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Quantification of Free Sterols, Sterol Esters, Sterol Glucosides and Acylated Sterol Glucosides in Plants by
Q TOF Mass SpectrometryQ-TOF Mass Spectrometry
Vera Wewer and Peter DörmannSeville Spain July 2012Seville, Spain, July 2012Institute of Molecular Physiology and Biotechnology of Plants (IMBIO)University of Bonn
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Phytosterols: Structural Variety
In plants, β-sitosterol and stigmasterol are predominent.Cholesterol: In plants, β sitosterol and stigmasterol are predominent.Cholesterol:the most abundant sterol in animals Campesterol is the precursor for brassinosteroid synthesis.
Ergosterol, a fungal sterol
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Conjugated Sterol lipids
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Sterol Distribution in the Plant Cell
Free Sterols (FS)
HO
Sterol Esters (SE)
Sterol Glucosides (SG) Acylated Sterol Glucosides (ASG)
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The need to develop a comprehensive method for sterol measurementssterol measurements
Changes of sterol lipids:
Abiotic stress: -P, -N, senescence,...Abiotic stress: P, N, senescence,...
Biotic stress: pathogens (bacteria, fungi), symbiotic interactions (mycorrhiza, nodulation)
Traditional method:
TLC separation + quantification of sterols or fatty acids by GC or GC-MS
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Strategy to measure all 4 sterol lipid classes
Direct infusion MS/MS similar to the lipidomics measurements by Ruth Welti
Avoid time consuming LC proceduresAvoid time consuming LC procedures
Nanospray (low sample amount): 1 µ/min
Lipid enrichment prior to MS: SPE with silica columns
Use two internal standards (small, large) to correct for size dependence of signalUse two internal standards (small, large) to correct for size dependence of signal
Use saturated standards to measure the plant sterols (which are unsaturated)
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Sterol Analysis: Workflow
TLC / GCQ-TOF MS
Wewer et al 2011Wewer et al., 2011J.Lip.Res.
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Tandem Mass Spectrometry
Direct infusion of lipid extract
Selection of desired ions in Q1
Fragmentation of selected ions in Q2 (MS/MS)
Time of Flight separation of fragment ions
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Problems to overcome: Standards
Free sterols: Saturated sterols: commercially available
Sterol Esters: cholesterol esters: commercial available
Sterol glucosides: synthesized from free saturated sterols and Ste o g ucos des sy t es ed o ee satu ated ste o s a dglucopyranosyl-bromide-tetrabenzoate
Acylated sterol saturated ASG: hydrogenation of ASG from soybeanglucosides
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Fragmentation Patterns of Sterols
Improve ionizationFree Sterols (FS)(betainylated)
pIntroduce good Leaving group
Use of cholesterol esters as internalSterol Esters (SE) esters as internal standards
Sterol glucosides (SG)g ( )
Saturated ASGs and SGs fragment differently than
Acylated sterol glucosides (ASG)
unsaturated ones
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ASG Molecular Species
ASG Arabidopsis leaf
16:0-Glc-Sitosterol
18:3-Glc-Sitosterol18:3-Glc-Sitosterol
18:2-Glc-Sitosterol
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SE Molecular Species
SEArabidopsis leaf
18:3-Sitosterol
18:2-Sitosterol
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Method Validation
GC vs. Q-TOF MS
GCGCQ-TOF
Analysis of Arabidopsis WT leavesby GC and Q-TOF MS.
Proof of concept
Only small amounts of plant tissue required Advantages of Q-TOF MS
y p qEasy sample preparation allows for a large number of samplesDirect measurement of conjugated sterols present at low abundances (esp. ASG)
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Method Application
Arabidopsis thaliana
P P+P -P
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Sterol Analysis in Arabidopsis under Phosphate Deprivation
WT pho11, WT leaf (+P)2, pho1 leaf (-P)
3, WT leaf +P4, WT leaf –P
N j h i h i i f l i
, ea
5, WT root, +P6, WT root, -P
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
No major changes in the composition of sterol species.
(here: free sterols, distribution similar in SE, SG and ASG)+ P -P
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Sterol Analysis in Arabidopsis under Phosphate Deprivation
*WT pho1
1, WT leaf (+P)2, pho1 leaf (-P)
3, WT leaf +P4, WT leaf –P, ea
5, WT root, +P6, WT root, -P
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
+ P -P
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Sterol Analysis in Arabidopsis under Phosphate Deprivation
WT pho11, WT leaf (+P)2, pho1 leaf (-P)
*
3, WT leaf +P4, WT leaf –P, ea
5, WT root, +P6, WT root, -P
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
+ P -PSterol Ester amounts increase during –P conditions, b t l i l t i t il ltbut also in plants grown in sterile culture:Sterol Ester accumulate during different stress conditions.
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Sterol Analysis in Arabidopsis under Phosphate Deprivation
WT pho11, WT leaf (+P)2, pho1 leaf (-P)
*
3, WT leaf +P4, WT leaf –P, ea
5, WT root, +P6, WT root, -P
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
+ P -P
Glycosylated sterols (SG ASG) are increased during P conditions in leavesGlycosylated sterols (SG, ASG) are increased during –P conditions in leaves
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Sterol Analysis in Arabidopsis under Phosphate Deprivation
ASGASG
WT pho11, WT leaf (+P)2, pho1 leaf (-P)
3, WT leaf +P4, WT leaf –P, ea
5, WT root, +P6, WT root, -P
16:0 ASG decreased and 18:3 ASG increased during Pi deprivation
+ P -P
16:0-ASG decreased, and 18:3-ASG increased during Pi deprivation.
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Conclusion
Arabidopsis thaliana
SG and ASG increased at – Pi
Glyosylated sterols replace phospholipids. (also see: Anderson et al. 2005 JBC,Tj ll t ö t l 2009 FASEB J)Tjellström et al. 2009 FASEB J)
18:3-ASG increased,16:0-ASG decreased at - Pi
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University of BonnInstit te of Molec lar Ph siolog and Biotechnolog of Plants (IMBIO)Institute of Molecular Physiology and Biotechnology of Plants (IMBIO)
Vera WewerIsabel DombrinkKatharina vom DorpPeter DörmannPeter Dörmann