4 PE 18:0/22:5 4000 PE 16:0/18:1 PE 18:0/20:4 LBPA 18:1/18:1 PI 18:0/2 2000 PA 18:0/18:2 PI!8:0/18:2 0 700 725 750 775 800 825 850 875 m/z Scanning MS1 Collision cell MS2 Detector
ow to improve selectivity? A. Lipid class -specific scanning (MS/MS) B. n-line chromatographic separation (LC-MS)
Lipid class -specific scanning Phospholipid class consist of species with the same polar head-group but different fatty acids Phospholipid class Specific scan Phosphatidylcholines Precursors of +184 Phosphatidylinositols Precursors of -241 Phosphatidylethanolamines Neutral-loss of 141 Phosphatidylserines Neutral-loss of 87
Precursor ion scanning Requires a characteristic, charged product ion PC => Diglyceride + phosphocholine (+184) Scanning Fragmentation Static (+184) MS1 Collision cell (elium or Argon) MS2
Precursors of +184 => PC + SM SM-16:0 -Alkaline hydrolysis can be used to remove PCs
Neutral-loss scanning..when the characteristic fragment is uncharged PE => Diglyceride (+) + phosphoethanolamine (141) Mass interval = 141 Scanning Fragmentation Scanning MS1 Collision cell (elium or Argon) MS2
Neutral-loss of 141 (= PE) 700 750 800 Intens. PE 18:0/18:2 MS-scan 6000 PS 18:0/18:1 PE 18:0/20:4 PE 18:0/22:5 4000 PE 16:0/18:1 PE 18:0/20:4 LBPA 18:1/18:1 PI 18:0/2 2000 PA 18:0/18:2 PI!8:0/18:2 0 700 725 750 775 800 825 850 875 m/z
MS analysis of Sphingolipids
C AcN C 3 S 3 - C 2 C N C C C 2 C N C C C 2 C N C C AcN C 2 C C C N C 2 C N C C 3 S 3 - Sphingosine Ceramide Lactosylceramide Ganglioside Sulfatide
Ceramide and Neutral Glycosphingolipids - Precursors of sphingosine (m/z +264) Glucosylceramides Ceramides 24:1
Sulfatides - Precursors of Sulfate (m/z -97)
Liquid chromatography-ms (LC-MS) Advantages - Increased sensitivity due to diminished suppression of minor species by - Major species - Impurities Disadvantages Takes more time (not UPLC) Data analysis more complex (?)
LC-MS analysis of mouse brain lipids Time ermansson et al. (2005) Anal Chem.77:2166-75
Data analysis => software A. Processing of the data => Identification => Concentrations B. Bioinformatics => Biomarkers? =>Biological significance?
Quantification not simple Signal intensity depends on: Lipid head-group Acyl chain length Acyl chain unsaturation Ions present (adduct formation) Detergent and other impurities (suppression) Solvent composition and instrument settings => Internal standards necessary!
LIMSA Excel add-on for Quantitative Analysis of MS data (aimi et al..2006. Anal Chem. 78:8324-31) LIMSA does: Peak picking and fitting Peak overlap correction Peak assignment (database of >3000 lipids) Quantification with internal standards Batch analysis
MS-imaging of Lipids by MALDI PI 38:4 Sulfatide 24:1 ydroxy-sulfatide 24:1
Analysis of Lipid Metabolism by MS Adds another, dynamic dimension to lipidomics Labeled lipids can be selectively detected! Precursors D 9 -PC => +193 (Unlabeled PC => +184) D 4 -PE => 145 (Unlabeled PE => 141) D 4 -PS => 90 (Unlabeled PS => 87) D 6 -PI => -247 (Unlabeled PI => -241) Water soluble precursors (D 9 -choline etc) Exogenous lipids
Phospholipid Remodeling: Exchange of acyl chains P 4 Glycerol Fatty acid Fatty acid Alcohol Glycerol Fatty acid Fatty acid Alcohol PLA 2 P 4 P 4 Glycerol Fatty acid Alcohol Acyl transferase
Analysis of phospholipid remodeling using soluble precursor is problematic D 4 -ethanolamine => cells => D 4 -PE species 18:1/18:1 100 16:0/16:1 16:0/17:1 16:0/18:1 18:0/18:1 0h Kinetics 100 50 1h Relative intensity (%) 100 100 5h 24h Relative abundance (%) 25 16:0/16:1 16:0/17:1 16:0/18:1 16:1/18:1 18:0/18:1 18:1/18:1 18:1/18:2 18:0/20:4 18:1/20:4 18:0/22:6 100 0 0 5 10 15 20 25 unlabeled Chase time (h) 680 700 720 740 760 780 800 m/z
ur approach: Use intact exogenous phospholipids with a deuterium-labeled head-group PRTCL Synthesize a phospholipid with a deuterium-labeled head group Make vesicles containing the labeled phospholipid Incubate cells with these vesicles and β-cyclodextrin (carrier) Extract and analyze lipids using MS/MS scans showing the labeled (or unlabeled) lipid only Determine the pathways and kinetics of remodeling
Unnatural 14:0/14:0-PE is remodeled very rapidly 100% 14:0/14:0-D 4 -PE 14:0/14:0 KINETICS 0h 100% 16:0/14:0 14:0/18:1 14:0/20:4 18:0/18:1 3h 50 Relative Intensity 100% 100 16:0/18:1 18:1/18:1 18:0/20:4 18:1/20:4 7h 24h 25 14:1/14:1 16:0/14:1 18:0/14:1 18:1/14:1 16:0/18:1 14:1/20:4 18:0/18:1 18:1/18:1 18:0/20:4 18:1/20:4 100% 0 0 5 10 15 20 25 Endogenous 700 750 800 850 m/z
Natural 18:1/18:1-PE is hardly remodeled 100% 18:1/18:1 0 h 100% Relative abundance 100% 100% 100% 1 h 3 h 5 h 24 h 100% endogenous 700 750 800
Positional isomers are remodeled with very different kinetics 14:0/18:1 18:1/14:0 100% 14:0/18:1 100% 18:1/14:0 18:1/18:1 18:1/18:1 0h 16:1/16:1 18:1/16:1 18:1/18:2 18:1/20:4 18:1/22:6 0h 100% 700 750 800 100% 700 750 800 Relative abundance 100% 18:1/18:2 18:1/18:1 3h 700 750 800 100% 700 750 800 3h 16:1/18:1 16:0/18:1 7h 7h 100% 700 750 800 100% 700 750 800 endogenous endogenous 700 750 800 m/z 700 750 800 m/z
Pathways of 14:0/14:0-PE remodeling 14:0/14:0 16:1 /18:2 14:0/ 18:2 14:0/ 16:1 14:0/ 18:1 14:0/ 20:4 14:0/ 22:6 16:0 /18:2 18:1 /18:2 18:1 /16:1 16:0 /18:1 16:1 /18:1 18:1 /20:4 18:1 /22:6 18:1 /18:1 18:0 /20:4 Kainu et al. (2008) J Biol Chem. 283:3676-87
Studies with >50 phospholipid species (and PLA inhibitors) indicate that => Multiple acyl chain specific PLAs are involved in remodeling of phospholipids in mammalian cells BUT which PLAs?..and what determines their specificity?..and which acyltransferases are involved?
Conclusions MS-based lipidomics is highly usefull in Biology Medicine Food industry...but needs to be integrated with other omics and functional assays eavy isotope labeling adds an important extra dimension to lipidomics
Contributors Martin ermansson Ville Kainu Perttu aimi