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

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

Conjugated Sterol lipids

Sterol Distribution in the Plant Cell

Free Sterols (FS)

HO

Sterol Esters (SE)

Sterol Glucosides (SG) Acylated Sterol Glucosides (ASG)

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

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)

Sterol Analysis: Workflow

TLC / GCQ-TOF MS

Wewer et al 2011Wewer et al., 2011J.Lip.Res.

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

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

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

ASG Molecular Species

ASG Arabidopsis leaf

16:0-Glc-Sitosterol

18:3-Glc-Sitosterol18:3-Glc-Sitosterol

18:2-Glc-Sitosterol

SE Molecular Species

SEArabidopsis leaf

18:3-Sitosterol

18:2-Sitosterol

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)

Method Application

Arabidopsis thaliana

P P+P -P

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

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

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.

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

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.

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

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